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315 Cards in this Set
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Compare and contrast beta-oxidaton and fatty acid biosynthesis.
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-beta-oxidation also occurs in peroxisome
>shortens a C-chain 2-C’s at a time -Biosynthesis is reverse of beta-oxidation >stick 2-C’s on at a time -CoA carries chain in beta-oxi, acyl carrier protein is unique to biosynthesis |
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What is the first step of beta-oxidation of FA's?
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-FA movement to mitochondria is highly regulated
-1st stpe:AMP attached to lipid >SH=thiol -acyl=generic hydrocarbon (acetyl=2 C) -acylCoA also substrate for FA biosynthesis |
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How is the acyl FA transported into the mitochondrion?
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-separate pools of CoA in mitochondria dn cytosol (pools remain separate due to carriers)
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What is the 1st step of beta oxidation within the mitochondria?
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**need to know alpha and beta carbons**
1)AD oxidizes chain (double-bond between alpha and beta) >reduces FAD to FADH2 >2 ATP’s generated by e- transport chain (ETC) 2)EH converts double bond to alcohol >intermed. Step 3) HAD converts alcohol to ketone (on beta carbon) [1-3 are still attached to CoA] 4) KT-> another mol of CoA attack beta ketone to form new FA chain 2 C’s shorter and generates acetyl (2-C) CoA -FA goes though cycle further to shorten 2 C’s each round -1-> dehydration=oxidation to double bond |
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What steps of the TCA cycle resemble beta-oxidation of lipids?
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What are the steps that break the carbon-carbon bond and reduce the fatty acid by 2 carbon atoms at a time?
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-KT has free thiol w/available proton
>attack ketone and breaks C-C between alpha and beta C >acyl attached to KT=intermed. >carbanion isomerizes to phenol -how to separate C-C bonds of acetyl CoA -almost 2X more ATP from FA’s than glucose |
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What is the first step of FA biosynthesis?
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-malonyl CoA=CoA+CO2
>take nrg to make=rate ltg state -ACC has to polymerize to be active -lipid biosyn. turned off when glucose needed for body’s needs -carbonate transferred to biotin which interacts w/acetyl CoA |
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What is the net result of peroxisomal beta oxidation?
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Peroxisomal b oxidation is on longer chain carbons (>C22). Once chains are short enough for mitochondial oxidation, they are transported there. Only 2 ATPs per 2 carbon cycle are generated in the peroxisome.
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What is the overall pathway of FA biosynthesis?
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What is the 1st step of FA biosynthesis?
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-need acetyl CoA
>MAT transfer it to a carrier protein >MAT also generates malonyl-ACP -malonyl- and acetyl-ACP combine to form acetoacetyl-ACP >condensation and loss of CO2 >loss of CO2 drives rxn forward |
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After production of acetoacetyl-ACP, what are the subsequent steps of FA biosynthesis?
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-KR reduces ketone to alcohol
-DH remove OH to form double bond between alpha and beta C -ER reduces double bond >NADH oxidized to NADP -6 mre cycles to yield palmitic acid |
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How do you finish a FA chain?
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-ACP=acyl carrier protein
-TE liberates palmitic acid from carrier protein |
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In addition to compartmentalization, what other way have organisms developed to localize metabolic enzymes?
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In what conformation are proteins translocated across membranes?
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-native conformation not necessarily lowest free nrg
>may be off-path formations with similar free nrg |
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What are the 2 modes of protein translocation?
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Figure shows co-translation translocation to rER and post-translational pathway to mitochondrion, peroxisome, and nucleus. Proteins without signals remain in the cytoplasm (default location) and are not trafficked to a specific cell compartment. In general, the same protein does not go to the nucleus, peroxisome, and mitochondrion as suggested by this figure. Proteins targeted to the nucleus, peroxisome, and mitochondrion have different signals.
-2 modes of translocation >w/or after translation -peroxisome capable of transporting folded proteins |
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What are the translocon of rER, mitochondria, and peroxisomes?
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Translocation means protein movement into or across membrane. The translocation complex that constitutes the specific site of protein translocation across the endoplasmic reticulum is a heterotetramer of alpha, beta, gamma and delta subunits (translocon-associated proteins or TRAPs). In yeast it is a heterotrimeric complex consisting of Sec61p, Sbh1p, and Sss1p. The Sec61 complex functions in cotranslational and posttranslational translocation events.
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What are the different types of mitochondrial sorting signals?
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Like other signals, matrix targeting sequences in different proteins do not have the same sequence, but share structural similarities. Peripheral membrane proteins of the mitochondrial matrix (e.g., subunit 4 of cytochrome C oxidase) have a cleaved matrix signal like soluble matrix proteins.
-red seq=TM domain >target to intermem -all 3 ex’s use same translocase machinery |
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Describe the mechanism of matrix import.
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Matrix proteins are unfolded - linearized - during transport process. GIP = General Import Pore (Tom40 trimer). Subunit of Tim23 complex has alpha helical extension that contacts/inserts into OM to keep TOM and TIM aligned at contact site. Role of handoff between receptors in performing quality control in recognition of mitochondrial proteins.
-protein maintained in import-competent state by hsc70 >unfolded state -TOM=translocase of outer mem. (TIM=inner mem) -receptor recognizes sseq and delivers to general import pore >pore=non-specific, receptor=specific >physical assoc. between Tom and Tim >polypep chain can thread thru GIP (gen import pore) and Tim when bound together -matrix targeting seq form amphipathic helix >but no consensus seq -GIP and Tim line up (GIP=Tom40) -polypep arrives in matrix linearly >matrix chaperones assist in folding -matrix targeting seq=positively charged >matrix= negatively charged >electrophoretic property of matrix targeting >may want to remove + charge signal to maintain matrix electronegativity |
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Describe Hsp70.
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Hsp70 family members (including Hsc70s) keep mitochondrial precursor unfolded.
-Hsp’s recogniz hydrophobic patches and weakly bind >hydrolyze ATP slowly >diff affinity for hydrophobic patches when ATP vs. ADP bound |
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Describe Mitochondrial Signal Recognition?
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The import receptor is a complex of Tom20 and Tom22. Only part of Tom20 is shown above. For a signal to carry information, the information in the signal must be interpreted by machinery that includes a receptor that recognizes the information. For a zip code to work, a person or device must be able to recognize and sort the zip code appropriately. From Pfanner, 2000, Curr. Biol., 10:R412.
-analogous to ½ of a Leu zipper |
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Describe protein translocation and processing across the mitochondrial membrane.
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Contact sites are often not shown in diagrams of mitochondria and require high magnification EM to be visible. Tim23 actually links the IM and OM and is one of the few membrane proteins in cells that spans two different membranes. From Pfanner, 2000, Curr. Biol., 10:R412.
-electrophoretic effect of import |
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Where does the nrg for mitochondrial import come from?
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Proton motive force is required for movement through TIM complex in IM. There is no proton motive force across OM. There are exceptions to each of the requirements above.
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How are proteins targeted to the inner mitochondrial membrane?
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The TIM complex has a “side door” to allow “exit” into lipid bilayer. Second signal is a type of 'stop transfer' signal.
-Tim complex recognizes a 20 aa stretch of hydrophobic residues >stop transfer/mem seq >targets that protein to bilayer vs. import to matrix |
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How are proteins targeted to the inner mitochondrial membrane?
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The TIM complex has a “side door” to allow “exit” into lipid bilayer. Second signal is a type of 'stop transfer' signal.
-Tim complex recognizes a 20 aa stretch of hydrophobic residues >stop transfer/mem seq >targets that protein to bilayer vs. import to matrix |
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How are proteins targeted to the intermembrane space?
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Path shown above is the major pathway. The older literature mentions a “conservative” pathway that involves targeting to the matrix followed by “re-export” to the intermembrane space across the IM. This pathway is likely to be a specialized pathway used by relatively few mitochondrial proteins. Heme is required for cytochrome b2 function.
-protease in inner mem. cleaves soluble targeting seq |
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Describe mitochondrial outer membrane proteins.
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ATP is required for porin import, but mechanism is not known.
-many outer me. pro’s insert relatively spontaneously >no processing protease available in intermem. space |
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What are some notable features of ER signal seq's?
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-hydrophobic core (vs. amphipathic helix of mito-targeted pro’s)
>no consensus seq |
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Explain Signal Hypothesis for Secretory Protein Targeting.
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Caesar Milstein later received the Nobel Prize with Georges Köhler for the development of monoclonal antibodies.
-signal translocated first (N-term) -gating/regulation of translocation -IP3 opens Ca channels in ER >constitutively opened translocons would allow Ca to leak out |
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Explain the data.
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-mRNA for pre-prolactin (pre=contains localization signal)
>in absence of rough microsomes just get pPL >in presence of rough microsomes also get PL -proteinase K added outside of mem >degrades what’s outside of mem >pPL is sensitive whereas PL is resistant >in different compartments -Triton solubilizes mem and all proteins are degraded >proves PL was in the microsome |
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Explain targeting to the rough ER and subsequent translocation.
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Post-translational translocation into the ER occurs in certain eukaryotes, but will not be presented. Membrane proteins exit sideways from the translocon.
-SRP=signal recognition particle=receptor >recognizes amino-terminal signal >attaches to receptor in ER mem to tether ribosome >ribosome attaches to translocon and opens it >SRP released -signal peptidase=mem-bound protein that cleaves signal pep -SRP and SRP receptor=GTPases >GTP regulates conformational changes needed for signal recognition -SRP recognizes multiple signal seq w/hydrophobic cores >SRP is soluble whereas Tom is mem bound >SRP allows ribosome to pause translation so it can be relocated |
Nascent polypeptide chain emerges from free ribosome
Signal recognition particle (SRP) binds to signal sequence on nascent protein chain and arrests/pauses translation SRP binds to SRP receptor (SR) in the rER membrane Ribosome binds to translocon, SRP dissociates from the ribosome (GTP used), translation resumes, SRP recycles Signal sequence is removed in lumen by signal peptidase In rER lumen, protein folding is assisted by chaperone proteins and N-linked glycosylation takes place Translation terminates and translocon closes Folded secretory protein is ready for export from the rER |
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Describe the signal recognition particle.
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For all signals there is a receptor. RNA “tail” interacts with ribosome to limit access to A site of ribosome (where amino acid - tRNA binds). P site in ribosome binds peptidyl chain. E site in ribosome is exit site.
-SRP has RNA assoc. w/it -p54 is highly conserved >Methionine hydrophobic “fingers” grab the hydrophobic core >opposite end reaches into amino-acyl end of ribosome (active site) >blocks elongation **signal recognition and translation pause** |
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Explain the data.
(pPL=pre-prolactin, PL=pro-lactin) |
Assay on right was used to screen fractions of purification scheme for SRP activity.
-RM stripped using high salt and EDTA >leaves mem in tact but strips off perpheral pro’s -cotranslational translocation assay using stripped mem and adding back SRP -stripped mem show a little bit of residual processing capability >adding SRP completely reconstitutes cotranslational translocation >protein protected from protease unless solubilized by Triton **test question based on this type of assay** |
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How is the translocon regulated?
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TRAM may be involved in translocation pausing.
-multi TM domains of multiple sec61 -open vs. closed conformations >many ER luminal proteins are glycosylated -panel 2=side exiting for mem incorp. pro |
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How is the ER signal peptide cleaved?
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Potential problems with residual transmembrane, hydrophobic peptides.
-SPase=active site on luminal side -SPPase cleaves mem-bound signal to prevent its accumulation |
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Describe the transfer of the glycosylation unit in the rER.
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-signal trapped in translocon
-Asn glycosylated >N-linked glycan >sugar built on dolichol and transferred en masse to N >for soluble (lumenal pro’s) >done by oligosaccharyltransferase >glycan further modified later |
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What kinds of membrane topologies are possible for ER TM proteins?
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Topology of proteins of ER, Golgi, lysosome, endosome, plasma membrane is established in ER.
-Type I=N=term inside, Type II=N-term outside |
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What topogenic sequences are used in ER TM proteins?
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How are Type I ER TM proteins inserted?
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Glycosylation not shown. Some type 1 membrane proteins do not have a cleaved signal.
-red=hydrophobic stretch >recognized by translocon and moved into plane of bilayer >translation finished on cytoplasmic face of ER **2 seq for Type I mem pro’s** |
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How are type II ER TM proteins inserted?
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Interaction between SRP, SR, nascent chain, and Sec61 complex may be involved in loop formation. Almost all membrane proteins are inserted into the membrane in the rER.
-signal anchor seq forms hairpin >charge dist. important for orientation >translation finished in lumen **if charge dist. flipped you’d und up w/Type I orientation |
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How are multi-pass ER TM proteins inserted?
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First signal-anchor is recognized by SRP; others may not require SRP. Hydropathy plots reveal the location of topogenic sequences in multipass (and single pass) membrane proteins.
-same topogenic seq rules can be used to make a multi-pass pro |
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Explain how to experimentally reconstitute a cotranslational translocon.
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-liposome=double-mem structure (bilayer w/2 leaflets)
>aqueous interior and exterior >micelles=single leaflet surrounding hydrophobic molecule -dialysis removes detergeant >allows lipid to spontaneously assoc. w/liposome >pro. can also spontaneously insert into liposome w/hydrophobic domain in mem in presence of mild detergent -pro+mild detergent + liposome=reconstitution of TM pro’s in a mem -TRAM - translocating chain-associating membrane protein is required for specific signals (i.e., some signals function independent of TRAM); side door to mem., TRAM may be involved in translocation pausing. |
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How are nearest neighbors in translocation machinery determined?
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-nearest neighbors questions addressed using this method
>achieved via cross-linking |
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How does yeast protein translocation to the ER differ from humans?
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-yeast break the rulescan post-translationally translocate (rt. panel)
tetrad analysis: -WT and mutant (SRP genes KO’d) allowed to go through meiosis >4 colony types result -WT yielded big colonies whereas mutant colonies grew VERY slowly >mutant slow growth due to inefficient synthesis and secretion of peptides due to loss of SRP (had to use SRP-independent pathway for pro transport across mem) -mammals don’t post-translationally translocate |
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Summarize the Protein Sorting Pathways.
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How is protein folding in the ER different than in the cytosol?
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rER is “entrance” to secretory (exocytic) pathway and delivers proteins to organelles in the secretory and endocytic pathways and the cell exterior. Proteins are made by membrane-bound ribosomes in the rER and fold and/or assemble in the rER lumen.
Problems: Proteins fold incrementally as they are threaded through the Sec61 complex and emerge in the ER lumen ER Lumen is an oxidizing environment that favors disulfide (S–S) bond formation (cytosol is a reducing environment) ER lumen contains a high concentration of incompletely folded proteins (creates potential for protein aggregation) Ca++ concentration is high in ER lumen (low in cytosol) Folding is impaired by mutations and stress (e.g., heat, cold, heavy metals, toxins, nutrient deprivation) Solutions: ER has mechanisms to assist protein folding and assembly ER senses unfolded proteins and communicates to nucleus ER has quality control mechanism that retains incompletely folded proteins to give them more time to fold properly ER has mechanism to degrade incorrectly folded proteins ER is a folding compartment. Polypeptides arrive and fold in the ER lumen in much the same way that they arrive and fold in the cytosol. However, the environment of the rER lumen is more similar to the cellular exterior than the cytosol. Concentration of calcium in the rER lumen is ~0.2 µM (cytosol is ~1 mM). rER lumen is oxidizing whereas cytosol is reducing. |
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What are the different fates of secretory proteins from the ER?
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VTC – vesicular tubular clusters. Not all delivery to lysosomes is for degradation of misfolded proteins. Lysosomal resident proteins are normally delivered there.
-soluble, secretory prot. delivered from Golgi to PM >usually glycosylated (N-linked) >modified in Golgi -prot. length of time in ER can be increased >KDEL receptor trafficked to and from Golgi (not on secretory prot.’s) >BiP is hsp70 family member foldase with KDEL seq >retrotranslocated proteins are targeted for degradation (from ER to get ubi.) |
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Describe topology of TM proteins in the rER as it pertains to their final conformation w/in the PM or other membrane?
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Transmembrane proteins of the plasma membrane are inserted into the membrane of the rER. During this process, the correct topology of the plasma membrane protein is created in the membrane of the rER. The rER is the only organelle for inserting proteins across or into cellular membranes. All cell membranes have 2 faces and no edges. Cytosolic face is in contact with cytosol; exoplasmic face is not. Exoplasmic face on cell surface is equivalent to exoplasmic face of lumen of organelles
There are no ribosomes associated with the plasma membrane (i.e., transmembrane proteins of the PM are not synthesized there). Soluble cytosolic proteins are not synthesized by ribosomes on the rER. -lumenal (ER) face of TM prot. equivalent to extracellular space |
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What are the rER lumenal proteins?
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Chaperones:
BiP - Hsp70 family chaperone Protein disulfide isomerases - PDI Peptidyl prolyl isomerase - PPIase Calnexin - transmembrane Ca++ binding protein Calreticulin - soluble Ca++ binding protein Glycosylation-related: Oligosaccharyltransferase (OST) - N-glycosylation Glucosyl transferase - UGGT Glycosidases - α-glucosidase I, α-glucosidase II, ER-mannosidase I, endomannosidase Retrieval: KDEL receptor (Soluble proteins in rER lumen; membrane proteins have rER luminal domain. UGGT – UDP-glucose glucosyl transferase. -OST part of Sec61 complex) |
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How is protein folding in the rER promomted?
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Retention
Prolonged association with chaperones retained in rER Disulfide cross-links with chaperones N-linked oligosaccharide signals Failure to present an export signal Failure to conceal a retention signal Insolubility Retrieval Return to rER after “escape” of unfolded proteins Quality control is based on structure rather than function! Treatment with reducing agents (e.g., DTT) can release proteins bound to chaperones via disulfide bonds. The RxR motif on the ATP sensitive potassium channel monomer is an example of a retention signal in the ER lumen. Difference between retention and retrieval is a matter of thinking in terms of steady-state or dynamic mechanisms. |
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What is BiP?
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BiP originally identified by co-IP with unassembled IgG HC. BiP is also known as a glucose regulated protein (GRP78). Cellular response to glucose deprivation includes: induction of GRP78 and other chaperones, and repression of N-glycosylation. Similar responses are upregulated by RNA tumor virus infection, tunicamycin (inhibitor of N-glycosylation), dithiothreitol, calcium ionophores, overexpression of unfolded mutant proteins (e.g., influenza hemagglutinin). Heat shock does not lead to the same response.
-assists with Ig folding -BiP=Hsp70 family member >ATP/ADP determines affinity for binding (but always binds weakly) >KDEL seq-containing >one of the most abundant proteins in ER of exocrine cells >no seq specificity of binding partner |
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How does BiP assist in protein folding?
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BiP functions in the same manner as cytosolic Hsp70 (which keeps mitochondrial protein precursors in an import-competent state for example) and mitochondrial Hsp70 (which assists with folding in the mitochondrial matrix). Gene for BiP has UPR element. In yeast, Bip has been implicated in “thermal ratchet” movement of the polypeptide chain during post-translational translocation into the rER lumen (i.e., cycles of Bip binding prevent “backsliding” of the polypeptide chain in the Sec63 complex). The Sec63 complex in yeast has additional proteins for post-translational translocation into the rER. Folding does not require covalent bond formation.
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What is protein disulfide isomerase?
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Disulfide bonds are covalent. A special molecular pathway is needed to prevent formation of incorrect covalent S-S bonds. PDI assists intermolecular disulfide bond formation. PDI is present in all eukaryotes. Gene for PDI has UPR element. Ero1 protein in ER lumen converts reduced PDI to oxidized form for reuse. Sulfhydryl groups are also known as thiol groups. Proinsulin forms three specific disulfide bonds before processing to yield active insulin. ERp57 is a member of the protein disulfide isomerase (PDI) family. Unlike archetypal PDI, ERp57 specifically interacts with newly synthesized glycoproteins. ERp57 forms complexes with calnexin and calreticulin. Calnexin is a type 1 membrane protein (single pass with amino terminus in lumen). ERp72 is another thiol isomerase family member.
-Ig’s need correct S-S bonds -PDI contains Cys residues that can form S-S >allows prot. to find free nrg valley while it reshuffles S-S >mechanism to allow exchange between oxidated and reduced states >can also catalyze intermolecular S-S |
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What is PPIase?
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Peptide bond has 'partial double bond' characteristics. Gene for PPIase has UPR element. Proline is sometimes called an imino acid. But, the imino group is >C=NH. So, proline is not really an amino or an imino acid because it lacks a C=N. Proline is a secondary amine containing amino acid. Cyclosporin A inhibits PPIase activity.
-facilitates rotation around a proline bond |
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How are proteins N-glycosylated in the rER?
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Glycan is preassembled on a special lipid, dolichol pyrophosphate. Addition of carbohydrate to protein forms a glycoprotein. Sequence Asn-X-Ser/Thr is addition site recognized by oligosaccharide transferase (OST). OST is near the Sec61 translocon. O-linked oligosaccharides are also added in ER and usually consist of <10 sugars (e.g., collagens, ABO blood group antigens added to glycoproteins and glycolipids on RBCs). Single letter code for asparagine is N. N-glycosylation does not happen anywhere else in the cell except the rER lumen.
-N-linked glycan=QC -OST catalyzes 1 (transfers from dolichol to Asn) -2 and 3 glucosess cleaved slowly >ultimately all glucoses and 1 mannose must be cleaved to leave ER |
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Describe glycan assembly in the rER.
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Transfer to protein by oligosaccharyl transferase takes place all-at-once. Dolichol pyrophosphate is a long chain polyisoprenoid lipid (can flip-flop).
-shows how glycan is assembled >dlichol is a specialized template/holder that can flip across mem. |
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Describe the N-linked glycan attached in the rER.
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Sequence Asn-X-Ser/Thr is addition site recognized by oligosaccharide transferase (OST). The high mannose form of the N-linked glycan is sensitive to digestion with endoglycosidase H. The complex form is resistant to digestion.
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What are the functions of N-linked glycosylation?
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Folding - N-linked glycan influences protein folding in ER lumen by limiting and/or favoring certain protein conformations
Quality control - N-linked glycan binds to calnexin, which mediates retention in the ER lumen during protein folding Protection - N-linked glycan may limit access of other proteins (e.g., proteases) to backbone of mature protein Folding, quality control, and protection may explain why N-linked glycans are added all at once. The glycan needs to be functional immediately after covalent attachment. Cell-cell interactions - mature N-linked glycoproteins at the cell surface may interact with carbohydrate-binding proteins (e.g., selectins) on the surfaces of other cells For many years, function was not clear, and a role in “stability” of protein was proposed. Selectins function in extravasation of leukocytes from blood into connective tissue during inflammation. Leukocytes secrete proteases that degrade the basement membrane, allowing them to escape the capillary endothelium – a process known as diapedesis. |
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Describe quality control in the rER.
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Like calnexin, calreticulin is also an rER luminal glycoprotein binding protein (not shown). Both are calcium-binding lectin-like proteins. Gl
-G=N-linked glycan -calnexin=lectin in ER lumen (as is calreticulin) >bind Ca >bind N-linked glycan that are mono-G >retain pro’s in ER -UDP-Glc adds back G if your still not folded to keep in ER |
Calnexin (transmembrane) and calreticulin (soluble) - rER luminal chaperones with lectin domains that bind monoglucosylated N-glycans (Glc1Man9GlcNAc2)
Glucosidase II cleaves glucose slowly, rendering the glycoprotein unable to bind calnexin or calreticulin Glucosyltransferase (UGGT, UDP glucose glycoprotein glucosyltransferase) specifically recognizes unfolded substrate glycoproteins and adds glucose to high mannose glycans to re-establish the calnexin / calreticulin interaction UGGT ignores proteins that are completely folded Positions of N-linked glycans may have evolved to direct the attention of UGGT to specific regions of particular proteins Co-IP (pull down) of nascent or unfolded proteins with anti-Cnx or anti-Crt antibody is inhibited by prior treatment of cells with tunicamycin (blocks N-linked glycosylation) or α-glucosidase inhibitors |
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Describe the unfolded protein response pathway.
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It is not clear where Hac1 mRNA is spliced. Evidence is for both splicing in nucleus as well as in cytosol. Two other transmembrane proteins signal from the ER lumen to the cytosol. PERK consists of a luminal domain that is activated by misfolded proteins and a cytosolic kinase domain that phosphorylates and inactivates translation initiation factors in the cytosol to reduce cotranslational translocation into the ER lumen. ATF6 is a transmembrane protein that has a luminal domain that sense unfolded proteins and a cytosolic domain that undergoes regulated proteolysis to release the cytosolic domain from the membrane to allow it to function in regulating gene expression.
Ire1 dimerization is inhibited until bound BiP is titrated by unfolded proteins. Splicing would occur in nucleus if Ire1 were localized to the nucleoplasmic face of the rER (i.e., nuclear envelope). -ER can respond to accumulation of unfolded proteins >signal nucleus to make more foldases >occurs during up-regulation of transcription (ex. immune response) -TM kinase is dimerized/activated by unfolded proteins >ribonuclear domain stimulates processing of pre-mRNA that become gene regulating proteins (TF) -BiP has UPRE |
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What do the UPR genes do?
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The UPR involves many genes and may be more appropriately considered to be a mechanism for regulating secretory pathway ‘capacity’. The implication is that there is a linkage between the UPR & ERAD. This is consistent with synthetic lethality between UPR and ERAD mutants (no great surprise here since both involve the presence of garbage in the ER, gratifying nevertheless).
-UPR genes increase robustness of secretory pathway >to handle increase in folded proteins that need shuttling |
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What is ERAD?
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The UPR involves many genes and may be more appropriately considered to be a mechanism for regulating secretory pathway ‘capacity’. The implication is that there is a linkage between the UPR & ERAD. This is consistent with synthetic lethality between UPR and ERAD mutants (no great surprise here since both involve the presence of garbage in the ER, gratifying nevertheless).
-UPR genes increase robustness of secretory pathway >to handle increase in folded proteins that need shuttling |
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Describe how the ER regulates protein degradation?
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Glucosidase II serves as a timer for revaluation by UGGT. ER mannosidase I serves as a timer for rER residence time. From Tsai B, Ye Y, Rapoport TA. 2002. Retro-translocation of proteins from the endoplasmic reticulum into the cytosol. Nat Rev Mol Cell Biol. 3(4):246-55. Review. PMID: 11994744.
Eukaryotes possess an ER-localized ER mannosidase-I like gene that contains an amino acid substitution that blocks its activity. Inactivation of the yeast gene does not affect N-glycan trimming, but reduces degradation of mutant secretory proteins. Overexpression of mammalian gene (EDEM) binds to and increases degradation of PI Z (misfolded a1-antitrypsin). Interpretation: EDEM is a Man8GlcNAc2-specific lectin involved in recognizing the Man8 isomer for ERAD. -enzymes in N-linked glycan pathway participate in degradation too -lectin recognizes minus-1 mannose proteins >mannosidase 1 in lumen acts as QC for proteins |
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Where do the different glycosidases cleave the N-linked glycan signal?
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Describe coated vesicle formation.
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-coat assembly regulated by G-pro’s
-cargo receptors determine what gets transported -coat pro’s allow positive sorting (decide what returns to ER) |
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Describe the different coats on transport vesicles.
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Clathrin coats Secretory pathway - form vesicles leaving the trans Golgi network for delivery to late endosomes
Endocytic pathway - forms vesicles leaving the plasma membrane for delivery to early endosomes Certain cargo proteins (but not all) from secretory and endocytic pathways are delivered to lysosomes COP I coats Retrograde transport - from cis Golgi network to ER, and retrograde transport within Golgi stack, including retrograde transport from the trans Golgi network COP II coats Anterograde transport - from ER to cis-Golgi network |
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Describe ER to golgi transport.
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The fact that a minus end directed motor moves VTCs is consistent with the location of the CGN proximal to the MTOC (centrosome).
-neg. end of MT coated w/gamma tubulin and attached to MOTC >golgi usually near MTOC >ER usually closer to PM (by pos end of MT’s) >dynein transfers protein from ER to cis-Golgi >kinesins take proteins from trans-Golgi to secretory vesicles or PM |
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How are ER resident proteins retrieved? What are some examples of ER-resident proteins that use this system?
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Examples of KDEL sorted ER proteins: BiP, PDI. What causes KDEL receptor to be trafficked backwards and forwards? Interactions of cytosolic domains of KDEL receptor with coat proteins. What is responsible for changes in pH in lumen across secretory pathway? V-ATPase. KDEL is a type of signal for retrieval sorting. KDEL receptor also monitors the the Golgi stack and TGN (not shown above).
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Describe glycan processing in the Golgi.
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Endo H cleaves between the two proximal GlcNAc residues. Endo H is not present in the Golgi. Collagen alpha chains are O-glycosylated in collagen. Nucleoporins in mammalian cells are also O-glycosylated by a distinct cytoplasmic pathway.
-tag goes from containing 2 types of sugars to multiple sugars as transported through the Golgi >multiples sugars considered “complex” form -no endo-glycosidase H in Golgi >used in experiments to determine modification stage of a protein (e.g. has it passed through the Golgi?) |
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Describe glycan processing in the Golgi.
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Endo H cleaves between the two proximal GlcNAc residues. Endo H is not present in the Golgi. Collagen alpha chains are O-glycosylated in collagen. Nucleoporins in mammalian cells are also O-glycosylated by a distinct cytoplasmic pathway.
-tag goes from containing 2 types of sugars to multiple sugars as transported through the Golgi >multiples sugars considered “complex” form -no endo-glycosidase H in Golgi >used in experiments to determine modification stage of a protein (e.g. has it passed through the Golgi?) |
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Interpret the data.
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VSV G protein becomes endo H resistance (i.e., acquires a complex N-linked glycan) ~20 min after synthesis indicating that the VSV G protein passes through the medial cisternae of the Golgi apparatus ~20 minutes after synthesis
VSV G protein is a viral membrane glycoprotein, containing two N-linked oligosaccharides that is synthesized in the ER and then transported to the host cell plasma membrane via the Golgi apparatus -pulse-chase experiment -VSV G=glyco protein >watch synthesis and look for changes as it moves through the Golgi >IP’s used to pull VSV G protein down >increase in MW due to N-linked glycosylation in ER -same pro then treated w/ Endo H >capable of cleaving the protein until it reaches Golgi and becomes a complex tag-bearing protein |
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Interpret the data.
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Normal Pi M A1AT is secreted into medium shortly after synthesis
Mutant Pi Z is secreted only slowly and is degraded after synthesis Fig. 1. Retained human AAT variants undergo degradation. Mouse hepatoma cells stably transfected with either the normal human AAT gene (Pi M), the Pi Z variant, or the Pi Null(Hong Kong) variant were subjected to pulse-chase studies as described in “Materials and Methods.” At various time points during the chase period, human AAT was quantitatively immunoprecipitated from cell extracts and medium from each dish. Immunocomplexes were fractionated by SDS-PAGE (8% polyacrylamide) and radiolabeled human AAT was detected by fluorography. An overexposed film is shown in panel PiZ. Abstract: The naturally occurring PiZ and Pi NullHong Kong variants of the human secretory protein alpha 1-antitrypsin (AAT) are retained within an early compartment of the secretory pathway. Intracellular degradation of these transport-impaired secretory proteins is initiated 30-45 min following their synthesis and translocation into the endoplasmic reticulum (ER). Interestingly, the overall rate of degradation of the retained mutant protein is significantly accelerated when all subcellular compartments are buffered at pH 6. In contrast, degradation is virtually abolished when intravesicular compartments are buffered at pH 8. However, despite this pH sensitivity neither lysosomotrophic amines, leupeptin, or leucine methyl ester have an apparent effect on the intracellular removal of the PiZ variant. The inability of a variety of inhibitors of ER-to-Golgi protein trafficking to hinder the degradative process suggests that degradation of the PiZ variant occurs prior to its delivery to the Golgi complex. To biochemically map the subcellular site of the degradation of the retained mutant protein, a recombinant truncated PiZ variant containing the tetrapeptide KDEL at its carboxyl terminus (a signal for sorting luminal proteins from a post-ER compartment back to the ER) was expressed in cells. Attachment of this ER-recycling signal to the recombinant protein prevented its intracellular degradation. These findings indicate that degradation of the PiZ variant occurs following its export from the ER. -PiM=norm (WT) -IP from cells and medium >PiM migrates at secreted glycoprotein band/weight >PiZ gets synthesized and remains in cell (mostly) >some appears in medium after 1 hr. >slow kinetics >amt. in cell declines >retrotranslocated for degradation (liver retrotransloc. can’t keep up with accumulation) >higher band shows some pro being glycosylated |
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Interpret the data.
(cells vs. medium; soluble vs. insoluble) |
PiZ is secreted slowly and degraded in cell following synthesis
Figure 1. Secretion, degradation, and N-linked oligosaccharide processing for synthesized variant PiZ in HEK/Z1. (A) Fluorographic detection after SDS-PAGE of variant PiZ (Z) immunoprecipitated from the NP-40-soluble (s) and insoluble (i) cell lysates (c) and medium (m) after a 15-min pulse of HEK/Z1 with [35S]methionine, and chase for up to 7 h. The discrete mobility shift (*) reflects asparagine-linked oligosaccharide modification during intracellular retention. (B) Results from the pulse-chase experiment are depicted as the percentage of radiolabeled variant PI Z remaining in the cells (open circles), the percentage secreted into the medium (closed circles), and the percentage degraded (shaded circles) at each time point. -quantification of IP -lysate in experiment 1st cleared at low speed -soluble and insoluble, cells and medium -secreted=medium -degraded=diff between cells and medium >of A1AT only |
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Interpret the data.
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Pi Z remains sensitive to digestion by endo H after 1 hour of chase indicating that Pi Z has a high mannose N-linked glycan and remains in ER at this time
-N-linked glycan removed by EndoH even after 1 hr. >stil in ER >at 50 kDa, should already be through Golgi (compare to VSV G) >most pro are through secretory pathway by 1 hr. |
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Interpret the data.
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Figure 3. Newly synthesized variant PI Z undergoes sequential physical interaction with calnexin and grp78/BiP. (A) Calnexin and KDEL immunoblots of variant PI Z (PI Z) immunoprecipitates (Immppt.) generated under steady-state conditions from HEK/Z1 (lane 2) and from the total cell extract (Extract) (lane1), the latter of which was used merely to show the relative migration of the endogenous immunoreactive protein. The immunological detection of calnexin (Cxn), grp78/BiP and grp94 is shown.
Grp94 does not associate with PiZ. -IP again using anti-A1AT -probed with calnexin -bottom blot probed with/anti-KDEL -could be 1 complex with BiP and Calnexin bound, or 2 separate complexes with each individually bound **mobility shift (indicate secretory path movement), Endo H, IP and Westerns for test |
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Interpret the data.
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Figure 3. Newly synthesized variant PI Z undergoes sequential physical interaction with calnexin and grp78/BiP. (B) SDS-PAGE and fluorographic detection of immunoprecipitated variant PI Z (Z) released from a calnexin (Cxn 3 Z) or grp78/BiP (Grp78/BiP 3 Z) immunoprecipitate after a 15-min pulse of HEK/Z1 with [35S]methionine and chase.
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What is an experiment to assay the turnover of endogenous proteins?
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-pulse chase experiment: basis of looking at protein turnover
>radio-labeled amino acid (like s35-methionine, tritiated valine or leucine <general degradation>) >pulse for 16-20 hours >chase w/cold leu (or a.a.) >prevents radio-labeled protein from being reused >do TCA precip. >count soluble radioactivity >count what’s in media and cells -biphasic release of radioactivity (reflects the pro. degradation) -long-lived proteins account for ~80% of proteins >1/2 lives of 16-24 hours >why chase is so long -short-lived proteins are being degraded and synthesized quickly -cells are steady-state in culture -long-lived proteins synthesized slowly (why the chase is so long) -short-lived pro: degraded by ubi-proteasome system and -long-lived pro: predominantly degraded in lysosomal system -pulse-chase gives you the entire pop. of pro. w/in the cell >WB would only tell you up or down-regulation and/or modification to a specific protein |
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What are the ways proteins in the cell are degraded?
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by the ubiquitine/proteasome mechanism or the lysosome
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What type of proteins does ubiquitin regulate? What enzymes are involved?
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What Functions does the Ubiquitin/Proteasome Degradative Pathway perform?
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-A1AT is ex. of misfolded pro.
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Give a brief overview of what the ubiquitn enzymes do.
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-E1 requires ATP
-E3=ubi-ligase (E2 conjugating enzyme) -2 proteasomes: nuclear and cytosolic >core is similar but caps are different >not present in any other organelles |
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What are the components of the proteasome and what function(s) do they serve?
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-proteinases in core
-ubi-pro. recognized by caps and threaded through >caps also stretch out the protein to make a linear pro |
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Describe the mechanism of ubiquitin targeting of a protein for degradation.
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-E1=activates ubi and transfers to E2
-ubi-ligase recognizes substrate |
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How does ubi regulate M-phase?
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-M-phase
-degradation of mitotic cyclins by anaphase promoting complex (APC) -APC=E3 >recognizes mitotic cyclins, cause poly ubi, and destruction of cyclin >APC also regulated >Cdh1 required for APC activity |
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How does ubi regulate S-phase?
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-control of S-phase
-ubi helps regulate cell cycle >predom. regulated by cyclins and kinase -S-phase=DNA syn. >Sic1 must first be degradaded >upon phos., then ubi’d and degraded by proteasome >degradation of inhibitor to allow DNA syn to occur |
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What is the dist. of ubi enzymes?
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-plethora of E2’s (conjugating enzyme)
>differential expression based on cell type -1000+ E3’s id’d! (responsible for pro recognition) -mono-ubi of multiple sites also occurs (vs. poly-ubi) |
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What are the families of E3's?
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-2 families of E3: HECT and RING
>based on domains found in families -HECT E3: anchors ubi to itself (ubi-bound intermed.); then transfers to pro -RINGS: recognize substrate and bind E2; allow ubi to travel from E2 to substrate -ubi is ligated thru C-term onto an amino gp of substrate (of lys or N-term.) >poly-ubi via carboxy gp of ubi and lys substrate -adapter regulates E3 >APC has adapter >adapters found on RING family |
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What are the regulatory mechanisms of Ubiquitin-Mediated Protein Degradation?
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Activation of a degradation signal on the substrate protein.
Activation of the E3 ubiquitin ligase. |
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Describe activation of a degradation signal by ubi.
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-1st step of regulation=modifying substrate
>expose motif, phos, or remove portion of N-term (expose destabilizing N-term aa) -met is first aa of syn pro >but not all pro prod have met due to post-trans modification -kinase phos, phos site recognized by SCF (E3) |
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Describe activation of a ubiquitin ligase.
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-modification of E3
>or mod adaptor -Cdc=yeast -degradation of M-cyclin >APC is an E3 ligases with RING and Cullin subunits that causes degradation of the M-cyclin when in active conformation |
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Briefly describe what each of the ubiquitinating enzymes does.
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Protein ubiquitination requires three enzymes: ubiquitin activating E1, ubiquitin conjugating E2, and ubiquitin ligase E3.
There exists a family of E1 enzymes that can activate ubiquitin and ubiquitin-like proteins in an ATP-dependent manner. The ubiquitin conjugating E2 receives the “activated” ubiquitin from the E1 and delivers it to the substrate or an E3 intermediate. There exists two major E3 groups: HECT and RING. Both recognize the protein substrate, but HECT is able to accept the ubiquitin from an E2 and enzymatically transfer it the the substrate. HECT E3s usually act alone, while RING E3s act alone or in combination with an adaptor such as the cullin family. The regulation of protein ubiquitination can occur by revealing or adding a degradation signal on the substrate protein or activating E3. -only E1 req ATP (rate ltg step) -families of E2s and E3s -cell cycle is one of the main regulated pathways of ubi enzymes |
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Briefly describe USP deubiquitination.
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-aka dub
-ubi specific protease -end up with free ubi (it’s not degraded) -ubi ~8 kDa |
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Describe the proteasome.
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-cap: binding site for ubi, unfolding properties, and 3 usp’s (only 1 actually labeled as a Usp, but all are deubi enzymes)
>usp’s cleave at diff sites >other pro’s anchor the usp’s -USP’s allow ubi to be recycled >cleave them so they’re not degraded by proteasome |
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How do USP's regulate EGF (and other receptors)?
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-another type of regulation
-EGF ubi to allow it to be internalized in pits -ESCRT allows invagination of vesicle -ubi regulates the targeting of the receptor and the endocytosis of the receptor |
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How is ubi involved in NFkB activation?
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Receptors for Toll ligands, TNF, and T and B cell antigens.
-pathway for NFkB activation -receptors interacts with TRAF complex >complex is an E3 >poly-ubi’s pro’s -don’t memorize all steps -ubi destroys inhibitor so NFkB can be in active form*** -A20 and CYLD are the USP’s (shut down 2nd messenger cascade and keep NFkB inactive) >redundancy in what A20 and CYLD do/bind to >A20 is a USP and an E3 (has RING finger domain) >polyubi RIP1 -K-48=Lys 48 that is ubi, as is K63 >mult. Lys moieties that can be ubi |
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What major pathway do USP's regulate?
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What disease processes is USP-regulation involved in?
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cancer and neurodegenerative diseases
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Ubiquitin regulates what major S-phase checkpoint protein?
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-p53 regulated by ubi-pathway
>major checkpoint for DNA syn (S-phase) |
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Interpret the data.
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-if DNA is syn and damaged p53 initiates apoptotic death
>death pathway due to DNA damage >p53 mutated in 70% of cancers >cancers accum DNA damage -2 cell lines w/ or w/o Mdm2 >the one w/o has a lot of p53 -lactasystin=inhibitor of proteasome B)p53 expressed in p53 null cells >lactasystin causes accum of p53 due to inadequate protein degradation (but could be increase syn) A)mutants of p53 that Mdm2 doesn’t bind -degradation of p53 requires proteasome and Mdm2 C) in vitro with GST-p53 and enzymes >UbcH5=E2 >E1 present in all lanes >only when both Mdm2 and UbcH5 present is there polyubi >Mdm2=E3 Degradation of p53 requires E3 Mdm2 ligase and Proteosome Activity |
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What role(s) does ubi play in p53 regulation?
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-poly-ubi of p53
-mono-ubi p53 induces apoptosis -HAUSP is deubi enzyme that maintains p53 in active form -grey=nucleus -downstream activities of active p53 include growth arrest, DNA repair, angiogenesis and apoptosis -MDM2, COPI, and JNK promote degradation of p53 via UPS |
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How does damaged DNA regulate p53?
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-damaged DNA=activation of kinase that stabilizes p53 and allows apoptosis
-Mdm2 also gets phospho so it won’t inhibit p53 -S-phase block (prevents G2) |
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What were the key concepts involved in deubiquitination?
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Deubiquination is critical in regulating protein turnover, protein degradation, cell cycle, endocytosis, second messengers, cell death, and histone functions.
Protein deubiquitination enzymes associated with the proteosome can remove ubiquitins at the distal and proximal ends of a polyubiquitin chain. Deubiquinating enzymes regulate endocytosis by modulating the ubiquitination of receptors and ESCRT. The TRAF complex is an E3 that regulates the activity of NFkB by causing the degradation of inhibitor IkB. The deubiquitination enzyme A20 inhibits the degradation of IkB and thus, downregulating the response. The activity of p53 is regulated by ubiquitination mediated by HECT and RING E3s and deubiquitination by HAUSP. |
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What are calpains and caspases?
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Calpains are a family of calcium dependent proteases that are associated with cytoskeleton, vesicles and plasma membrane.
m-calpain (3 – 50 mM Ca) m-calpain (400 – 800 mM Ca) calpastatin is a calpain inhibitor Caspases are a family of cysteine proteases that have essential roles in apoptosis. -cytosolic enzymes -calpain=ca-dependent poteases >regulated by calpastatin -both are endopeptidases (cleave w/in the pro., neither cleave free aa’s, end up w/pro fragments that must be further degraded…maybe by proteasome) |
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What is the main function of caspase?
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cell death
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What aspect of cancer is the proteasome implicated?
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Cachexia is defined as physical wasting of body mass including the loss of muscle protein.
The loss of muscle protein due to enhanced proteolysis during cancer cachexia is mediated by tumor necrosis factor (TNF a). The expression of ubiquitin and proteasome subunits is increased in skeletal muscle during cachexia. |
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What are the pathways of autophagy?
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microautophagy, macroautophagy, pexophagy, and mitophagy
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What are 2 major regulators of autophagy?
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PI3K and mTor
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What are the molecular events of autophagy?
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sequestration, maturation, and degradation
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Interpret the data. (Inhibitor is a lysosomal depressant.)
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-LC3-II=marker for autophagic vacuoles
-starvation conditions turn on autophagy -inhibitor=lysosomal inhibitor (like chloroquine) >inhibits lysosomal degradation that causes accum. of LC3 -bottom: tandem-tagged LC3 >GFP doesn’t fluoresce under acidic conditions (like lysosomes) but RFP does >GFP localized to autophagic vacuoles that aren’t acidic >RFP localized to acidic and non-acidic vacuoles |
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What is 3-methyladenine?
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an inhibitor of autophagy (inhibits degradation of long-lived proteins that are degraded by lysosomal system); acts by inhibiting PI3K class III (which is an autophagic activator).
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Interpret this data.
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Wortmannin inhibits protein turnover and the formation of the autophagosome.
-worthmannin inhibits PI3 kinase >inhibits presence of autophagic vacuoles and degradation |
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Describe the process of autophagosome maturation.
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Formation of the early autophagosome from the rough endoplasmic reticulum (or phagophore) requires ATP, actin, and the synthesis of proteins.
The formation of the early autophagosome requires a number of autophagy-related genes (ATG). The formation of the autophagosome requires the conjugation of Atg12 to Atg5 in a ubiquitination-like process with E1 Atg7 and E2 Atg10. The expansion of the autophagosome requires the conjugation of LC3 to phosphatidylethanolamine (PE) which is accomplished by the protease Atg4, E1 Atg7 and E2 Atg3. This sequestration event may be selective or nonselective. |
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What are some of the regulators of the sequestration step of macroautophagy?
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-sequestration step is first and regulated
-PI3K activates and mTor inhibit (both have inhibitors too) -phagophore mem comes partially from ER -LC3 labels the mem. -double-mem bound autophagosomes (mito and nuclei are also double –mem) |
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Describe the Atg7 conjugation pathways.
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-similar to the ubi pathway
-Atg 7 activates Atg12 -Atg12 gets conjugates to Atg5 by K >Atg10 is conjugator -LC3 has precursor form >have to have Lys moiety at C-term to be activated >Atg4 removes aa to expose K -LC3 becomes part of autophagic vacuole mem. -top pathway has to proceed before the bottom one >need Atg5 -top step required for formation of autoph. vacuole, 2nd path (LC3) required for expansion of autoph. vacuole >can still get vacuoles w/o LC3 but they’ll be small (can’t engulf organelles and lg. things) -LC3+PE (LC3-II migrates faster on gel due to lipid moiety (PE) |
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Describe the maturation and degradation phase of macroautophagy.
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Early autophagosomes acquire lysosomal membrane proteins including the proton pump thereby becoming acidic late autophagosomes.
Late autophagosome acquire hydrolytic enzymes thereby becoming autolysosomes. Acquisition of these hydrolytic enzymes and formation of the autolysosome require microtubules. |
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What components are associated with the different stages of autophagosomes?
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-V-ATPase=proton pump, LAMP=lysosomal assoc. mem. pro.
-late autoph. has 1 mem. and is acidic (early autophagosome isn’t) -why doesn’t late autoph. vac. fuse w/lysosome >1st bring in LAMPS then hydrolytic enzymes (or else they’d destroy the mem.) -GFP expressed in early autophagosome but not in late or autolysosome (due to acidic nature) |
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Interpret the data.
(LAP=late autophagosome and EAP=early autophagosome) |
Microtubules are required for maturation of LAP to AL
-nocodazole: causes depolymerization of microtubules (taxol freezes polymerized MT’s) -bottom: early, late, and autophagosome >can be defined by EM >starved=mostly EAP >adding nocadozole increases the LAP’s (block in maturation that requires MT’s) |
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What are some of the key concepts about autophagosomes?
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Autophagosome formation requires the ubiquitin-like conjugation of Atg12 to Atg5 done by E1 Atg7 and E2 Atg10.
The expansion of the autophagosome requires the conjugation of LC3 to PE done by Atg4, Atg7, and Atg3. The autophagosome will mature into an acidic autolysosome by fusing with a lysosomes thus delivering its contents for degradation. This maturation requires microtubules. Autolysosomes contain all the hydrolytic enzymes needed to completely degrade lipids, phospholipids, proteins, nucleic acids, and oligosaccharides. The monomeric subunits will be recycled back to the cytoplasm via lysosomal membrane permeases. |
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What are some suppressors and enhancers of autophagy?
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Suppressors of Autophagy:
Insulin and growth factors Amino acids mTor Drugs (e.g., 3-methyladenine, wortmannin) Enhancers of Autophagy: Glucagon and TGFb Starvation Class III PI3 kinases Heat Bacterial pathogens (e.g., P. gingivalis) Protein Aggregates (e.g., PMP22 and Rhodopsin) Drugs (e.g., rapamycin, vinblastine) -autophagy critical for cell survival but needs to be tightly regulated or else it will eat cell up -Akt activates mTor -the drugs mentioned (m-ad and wortmannin) inhibit PI3 kinases |
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How is autophagy expressed in cancer cells?
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It's inhibited to allow the cell cycle to continue unabated.
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What effect do p53 and mTor have on autophagy?
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-don’t memorize
-mTOR is major regulator of autophagy and has many inhibitors and activators >well regulated 2nd messenger cascade >any change in mTor has effect on autophaygy -p53 blocks autophagy |
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How do the PI3K's fit into the autophagic pathway? What proteins regulate them?
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(pictured above is PI3K class III complex)
-class I PI3K inhibits autophagy, class III activates it -assoc. pro. regulate activity -Beclin bound=induction of autophagy >Beclin 1 bound to Bcl2=can’t bind to PI3K to activate it and autophagy is inactive -wortmannin and 3met-ad inhibit PI3K complex |
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Interpret the data
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-luciferase assay
-LC3 WT or mutant that can’t be lipidated hooked to luc >looking for loss of luc -3 me-adenine blocks loss of luc -rapamycin inhibits mTor (mTor inhibits autophagy) -HBSS=starvation (buffered saline) -luc assay is a good marker of autophagy flux |
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What drugs activate autophagy and what are the potential targets that these drugs use?
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-luc assay used to id specific drugs that turn on autophagy
-under fed conditions when autophagy is low |
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What effectors inhibit autophagy?
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-PI3K class I, Akt, and mTor inhibit autophagy
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Summarize the key molecules that regulate autophagy.
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Formation of the early autophagosome is suppressed by amino acids and growth factors (eg, insulin and EGF), and enhanced by amino acids deprivation, glucagon and TGF-b.
PI3K-I / Akt signaling pathway activate mTor to suppresses autophagy. S6Kinase stimulates protein synthesis thereby inhibiting autophagy. mTOR stimulates S6Kinase. When mTOR is inhibited by rapamycin, autophagy is enhanced. Class I PI3 kinases inhibit autophagy while class III PI3 kinases stimulate autophagy. Inhibition of class III PI3 kinases with wortmannin will inhibit autophagy. |
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Explain selective autophagy and key players involved.
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Autophagy can selectively sequester mitochondria, peroxisomes, protein aggregates, intracellular pathogens, ER, etc…
LC3 and p62 are necessary for selecting ubiquitinated damaged mitochondria and protein aggregates. Parkin recruitment leads to mitophagy: Drp1 initiates the sicission of the damage mitochondria. Cytosolic parkin is selectively recruited to uncoupled or dysfunctional mitochondria by the outer membrane protein kinase Pink1. The E3 ubiquitin ligase Parkin ubiquitinates mitochondrial surface proteins. The ubiquitinated proteins are recognized by p62 which binds to LC3 to sequester the mitochondria within autophagosomes. |
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What is Parkin? What does it do?
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-Pink1 recruited to damaged mito and recruits Parkin (missing in Parkinson’s dis.)
-Drp1 req. for fission (releasing damaged part) -Parkin=E3 that ubi surface of mito >ubi recruits p62 (has binding site for ubi and LC3) >LC3 on sequestering mem recognize p62 and engulf mito |
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How are aggregated proteins removed/degraded?
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LC3 and p62 are necessary for selecting these ubiquitinated aggregates for autophagic degradation.
p62 binds to both the ubi and LC3 on the lysosome so that the protein can be engulfed and subsequently degraded within the organelle. |
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How do lactasystin, heat shock, starvation, and 3-me-adenine effect protein aggregation?
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-protein aggregates of ubiquitinated proteins
>lactasystin added to cause aggregation (shuts down proteasomal sys.) -heat-shock and starvation reduce agg. >heat-shock increases number of chaperones to stimulate folding >starvation stimulates autophagy -aggregates don’t disappear with 3me-ad -prevention and removal of aggregates |
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What is the fate of aggregated proteins?
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-pro must be unfolded to go into proteasome
>can’t handle large aggregates >autophagy handles large agg. >p62 binds to agg. (good target of selective autophagy) |
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How is autophagy related to cancer?
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Cancer is a malignant tumor of unlimited growth that expands locally by invasion and systemically by metastasis.
Some tumor cells are resistant to nutrient deprivation and chemotherapy because of their ability to activate cell survival pathways such as autophagy. Such tumors have elevated LC3 and Atg4. Autophagy provides the cell with an avenue to remove damaged organelles such as mitochondria and to recycle nutrients such as amino acids and sugars. Other tumor cells have mutated ATG genes, such as the tumor suppressor protein Beclin-1 that is a functional homologue of Atg6. Enhanced autophagy can result in suppressing growth and promoting death in some tumor cells. -Atg16L binds to Atg5/12 complex -Autophagy can suppress or promote tumor growth. |
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What are some Autophagy-related Proteins (Oncogenes vs Oncosuppressors)?
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Oncogenes (autophagy inhibitors)
Bcl-2, Bcl-XL PI3K-I PTEN Akt1 TSC-1, TSC-2 Oncosuppressors (enhancers of autophagy; may be able to check prodigious cell growth) Beclin-1 Bif-1 UVRAG DAPK-1 p53 |
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How is autophagy implicated in cancer cell survival?
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-cell survival in nutrient conditions
>the cells that survive the best are the ones that can turn on autophagy (critical to their survival as a tumor) >not a lot of nutrients in tumor before vascularization |
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Describe epithelial cells.
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-apical surface=free portion
>can have a no. of specializations (like microvilliabsorb, ciliamove things) -basal surface sits on basement mem. -epithelia adhere to each other (via junctions) -adherens junctionsactin, desmosomesIF’s -connective tissue underlies the epithelium -a lot of transport occurs across endothelial cell -skin is stratified squamous that’s keratinized on top |
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Describe the basement membrane.
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-interaction of proteins (esp. collagen type IV)
-laminin=ECM (vs. lamin which is nuclear) >has many binding sites (above and below the basement mem.) >basal lamina is subdomain of basement mem. |
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Describe the skin.
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-loose conn. tiss. followed by dense
-dermis anchors epidermis -Epidermis and dermis are stratified squamous -cells in dermis have nuclei but epi. doesn’t >epis have been keratinized >keratin=IF >most organelles are gone (via apoptosis) -only thing anchored to basement mem. are the basal cells -predom. attached to each other via desmosomes -ducts are the protrusions (sweat glandssecretory epi) -dynamic tissue >cells are constantly made, mature, and get sloughed -intermed. layer between the dermis and epi is the water barrier (thin line between nuc. and non-nuc. cells) >cells secrete oily substance |
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What is the main route of epithelial repair/regeneration?
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-epithelia have stem cells
>may not be pluripotent (somewhat differentiated) >sit on basement mem. >divide by maintaining attachment to basement mem. -green=desmosomes -yellow=water barrier layer -differentiate as they move up, and eventually die at surface |
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What is the alternative pathway of skin repair/regeneration?
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-stem cells in hypodermis as well
-glands and ducts have stem cells and can replenish skin with these stem cells >important for burn victims >ducts and follicles are contiunous with epidermis |
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What are some classification and functions of connective tissue?
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-embryonic=mesenchymal type tiss. (disappears by birth)
-lymphocytes and macrophages considered CT |
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What are some connective tissue components?
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-fibroblast is primary type of cell
-reticular fibers=collagen -elastic fibers=elastin (pro.) |
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Describe the ECM ground substance.
|
-GAG’s (chondroitan sulfate)
-aggrecan and versican=entire structure >bind to hyaluronan molecule >become hydrated to yield a jello-like mass >collagen acts like a steel rod to provide support to this jelly |
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What are ECM fibers?
|
-I-IV are major types of collagen
-I is found everywhere (dermis, bones, etc.) -II is found ONLY in cartilage -III is reticular fibers (predom in organs of lymph sys.) -IV=basement mem. |
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How do loosed and dense connective tissue differ?
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-LCT has more cells and less ECM
>lots of lymphs (move between blood and LCT) -DCT has more ECM >lots of collagen (fibrous and strong) -LCT has more ground substance and less fibrils in the ECM |
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What is the main connective tissue cell? Describe it.
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-capable of assembling ECM
>makes the components of the ECM |
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Describe the formation and transport of collagen.
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-3 subunits of collagen assembled in rER into procollagen
-packaged and secreted -pro-pep cleaved outside of cells -then assembled into fibril >striation due to staggering -fibrils organized into fibers -use procollagen intracellularly to prevent assembly w/in the cell >many –OH prolines on pro-collagen >important for assembling pro-collagen and fibrils -hyperbaric chambers increase Oxygen in tissues to increase this process >assists in healing (bone or other tissue loss processes) |
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Describe cartilage.
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Specialized connective tissue
Composed of chondrocytes (5%) and extracellular matrix (95%) Chondrocytes occur singly or in isogenous groups (cell nests) synthesize extracellular matrix housed in lacunae (small cavities in ECM Tension-resistant collagen and elastic fibers combined with hydrated proteoglycans (hyaluronan, chondroitin sulfate, and keratan sulfate). Interstitial and appositional growth Avascular and aneural (no nerves or vasculature) Function in support and development of the skeleton. (main fn) Hyaline and elastic cartilage have very limited repair capability. When damaged these tissues will be replaced by bone. Fibrocartilage such as a meniscus can repair itself. |
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What are the types of cartilage?
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-joints have hyaline cart.; helps to cushion; slightly more pliable than bone
>no nerves in hyaline cart., but nerves in bone >decreases with age -elastic has Type II collagen -fibrocartilage is Type I collagen >assists in bone healing >forms callus on broken bone and disappears once bone is in place |
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Describe bone.
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Specialized connective tissue
Composed of osteocytes and matrix of collagen type I fibers and ground substance including proteoglycans, glycosaminoglycans, and noncollagenous proteins cells housed in lacunae and connected by gap junctions osteocytes synthesize extracellular matrix Bone contains mineral crystals of calcium phosphate Appositional growth Vascular and neural Function in support and protection and provides a storage site for calcium phosphate. Bone will be repaired by first forming a fibrocartilage callus which will be replaced by bone. -cartilage can grow from w/in or from outside -bone can only grow from outside (appositional) -bone can be repaired (vs. cart.) |
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How do bones develop?
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-near growth plate of growing bone
-starts with cartilage model >growth of long bones is from growth of cart. model that’s replaced with bone -bone marrow on right -osteoblasts become osteocytes >blasts lay down bony matrix around themselves |
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Describe the lamellae of bone as far as the organization around the vasculature and how nutrients are supplied to distal cells.
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-osteoblasts and –cytes are all interconnected by gap junctions
>allows nutrients to pass thru (cart. has a lot of water and thus gets nutrients easily) >bone has vasculature that supply nutrients to osteocytes that pass on the nutrients to distal cells via gap junctions -circles=haversion systems >constantly torn apart and rebuilt due to phys. stress >gravity directs formation of haversion sys. |
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What are the muscle types?
|
-skeletal muscle
>multi-nucleated cells (aka myofiber) at periphery >traverse tendon-to-tendon (very large) >sarcomere is functional unit >myofibrils >line up end-to-end in myofibers |
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Describe the sarcomere.
|
-anchors at Z-disk on either side
-contractile unit |
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What is the "triad" component of the skeletal muscle cell?
|
Ross 6th Ed., Figure 11.8, p. 321
-t-tubule and sarcoplasmic reticulum (ER) compose triad >t-tubule carries depolarization (sarcolemna) >part of PM of skeletal muscle >causes release of Ca from ER when depolarized >every sarcomere has 2 (one at either end) -mito packed into bundle |
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Describe cardiac muscle.
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-not a fused cell but multiples lined up and connected by intercalated discs
-cells tend to branch -nuclei in middle vs. periphery |
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Describe smooth muscle.
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-no sarcomeres
-contractions coordinated by gap junctions |
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What are the types of neural cells?
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Motor – control effector organs like muscle or glands. Somatic or autonomic
Sensory – reception of sensory information from inside body and environment Interneurons – connect other neurons to each other to allow for intercommunication among complex networks |
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What is the primary supporting cell of the peripheral nervous system?
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Schwann cell
-myelin completely surround axons -basal lamina is grey line surrounding the schwann cell |
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How is the nervous system incorporated into the skin?
|
epidermis sits above meissner’s corp.
-pacinian corp. is in hypodermis (deep within) -nerve endings |
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Briefly describe transepithelial transport of ions and nutrients.
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Membranes are barriers to ions and many biological molecules. Thus, transporters are needed to move ions into and through cells.
HCL secretion requires cytosolic carbonic anhydrase, two ion channels at the apical surface, one ion antiporter at the basal surface, and a H/K ATPase at the apical surface. Glucose uptake requires one symporter at the apical surface, a GLUT2 uniporter at the basal surface, and a Na/K ATPase at the basal surface. |
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Describe the PM ion gradients.
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-Na high outside, K high inside
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Which types of membrane transport systems uses passive or active transport?
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Passive - transport down concentration gradient, no energy required (e.g., channel and carrier)
Active - substrate moves up gradient, energy required, special type of carrier (e.g., ATP-dependent “pump”) Specificity - strong preference for transported molecule |
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What modes can permeases utilize?
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Facilitated transporters can work in either direction, depending on concentration gradient. Some cotransporters can work in reverse, depending on the concentration gradients.
(Conformations - outward-facing and inward-facing; conformations alternate during transport (of each molecule) Mediate passive transport (movement down gradient) Structure - membrane-spanning proteins create pathway for substrate that is protected from hydrophobic core Nomenclature - also known as “transporter” or “permease”) |
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Describe the ion pump.
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Describe how the stomach is acidified.
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-carbonic anhyd. prod. H in cell
>H gets pumped out of cell (against gradient) >K gets pumped inside against gradient which gets pumped back out via a channel -carbonate pumped out via antiporter (with gradient) >cl gets pumped against gradient into cell, which then gets pumped back out via channel -pumps and transporters are polarized >don’t want carbonate to get pumped into lumen where it will buffer the acid -tight junctions prevent H or other ions from leaking thru between cells |
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Describe the Transepithelial transport of glucose.
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-microvilli increase number of transporters at surface
-pump est. gradient (high K and low Na within the cell) -glu pump: pumps Na down gradient and glu against gradient -GLUT2: permease that transports glucose from high to low conc. -polarization of transporters and pumps |
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How does the Na/K pump work?
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-don’t memorize
-both Na and K are pumped against their gradients -the pumping involves phosphorylation and dephos. event coupled w/conformational changes |
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Describe transepithelial transport of proteins.
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Transport of proteins across an epithelium begins with receptor-mediated endocytosis.
Proteins and receptors are internalized into the early endosomes and sorted to transport vesicles. Transcytosis of immunoglobulins occurs in polarized enterocytes. |
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Describe the transport of IgG from the intestinal lumen of the infant (from mother's milk).
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-from apical (intestinal lumen) to blood supply
-passive immunity from mother’s milk to blood -receptor-mediated -binding and release are pH-mediated -receptors recycled back to apical surface |
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Describe transport of IgA into the intestinal lumen.
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-movement of IgA from basal to apical surfaces
-receptor cleaved on PM that releases receptor and IgA complex >cleaved receptor is secretive component -IgA is digestive ab and IgG is circulatory |
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What protein assists in endosome traffic in a polarized Epithelial Cell?
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-vesicle movement governed by Rab’s (G-pro)
>found on endosomes -don’t want transcytosed materials to be recycled instead |
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What are 2 aspects that help maintain cell polarity?
|
Cell polarity maintained by tight junctions is essential for epithelial functions.
Membrane transporters at apical and basal surfaces are needed for the directional movement of ions into and through cells in |
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What maintains epithelium polarity?
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The epithelium polarity is maintained by the tight junctions, which prevents lateral movement of the plasma membrane proteins between apical and basal surfaces.
Plasma membrane proteins are sorted within the Golgic apparatus whereby basal plasma membrane proteins are targeted to the basal surface and apical plasma membrane proteins to the apical surface. Some apical plasma membrane proteins traffic to the basal surface where they are transcytosed to the apical surface. |
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Describe tight junctions and its components.
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-almost a fusion of the bilayers
-prevent movement of ions between cells and also lateral movement of PM proteins between cells |
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What are the main pathways of epithelial TM protein sorting?
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-polarized epi
>apical, basal, and lateral (sides) surfaces -summation of sorting pathways -SNARE’s imortant for fusion events -2 major paths to apical surface: lipid raft pathway (lipid rafts directly to apical surface); or sorted to basal surface first, retrieved to common recycling endosome, and then transported to apical surface >also a recycling pathway (keeps proteins apical) -basal surface has 1 avenue: direct route from golgi to basolateral surface >diff. signals and accessory proteins >exocyst=complex of proteins that directs fusion events and docking of vesicles of basal surfaces |
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Which motor protein and cytoskeletal elements are involved in vesicle trafficking within a polarized epi?
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MTs are organized into two populations in polarized epithelial cells: stable cortical MTs with the minus ends facing apically, and dynamic MTs that originate at the MTOC, with the plus ends facing apically.
-need the right motor to get vesicles to mem. -myosin=actin motor; kinesin and dyneins=MT motors -basolateral trafficking: myosins >myoVb for apical traffickin >kinesins and dyneins used predom. for apical trafficking |
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Interpret the data in terms of the dist. of the aquaporins.
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-not efficient to get water through the cell
>aquaporin=water channel (esp. in kidney) that facilitates water transport -PT=prox. tub., LH=loop of henle, cd=collecting duct -conc. urine (for water retention) >aquaporins used for concentration purpose -AQP3= basolateral, AQP2=apical >in collecting ducts >allows water to be moved from apical to basal >aqp3 constitutively at bl surface, aqp2 regulated (apical) by hormone (ADH) |
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Describe the activation of aquaporins.
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ADH secreted from the supraoptic nuclei of hypothalamus.
-ADH=vasopressin >made in hypothalamus and secreted into pituitary and released into circulation >G-pro coupled hormoneactivates adenylate cyclase which activates PKA >PKA may phosphorylate C-term of aquaporin -the more aquaporins the more water moved |
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What is the apical homolog to the exocyst in a polarized epi?
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-Crumb’s complex: complex of pro. at apical surface
>may be transported independently of aquaporin or with it >similar to exocyst but poised to apical surface (exocyst=complex of proteins that directs fusion events and docking of vesicles of basal surfaces) >-variation in accessory proteins of Crumbs |
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Compare and contrast apical and basolateral sorting.
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-in Trans-golgi: proteins packaged and sorted to basal or apical (via lipid rafts) surfaces
-at basal surface: proteins get anchored and stay >endocytosis of both bl and apical pro >apical pro get transcytosed to apical surface and bl pro. get recycled back to basal surface -GPI anchor=lipid anchor indicative of lipid rafts |
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What are the diseases of disordered protein trafficking that we learned about?
|
-mis-sorting of proteins disorders
-MVID: missing/defect in myosin Vb (motor for getting vesicles to apical surface) -defect in VPS33 (required for getting pro. to apical surface; some pro end up at basal surface p75 is neurotrophic receptor and BSEP is involved in bile prod.) >bile prod. at apical surface of hepatocyte |
Microvillus Inclusion disease presentation:
Mutation in Myo5b Disorder involves the small intestine due to the absence of microvilli Diarrhea Metabolic acidosis Dehydration Death Arthrogryposis-renal dysfunction-cholestasis syndrome presentation: Mutation in VPS33B Disorder involves the kidney, liver, skin, CNS, and musculoskeletal systems Stiffness of joints Renal dysfunction No liver bile flow Death 7-23 months of age -both are lethal and rare -MVID=disease of small intestine -arthrogryposis= many apical proteins affected >no bile flow |
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What is required for the basolateral sorting of proteins?
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The sorting of proteins to the basolateral surfaces of polarized epithelial cells require clathrin, myosin, and basolateral SNAREs.
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What is required for the direct route of sorting proteins to the apical surface?
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The direct route of sorting proteins to the apical surfaces of polarized epithelial cells proceeds by lipid rafts and requires myosin, kinesin, dynein, and apical SNAREs.
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Describe the indirect route of sorting proteins to the apical surface?
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The indirect route of sorting proteins to the apical surfaces requires proteins to be trafficked to the basolateral surface where it is transcytosed to the apical surface.
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How is trafficking of Aqp-2 regulated?
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Trafficking of AQP-2 is regulated by ADH, which activates PKA.
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What is the Crumbs complex?
|
Crumbs is a protein complex that guides membrane proteins to the apical surface.
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Describe the distribution of cells in a villus. Where are the stem cells located?
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-epi in constant state of flux
-stem cells have to differentiate and move after division -at tips of villi is where cells die |
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Describe the growth factor gradient within the crypt.
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-stem cells Lgr4 and 5 at bottom
-gradients of cytokines/growth factors >Wnt induces differentiation and division >Grem is antagonist of BMP (prevents initiation of division from starting prematurely) -entire structure is crypt |
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Describe the Wnt signalling pathway.
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-beta-catenin= TF that’s normally degraded by UPS (maintained at low conc.)
-WNT shuts down degradation of b-catenin (not getting phospho. and released into nucleus where it turns on genes to initiate cell division) -frizzled is receptor for WNT -b-catenin levels go up and sequester to nucleus upon addition of Wnt to cells |
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What cells are responsible for maintenance of the stem cells in the crypts? Name an important set of receptors and their ligand on crypt stem cell?
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-paneth cells are secretory; main fn is to kill bacteria in intestine to keep pop. under ctrl.; release antimicrobial substances; also secrete Wnt that maintains stem cells
-R-spondin is the ligand for Lgr4/5 (stem cell pro. markers) -majority of crypt is TA cells |
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Interpret the data.
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-Cell surface binding assay shows LGR4 and LGR5 are receptors for R-spondins.
-Clathrin endocytosis is required for Rspo3-induced nuclear accumulation of b- catenin. -what does Lgr4/5 do? -transfection of Lgr4/5 expressed in non-stem cells >fused with alk-phos (red rxn prod) >Lgr4/5 bind to all r-spondins >cell surface binding assay -also looked at signalling of Wnt pathway >add Wnt and looked for b-catenin in nuc. (When Wnt binds to frizzled (Fz), its receptor, dishevelled (Dsh), is recruited to the membrane. GSK3 is inhibited by the activation of Dsh by Fz. Because of this, β-catenin is permitted to build up in the cytosol and can be subsequently translocated into the nucleus to perform a variety of functions.) >both ligands (Wnt and Rspo) induce Wnt signalling pathway >calveolin important for budding of lipid rafts (type of coated pit) >Wnt path: requires clathrin to initiate signalling >Wnt requires clathrin but Rspo3 requires calveolin for endocytosis |
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Interpret the data.
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R-spondins increase b-catenin levels and promote epithelial proliferation in small intestine and colon.
-injected into mice -amt of b-catenin in small intestine (Li Cl=pos. ctrl) -A) looking at cypts >1,2, and 3 have deeper, elongated crypts (seen better in colon) due to prolif. of endothelial cells (injections further activated cellular differentiation and division) -model: ?=Lgr4/5 |
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How is polarity maintained even during cell division?
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via attachment to basement membrane.
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What keeps paneth cells localized to the bottom of the crypt?
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-EphB ensures that Paneth cells stay at bottom
>mutant causes Paneth to be found in multiple sites (vs. at bottom of crypts) |
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How are apoptotic inhibitors and inducers used to maintain the villi? What are the 2 major components and their location?
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Interpret the data.
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Tight junctions are maintained in shedding of apoptotic epithelial cells.
-ZO-1= occludin pro. -actin labels apical surface of cell >microvilli are packed with actin -dying cell getting sloughed off -Zo-1 initially at apical surface, but as cell does it gets deposited all around the lateral sides of the cell >maintains tight junctions of the remaining cells (graphed in following slide) |
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How is stem cell division within the crypts regulated?
|
Stem cell division is regulated by the Wnt and R-spondin signaling pathways that suppress the degradation of b-catenin allowing it to move to the nucleus to regulate transcription.
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How is epithelial integrity and function maintained during epithelium renewal?
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Epithelium renewal maintains the apical and basolateral surfaces by tight junctions and thus sustains epithelium integrity and function.
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What is an autoimmune disease in which apoptosis is implicated?
|
-destruction of villi by autoab’s
>apoptosis proceeds faster than regeneration |
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How is apoptosis involved in colorectal cancer?
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-don’t memorize steps
-p53 involved (cells are losing it so apoptosis is suppressed) -adenoma has high conc. of b-catenin (Wnt or Lgr4/5 may be involved) >massive stimulation of stem cells and reduced apoptosis -Colorectal cancers begin as a benign adenoma that undergo loss of tumor suppressor genes such as p53 and become a malignant carcinoma. |
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What are disease presentations of both cell death inhibition and stimulation?
|
Cell Death Inhibited
Cancer Cell Death Stimulated Alzheimer’s disease Farber’s disease Friedreich’s disease Batten’s disease |
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What are the major classifications of cell death?
|
-3 major types of cell death
>apoptosis is main one -necrosis does NOT have blebbing >cellular swelling (vs. condensation seen in apop. and autoph.) >diff. osmolarities -Annexin V: labels surface; Trypan blue also used for autophagy |
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Describe the cellular presentation of necrosis and apoptosis.
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-cellular swelling and explosion of necrosis
>Trypan blue and LDH illustrate internal release -apoptosis: cell falls apart via budding and all the intracellular components remain mem. bound >may see an increase in LDH with this mode as well (multiple markers necessary) >caspase is the best marker -caspase inhibitors and autophagy inhibitors would shut down either of these pathways (can be used to define necrosis) |
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What are the main events of apoptosis?
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-chromatin condensation and nuclear fragmentation
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What are some components of the 2 apoptotic paths?
|
Extrinsic Pathways
Fas/FasL TNF-a/TNF-R1 TGF-b/TGF-R APO-3L/DR3 Integrins Intrinsic Pathways Ceramide DNA damage Calcium Reactive Oxygen Species -2 paths >extrinsic: outside ligand binding to cell and triggering cascade within >intrinsic: mito and/or ER triggered |
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Which caspases are activated in each apoptotic pathway?
|
-bottom line: activation of caspases (proteases)
-extrinsic: caspase VIII -intrinsic: caspase IX |
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How do trophic factors regulate apoptosis?
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-lack of growth factors initiates cell death (lack of ligand binding to receptor)
-Akt kinase phospho. Bad which pulls it away from Bcl’s (anti-apoptotic pro.) |
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What are some components of the extrinsic apoptotic path?
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-extrinsic path
-activating caspase 3 -PARP cleaved by caspase to become inactivated (leads to DNA fragmentation) -some extrinsic receptors also interact with mito (intrinsic path overlap) |
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Interpret the data (TRAIL is a drug that initiates cell death).
|
Mutant K-Ras infers resistance to TRAIL in pancreatic cancer cell lines.
-PanC cells are resistant to TRAILS (mutated K-Ras) -caspase-8 cleavage fragment (cleaved=activated) elev. after TRAIL application under norm conditions >full-length Caspase 3 starts to disappear >PARP gets cleaved |
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What are the survival factors that inhibit apoptosis?
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-ultimately lead to Bcl-2/xl activation
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How does the Mitochondria Control
Apoptosis? |
-mito: Bcl2/xl block the path
>Noxa and PUMA inhibit Bcl2 (turns on cell death) -ultimately get release of CytC and Smac/diablo |
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Differentiate between the mitochondrion of a healthy vs. apoptotic cell.
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-Bax channel allows release of CytC and diablo
>CytC forms apoptosome; diablo interacts with IAP (inhibitor of apoptosis) |
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Describe the assembly and regulation of the apoptosome.
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-intrinsic
-apoptosome on right >accumulation of Apaf-1 pro. -IAP’s (on left) inhibit caspase 9 and 3 >Smac/diablo inhibit IAP -Bcl-2 inhibits Smac/diable and CytC |
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Describe Endoplasmic Reticulum
Control of Apoptosis. |
-ER pathway is Ca release path
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What are the domains found in key pro- and anti-apoptotic factors?
|
b)channel forming pro’s
c) interact with A to inactivate them |
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What are some paths of blocking apoptosis?
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-Smac/diablo is anti-IAP
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Interpret the data about hyperforin (HF).
|
Hyperforin promotes caspase-mediated apoptosis in myeloid tumor cells.
Apoptosis measured by annexin-V and propidium iodide kit with flow cytometry. -caspase-mediated cell death process 1st panel=DNA fragmentation (flavopiridol is pos. ctrl) 2nd panel=caspase activity with application of drug 3rd panel=PARP cleavage 4th panel= dist. anti- and pro-apoptotic pro (Noxa inactivates Bcl-2) 5th panel ©= inhibition of caspases >caspase 9 cleaves caspase 3 (drug seems to be utilizing caspase 9) |
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What is the Intrinsic pathway of hyperforin-induced cell death?
|
-model of previous slide
-turns on Noxa-->intrinsic pathway |
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What is a common target of drugs utilizing the intrinsic apoptotic pathway?
|
Bcl-2
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Describe the apoptosome (1 sentence).
|
Apoptosome is a protein complex activated by cytochrome C to recruit caspase 9 that activates caspase 3.
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What is the function of IAP's?
|
IAPs inhibit apoptosis by suppressing caspase activity while XIAP is inhibited by Smac/Diablo.
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What are the modes of survival factors?
|
Survival factors can increase production of anti-apoptotic factors, inactivate pro-apoptotic factors, or inactivate anti-IAPs.
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What do the Bcl proteins do?
|
Bcl-2 and Bcl-XL inhibit apoptosis by binding to and inhibiting pro-apoptotic Bad and Bax and preventing cytochrome C release from the mitochondria.
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Interpret the data (SS=sulindac sulfide).
|
Autophagy delays
sulindac sulfide-induced apoptosis in HT-29 cells. -PI=propinium idode (should be neg in viable cells; meas. of “leakiness”) -3-MA=inhibitor of autophagy >if cell death goes up with addition of 3-MA, autophagy is a protective mechanism |
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Interpret the data. (CQ=chloroquine; gefitinib and erlotinib=autophagy activators in lung cancer cells)
|
EGFR kinase inhibitors, gefitinib and erlotinib, activate autophagy in lung cancer cell lines:
-Tyr kinase inhibiting drugs -turn on autophagy (autophagic vacuoles) >is it for cell death or survival? Autophagy is a cytoprotective response to EGFR kinase inhibitors in lung cancer cell lines. MTT assay -CQ=chloroquine: inhibitor of late stage lysosomes -cell viability >decreased with CQ (increase in cell death) -same results with siRNA (Atg5 and 7 important to autophagy) >more specific than drugs |
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Interpret the data.
|
Bcl-XL inhibitor, Z36,
promotes autophagy-mediated cell death. -C) GFP-LC3autophagy turned on (dots in rt. panel) -c bottom: DMSO=ctrl, zDEVD=caspase-inhibitor >drug may be acting through autophagy but not caspase D) knock down of Beclin and ATG7 (autophagy pro’s) |
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Describe some ways there is cross-talk between autophagy and apoptosis.
|
Bcl-2 and Bcl-xl are anti-autophagy and anti-apoptosis proteins
Bcl proteins inhibit autophagy by binding to Bcn-1 Bcl proteins inhibit apoptosis by binding to Bad and Bax Atg5 is a pro-autophagy and pro-apoptosis protein Atg5-33K stimulates autophagy by conjugating to Atg12 Atg5-24K stimulates apoptosis by binding to Bcl-xl Beclin is a pro-autophagy protein that can become pro-apoptotic. Caspase cleavage of Beclin destroys its pro-autophagy activity C-terminal fragment of the cleaved Beclin amplifies apoptosis -Atg5 and Beclin are pro-apoptosis when cleaved |
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How does Beclin regulate autophagy?
|
-Beclin inactivates kinase when released from Bcl’s
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How is Beclin implicated in apoptosis?
|
-Beclin can be cleaved by caspase 8
>allows for activation of apoptosis |
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How is Atg-5 implicated in apoptosis?
|
Calpain-induced cleavage of Atg5 promotes the activation of caspase 3 and enhances apoptosis in HeLa cells.
-staurosporine= ctrl that kills cells via apoptosis >little cell death if Atg5 knocked down -staurosporine clips Atg5 due to Calpain -if 24K Atg5 added back apoptosis turned back on (but not for full-length) |
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How is calpain involved in the crosstalk between autophagy and apoptosis?
|
-left side is aberrant cleavage due to calpain
>binds up Bcl-XL (inactivator of apoptotic which turns on apoptosis) >staurosporine activates caspases via Atg5 (activates caspase3) |
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Summarize the key concepts of apoptosis/autophagy.
|
Autophagy is an effective cell survival pathway that can remove damaged organelles and mitochondria that would cause apoptosis.
Chemoresistance of some cancer cells is in part do to their ability to regulate autophagy. Autophagy in excess can cause cell death. Bcl-2 and Bcl-XL suppress both autophagy and apoptosis. Pro-autophagy Atg5 and Beclin can when cleaved become pro-apoptotic. -autophagy gets rid of damaged organelle due to chemotherapy and act as a chemoresistance factor -autophagy pathway can be anti-apoptotic |
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What are the main events/stages of the 1st 4 weeks gestation?
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1. Gametes and fertilization
2. First week A. Cleavage B. Blastocyst C. Implantation 3. Second week A. Bilaminar germ disc (epiblast and hypoblast) B. Embryonic membranes and cavities (aminon and amniotic cavity, chorion and chorionic cavity, and yolk sac) C. Uteroplacental circulation 4. Third week A. Gastrulation (primitive streak) B. Trilaminar embryo (endoderm, mesoderm, ectoderm) C. Notochord and mesoderm dynamics D. Somites and body segments 5. Fourth week A. Somite differentiation (myotome, dermatome, sclerotome B. Neurulation (neural tube and secondary neurulation) C. Neural crest and derivatives The embryonic (or embryogenic) period is the first 8 weeks of development. During this time the embryo establishes the body plan and most tissues and organs begin to form. The fetal period begins at week 9 and is characterized by growth and development of the fetus. The fertilization age is used in this lecture and corresponds to the time since conception. Gestational age is the time since first day of last normal menstrual period (usually 2 weeks longer than fertilization age). -most of the organs are patterned by 4th week (1/2 way thru embryonic period) |
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Describe the sperm.
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Spermatogenesis is the production of sperm and consists of three cell phases: spermatogonia, spermatocytes, and spermatids. Spermatogonia are stem cells and transient amplifying pool cells. Spermatocytes undergo meiosis I & II to reduce chromosome number (n) and DNA content (C) to give rise to four spermatids that are 1n 1C. Spermatids are immature gametes (1n 1C) and do not divide. Spermiogenesis is the maturation of spermatid into mature sperm. Spermeation is the release of spermatozoa from Sertoli cells into the lumen of seminiferous tubule.
The axoneme has a standard 9+2 arrangement of microtubules and microtubule doublets. The outer doublets are associated with outer dense fibers that are visible at the ultrastructural level. The axoneme contains thousands of dynein motors that generate force and movement. Mature sperm are 60 µm long. The sperm head is 4.5 X 3 X 1 µm (L X W X T). The middle piece of the tail is 7 µm long and consists of an axonemal complex surrounded by mitochondria. The principal piece of the tail is 40 µm long and has a fibrous sheath around the axoneme. The end piece of the tail is 5 µm long and contains only the axoneme. -spermatid:meiotic product (top left) >regular looking cell >cell becomes specialized for motility in later stages -mature sperm is highly differentiated -stages reflect compaction of chromatin, syn. of lots of mito. (for tail motion) -acrosome is secretory granule under PM at head of sperm (sep. PM from nuclear envelope) >secretory vesicle that releases digestive enzymes -cross-section of axoneme: additional densities assoc. with outer MT doublets, help contribute to contraction of tail >many dynein motors in tail that consume lots of ATP -histones are teste specific; protamines are histones that are amine rich that allows DNA to be ultra-compacted >sperm nucleus is 1/12 the size of oocyte nucleus (due to high compaction) |
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Describe the sperm biology.
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Male gametes are derived from germ line stem cells (spermatogonia) via meiosis.
Spermiogenesis - Process by which spermatid is converted into sperm. Spermatids are immature haploid (1n 1C) gametes produced by meiosis (see figure in previous slide). Takes place in specialized cell niche created by Sertoli cells in seminiferous tubules of testes. Ends when Sertoli cells release morphologically mature sperm. Chromatin - majority of DNA is packaged by protamines. Testis specific histones are replaced by transistional proteins and finally by protamines for most spermatid DNA. Protamines are small arginine-rich proteins that neutralize electrostatic repulsion between DNA strands resulting in formation of toroid loops of ~50 kb of DNA that are stabilized by disulfide bridges and condense the genome into semi-crystalline state. The mature human sperm nucleus is only 7.7% the size of the oocyte nucleus. Acrosome - Specialized, large secretory granule that contains soluble hydrolases and proteases. Positioned between plasma membrane and nuclear envelope. Acrosome reaction – Regulated secretion event causing fusion of the acrosomal and plasma membranes and release of acrosomal enzymes. Triggered by binding of the sperm plasma membrane to protein ZP3 (in the zona pellucida of the oocyte), which activates a G-protein coupled receptor signaling pathway and an influx of calcium. Motility - Flagellum of human sperm has ~9000 dynein motors and consumes ATP generated by densely packed coiled mitochondria to generates 1-6 pN of force. In the epididymis, sperm become motile and gain ability to fertilize the egg. Plasma membrane proteins segregate to specific regions of the sperm head where they are later needed for fertilization. Coating (decapacitating) factors are added to protect sperm in female tract and prevent the premature release of acrosomal enzymes. Capacitation is the final step of sperm maturation that takes place in the female reproductive tract. Capacitation involves the removal of glycoconjugates from the sperm surface. Removal of these coating factors exposes egg-binding proteins on the sperm plasma membrane that bind the ZP3 receptor in the zona pellucida of the egg. Acrosomal hydrolases include include hyaluronidase, neuramnidase, acid phosphatase, and a trypsin-like protease acrosin. |
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Describe sertoli cells.
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Sertoli cells are specialized epithelial cells that support sperm maturation by housing immature sperm (spermatids) in deep infoldings of plasma membrane. Specialized junctions (arrows between arrowheads) between adjacent Sertoli cells create the blood-testis barrier. Junctions also connect spermatids to Sertoli cells (arrows). Sertoli cells phatocytose and degrade residual sperm cytoplasm (residual bodies)
Figures 22.16 and 22.17 from Histology - A Text and Atlas, by Ross and Pawlina, 6th edition. Junctions between Sertoli (sustentacular) cells creates two compartments in the seminiferous tubules - basal and adluminal (or lumina). Sertoli-Sertoli junctions contain abundant zonula occludens (tight junctions). Two Sertoli cells are shown above (S1 and S2). The nucleus (N) and acrosome (A) of the spermatid are also indicated. -true epi cell in seminiferous tubule; house meiosis -primary spermatocyte is the germ cell that enters meiosis and spermitid is first meiotic product (4 products generated from male meiosis) -spermatogonium is stem cell pop. that gives rise to those that enter meiosis -empty pockets between sertoli cells would be filled with cells undergoing meiosis -junctions hold developing sperm to sertoli cell which nourishes it (supporting cell) >also junctions between sertoli cells (rich in desmosomes and tight junctions…mostly desmosomes between sertoli and sperm) >tight junctions creat barrier -mature sperm released from sertoli into lumen of seminiferous tubule |
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Describe the oocyte.
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Ovulation releases a secondary oocyte arrested in metaphase II (meiosis II) and surrounded by a thick (~6 µm) extracellular matrix layer (zona pellucida) and nurse (follicle or granulosa) cells derived from the ovarian follicle. Nurse cells support the rapid growth of the oocyte during folliculogenesis in the ovary prior to ovulation. Whereas sperm are among the smallest human cells, oocytes are among the largest.
Cumulus mass is the term for the ovulated secondary oocyte, zona pellucida, and the layer(s) of follicle / granulosa cells. The zona pellucida is ~6 µm thick and is composed of proteins ZP1, ZP2, and ZP3. Fertilization in human involves a secondary oocyte rather than a definitive ovum. In addition to size, the oocyte differs from the sperm in terms of cell polarity and symmetry. -large cell (~150 um), not polarized like sperm -permanent set of nurse/companion cells assoc. with from meiosis at birth (3rd month development in utero) >send processes into zona pollusa that protects and supports size of egg >continuum of communication between granulosus cells and oocyte >entire structure is ovulated (including follicular cells) -arrested at metaphase 2 (arrested at prophase of meiosis one until selection cycle) >at point of ovulation >meiosis completes AFTER fertilization -cytoplasm has RNA’s stored for later usage (specializedshort poly A tails or with regulatory factors that inhibit expression until fertilization) |
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Describe what happens when the sperm reaches the zona pellucida.
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After the sperm penetrates the zona pellucida it enters the perivitelline space. Most of the tail remains associated with the oolemma and does not enter the cytoplasm. Importantly, however, the centrosome (containing its centriole) from the sperm enters the egg.
-need dozens of sperm to penetrate follicle cell layer >layer may make egg more immobile for better access by sperm -zona pollucida contains proteins and carb’s (hyaluronon- long polysacc.) >ZP-1,2, and 3 are proteins that interact with sperm mem. -acrosomal contents released (hydrolases attack zona pollucida); many are needed to penetrate to perivitelline space -depolarization and release of Ca once mem. fuse >granules release contents: changes composition of PM, enzymes cross zona pollucida, all to block more than one sperm from entering egg (cortical rxn) >egg is more than diploid at this point since it hasn’t finished meiosis (3n3c) -centrioles also need to enter oocyte (sperm delivers DNA and mitotic spindle) -mito in sperm are degraded (forces maternal inheritance of mito DNA) |
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Describe the process of fertilization.
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A few dozen sperm reach ovulated oocyte in the fallopian tube. Multiple sperm are required to penetrate follicle cell layer (zona radiata) prior to step 1. Extracellular matrix associated with granulosa cells is broken down by enzymes in semen and sperm hyaluronidases (on sperm plasma membrane), not acrosomal enzymes.
Capacitation - biochemical changes in sperm plasma membrane take place in the female reproductive tract and are necessary for sperm to be able to fertilize oocyte. Step 1 - Binding of sperm plasma membrane to zona pellucida (protein ZP3). Step 2 - Regulated exocytosis of acrosomal vesicle contents (acrosome reaction). Stem 3 - Soluble hydrolases and proteases released from multiple sperm weaken the zona pellucida enough to allow sperm to penetrate. Step 4 - First sperm fuses with the oolemma and triggers a cortical reaction in the oocyte that blocks polyspermy (fertilization by multiple sperm). Step 5 - Sperm nucleus enters cytoplasm. Mitochondria and tail are degraded. Mitochondrial DNA is inherited maternally from oocyte. Oocyte meiosis II resumes to complete formation of definitive ovum (female haploid gamete, 1n 1C). Second polar body is produced. First polar body does not undergo second meiotic division. Only centrosomes from father create mitotic spindle for subsequent cleavage divisions. Sperm and egg are released from sex organs in a form that is not fully mature. In vitro fertilization (IVF) requires special treatments to make them capable of fusion in vitro. Usually, fertilization occurs in the ampulla of the oviduct. Capacitation involves signaling via RTKs and adenylate cyclase to increase cAMP levels and calcium levels in sperm cytoplasm. Also involved are changes in the sperm plasma membrane, including reduction in cholesterol content and redistribution of phospholipids and membrane associated carbohydrates. -sperm mature in female reproductive tract (biochem. changes in PM)--> capacitation |
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Describe gamete fusion.
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The integrin alpha6beta1 is a potential receptor for ADAM (a disintegrin and metalloprotease) family members fertilin beta and cyritestin. CD9 is an integrin family member cell adhesion protein on the oocyte plasma membrane (not shown). Because ADAM protein binding to the egg is blocked by CD9 mAb, it is likely that the ADAM receptor is a CD9-associated. Izumo is an immunoglobulin (Ig) domain cell adhesion protein on the sperm plasma membrane. The association of the integrin alpha6beta1 and a putative Izumo receptor with tetraspanins is speculative.
-molec. interactions confer specificity to binding event -Izumo is essential for gamete fusion -GPI anchored hyaluronidases on sperm important for fusion |
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Describe how you would structure an experiment to determine the required factor for gamete fusion.
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Left: Hamster eggs with zona pellucida removed were mixed with sperm from Izumo+/- or Izumo-/- mice. Sperm nuclei that enter the oocyte cytoplasm decondense, expand, and stain light blue with Hoechst 33342 dye (arrowheads). Sperm nuclei from Izumo-/- mice do not enter oocyte cytoplasm. Right: Hamster eggs without a zona pellucida were mixed with human sperm in the presence of control antibody (IgG) or anti-human Izumo antibody. The anti-Izumo antibody blocks sperm-egg fusion. Sperm were treated to prevent the polyspermy block. (MCB 6E Fig 22-6)
-KO and anti-inhibition studies -oocyte from hamster and sperm from izumo heterozyg. mouse -zona pollucida removed in vitro -left Hoechst: sperm DNA in oocyte have decondensed (pale blue); under conditions that don’t block polyspermy >KO: sperm have not fused with egg >Izumo required for fusion -Rt: hamster oocytes mixed with human sperm >right top: ctrl ab’s (some sperm have gained entry) >anti-izumo blocks fusion of sperm with oocyte |
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Describe the post-fusion of gametes events.
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Calcium flux – initiates at site of sperm entry and passes through oocyte cytoplasm as a wave at 5-10 µm / sec triggering biochemical changes and the cortical reaction
Cortical reaction – regulated exocytosis of cortical granules that change properties of oolemma. Proteases released by granules degrade oocyte surface glycoproteins that bind sperm. Cross-linking proteins released from granules interact with the zona pellucida to create a perivitelline barrier. This prevents entry of additional sperm. Meiosis completion – initiated by calcium flux, formation of second polar body Cortical granules are darkly stained in this EM. Oocyte also contains mRNAs that are used following fertilization to program translation of proteins needed for zygote DNA synthesis and cell division (e.g., histone proteins). Oocyte is large, measuring ~150 µm in diameter. Thus, the calcium wave takes 15-30 µsec to initiate changes. Oocyte microvilli in the ZP are visible in the EM. -oolemna with microvilli seen -cortical granules fuse during rxn with PM -meiosis resumed w/in min. of fert. |
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Describe the pronuclear stage of fertilization.
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The 2n 2C zygote is the final product of fertilization and initially enters a pronuclear stage. Sperm chromatin undergoes decondensation and oocyte histones replace protamines and sperm histones. Maternal and paternal chromosomes undergo DNA replication. Mitosis begins. Both centrosomes for the formation of the first mitotic spindle of the first cleavage division are derived from the sperm. Cleavage divisions alternate S and M phases without normal intervening G1 and G2 phases.
Does the oocyte nuclear envelope reform after meiosis or not? -only 1 meiotic prod. gen. in women >1st polar body doesn’t divide -pronucleus=1n (maternal chrom. only) >DNA’s undergo division in pronucleus >male chrom. are packaged in norm histones at this point |
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Describe the cleavage divisions post-fertilization.
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Cleavage divisions subdivide zygote without increase in size. Zygote travels toward uterus by ciliary action of epithelial cells in oviduct. At 8-cell stage all cells remain totipotent. At 16-cell morula stage blastomeres express E-cadherin and reorganize during the process of compaction. Cell adhesion diminishes and a fluid filled cavity forms in the blastocyst, which hatches from zona pellucida before implantation.
Fertilization usually takes place in the ampulla of the oviduct. There are only two polar bodies because the first polar body does not undergo division during meiosis II. The stage immediately prior to the zygotic stage (one nucleus) is the pronuclear stage with distinct male and female pronuclei from the gametes. Cleavage divisions consist of alternating S and M phases, but no G1 and G2 phases. Morula from “morum” Latin for mulberry. Oviduct is subdivided into fimbrae, ampula, oviduct proper (Fallopian tube). Blastomeres are totipotent (can form any tissue of embryo). Following IVF, embryos are allowed to grow to the 8 cell stage and are transferred to the uterus. Embryonic stem (ES) cells are isolated from the blastocyst stage and can be used to generate many different cell types. Compaction is the rearrangement of blastomeres to form a blastocyst. -called cleavage b/c subdividing the zygote (alternating S/M cycles) >zygote still protected by zona pellucida -fert. in fallopian tube -8 cell stage is totipotent (can give rise to any type of cell) -compaction at morula stage (clumped) -blastocyst stageorganized into layers with fluid-filled cavity (formed near end of 1st week when implantation occurs) |
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Distinguish between pluripotent and totipotent.
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Specific genes and signaling pathways (Wnt, Notch, TGFbeta, Oct4) are active in different blastomeres at different points during cleavage divisions.
-compaction visualized >specific cadherins important for this process -cells pluripotent at 16 cell stage (totipotent at 8-cell stage) |
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Describe the blastocyst.
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Beginning at the 64-cell stage cells differentiate into the inner cell mass or embryoblast and the tropoblast or trophectoderm.
Embryoblast or inner cell mass will form the embryo. Trophoblast or trophectoderm forms the placenta, which binds to and invades the endometrium. Fate of differentiated cells is determined by its location within the early embryo. Transplantation of labeled cells to embryoblast or trophoblast acquire fate of surrouding cells. The surface epithelium of the endometrium (stratum functionalis) is shown at top of figure. The blastocyst hatches from the zona pellucida and adheres to the endometrium (mucosal lining of uterus) by the end of week 1. Tropho is a prefix that denotes a relationship to feeding. The cavity above is the blastocyst cavity. Cells from the inner cell mass are isolated to obtain embryonic stem (ES) cells. -trophoblast invades epi of endometrium to begin formation of placenta (feeding cells) |
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Describe implantation.
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Implantation begins at end of week 1 and continues into week 2. Cytotrophoblast cells bound to endometrium divide and fuse to form syncytial syncytiotrophoblast that invades uterine wall. The embryoblast gives rise to epiblast and hypoblast. Thus, both layers of the early embryo give rise to two derived layers by days 7-8.
Syncytiotrophoblast secretes human chorionic gonadotrophin (HCG) which stimulates the corpus luteum to secrete progesterone which maintains pregnancy. Nutrition is by diffusion (no placenta formed yet). Ectopic pregnancy occurs in about 1/80 pregnancies and may be life threatening to mother and embryo. Ectopic pregnancy is defined as implantation of the blastocyst at a site other than the upper portion of the uterus. The most frequent abnormal implantation occurs within the uterine tube (tubal pregnancy). Other sites of ectopic implantation are in the abdominal cavity or cervix. Ectopic pregnancies are not maintained. Invasion of the blastocyst into non-uterine tissue typically causes internal blood loss in the mother. The decidual reaction is the term for cellular and vascular changes occurring in the endometrium at the time of implantation. Substances released by the syncytiotrophoblast cause degeneration of endometrial cells which are ingested by the trophoblast to nourish the embryo. -forms in portion of uterus with thickest endometrial lining -cells of trophoblast fusing to form syncytiotrophoblast >highly motile and invasive >breaks down cells via protease secretion >embryo nourished by diffusion -cells differentiating and acquiring diff. fates |
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Describe the bilaminar germ disc.
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Implantation continues as syncytiotrophoblast expands and invades connective tissue beneath endometrial epithelium. Embryoblast cells differentiate to form epiblast and hypoblast, the two layers of the bilaminar germ disc. These two layers of cells become epithelial in nature. The amniotic cavity begins to form (≠ blastocyst cavity).
Diagram is about day 8. Epiblast is primary ectoderm. Hypoblast is primary endoderm. Endometrial glands (upper left) nourish embryo in absence of uteroplacental circulation. Nutrients (e.g., glycogen) diffuse from epithelial cells of gland (simple columnar epithelium). Syncytium promotes better mobility for the blastocyst, which is relatively large. Blue is ectoderm precursor. Yellow is endoderm precursor. -small cavity in epiblast becomes amniotic cavity -syncitotrophoblast continues invadinc conn. tiss. |
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Describe the primary yolk sac.
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Diagram is about day 9. Heuser’s membrane is an outgrowth of the hypoblast (yellow) that lines the blastocyst cavity. Heuser’s membrane is also called the exocoelomic membrane. It forms the primary yolk sac and exocoelomic cavity (once formation of the exocoelomic membrane is complete). Heusers's membrane consists of extraembryonic endoderm. The blastocyst cavity becomes the primary yolk sac after it is lined by hypoblast cells (primary endoderm). The blastocyst is also termed the umbilical vesicle. Uterine glands extend toward and into the syncytiotrophoblast.
-true amniotic cavity lined by epiblasts -maternal circ. getting close to syncytiotroph. -cells of hypoblast growing out to form hypocele |
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Describe the extraembryonic mesoderm.
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Ectoderm means outer skin; endoderm means inner skin; mesoderm meand middle skin.
Origin of mesoderm cells that migrate into the extraembryonic reticulum is controversial. Extraembryonic mesoderm may also arise from cytotrophoblast or hypoblast. Embryoblast is still bilaminar. Extraembryonic mesoderm forms extraembryonic coelom. Maternal blood fills lacunae to support high growth rate of cells in early embryo. The placental circulation is beginning to be established. -blastocele lined with hypoblast -maternal blood in lacunae -reticulum develops >extra embryonic mesoderm is third layer in right (in red) >gives rise to vasculature |
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Describe the chorion.
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The chorion synthesizes HCG, which is detected by pregnancy tests. Extraembryonic mesoderm forms as an evolutionary reflection of the need for the embryo to derive nutrition from yolk in oviparous vertebrates. The chorion develops villi (vascular fingers) and gives rise to the placenta. In Greek, the word "chorion" means "skin or leather.” The somatic (somatopleuric) extraembryonic mesoderm lines the trophoblast, covers the amnion, and forms the connecting stalk (visible at the end of week 2). The visceral (splanchnopleuric) extraembryonic mesoderm covers the yolk sac.
-chorionic cavity imortant for first 2 weeks |
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Describe the definitive yolk sac.
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The primary yolk sac is replaced by the definitive yolk sac due to the outgrowth of a second wave of endodermal cells. The second wave of primary endoderm cells push the endoderm of the primary yolk sac away from the embryonic pole. The primary yolk sac endoderm and associated mesoderm are pinched off of the definitive yolk sac and are left as remnants in the chorionic cavity.
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Describe the end of week 2 post-fertilization.
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Maternal blood vessels fuse with lacunae, but maternal endothelium does not grow into lacunae. Thus, maternal blood comes into direct contact with fetal tissue (cells of cytotrophoblast). Once the embryonic circulatory system develops, embryonic vessels organize into villi that project into lacunae. After the placenta is established, there are 4 layers of separation between maternal and fetal blood: embryonic blood - embryonic endothelium - connective tissue - cytotrophoblast - syncytiotrophoblast - maternal blood.
Week 2 is “week of twos” Trophoblast - differentiates into 2 layers Syncytiotrophoblast and cytotrophoblast Embryoblast - differentiates into 2 layers Epiblast and hypoblast Extraembryonic mesoderm - differentiates into 2 layers Somatopleuric and splanchnopleuric Embryonic cavities - 2 form Amniotic and chorionic Yolk sacs - 2 form Primary and secondary (definitive) Somatopleuric mesoderm - associated with epiblast. Splanchnopleuric mesoderm - associated with hypoblast. |
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Describe gastrulation.
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One way to think about gastrulation in humans: 3D process in other organisms (e.g., sea stars) is converted into a 2D process in human. First m
esoderm (red) is extraembryonic and is not part of the bilaminar germ disc consisting of epiblast (blue) and hypoblast (yellow). -top-down views -2 depressions are where mesoderms will not go (cloacal and bucco. membranes) -pit: cells branch out |
Function: Formation of definitive endoderm, mesoderm, ectoderm
Primitive streak: Primitive groove - depression in epiblast forms at midline Primitive pit - deep depression at cranial end of groove Primitive node - mound of cells located around pit Body Axes: Dorsal - ventral Cranial - caudal Left - right Membranes: Buccopharyngeal (cranial) and cloacal (caudal) membranes form by association of epiblast layer with hypoblast layer Buccal refers to oral cavity. Both membrane regions exclude mesoderm. Later they become perforated to form openings of the oral cavity and cloaca. |
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Describe the importance of the primitive streak.
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Cellular mechanisms of gastrulation are not well understood. Epiblast cells ingress to form the definitive endoderm, but it is not clear if hypoblast cells are replaced or supplemented. That is, it is not clear if hypoblast cells undergo apoptosis, or are 'diluted out' by many migrating cells. Endoderm = entoderm (older terminology). The mesoderm formed by ingressing epiblast cells is intraembryonic mesoderm, which forms after the formation of extraembryonic mesoderm at about day 11.
-cells splitting from primitive streak >creates mesoderm |
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Describe mesoderm dynamic.
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Once mesoderm ingresses, epiblast becomes known as “ectoderm”. Ingressing cells undergo epithelial-mesenchymal transformation (EMT). Intermediate mesoderm in the cranial area induces overlying ectoderm to differentiate into neural ectoderm. Following migration (arrows), intraembryonic mesoderm meets extraembryonic mesoderm. Slug is a transcription factor that controls of transcription of E-cadherin.
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Describe formation of notochord.
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Notochordal process is a tube of mesodermal cells that sprouts from the primitive node. The notochord process is formed as the primitive pit regresses. Notochordal process becomes a solid cylinder - the notochord. The transition between hollow notochord process and solid notochord involves fusion of notochord process with underlying endoderm and subsequent re-emergence and separation of a solid notochord. Notochord becomes the nucleus pulposus (center area) of intervertebral discs in fetus. These notochord-derived cells are eventually replaced by mesodermal cells in the adult. Notochord cells don't contribute to vertebrae, but are required to induce their formation in the early embryo. Notochord may stiffen cranial-caudal axis of early embryo.
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Describe the neural plate.
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Precordal plate and notochordal process cells are a source of inducing factors. Inducing substances include the protein encoded by the chordin gene, which induces the expression of transcription factors required for formation of neuroepithelial cells. Neuroectodermal cells = neuroepithelial cells. Neural plate forms a groove, which deepens and involutes during the process of neurulation. Neural crest cells will originate from ridges that line the neural groove. Migrating mesoderm induces overlying ectoderm to begin differentiating into neuroectoderm when ectoderm is still epiblast (i.e., as intraembryonic mesoderm forms).
-primitive streak seen at bottom -neural ectoderm form CNS |
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What are the fates of the different layers in the embryo?
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Ectoderm - epidermis and its derivatives
Epithelium of skin (epidermis), hair, nails, glands, ducts Glands - sweat, sebaceous, mammary, salivary (parotid) Oral cavity - gingival epithelium, ameloblasts (enamel) Neuroectoderm - nervous system and neural crest (NC) cells CNS & PNS - neurons and supporting non-neuronal cells NC - tissues of head, face, jaw, oral cavity, odontoblasts (dentin) Mesoderm - muscle, connective tissue, cartilage, bone Muscles - trunk, viscera, appendages Skeleton - almost all except most of skull (i.e., membrane bone) Connective tissue - dermis of skin, CT of internal organs Cardiovascular - heart, vessels, blood, lymphatics Endoderm - epithelia of lungs, GI tract, glands, urogenital system Epithelia of pharynx and digestive tract Epithelia of respiratory tract (e.g., bronchi, bronchioles, alveoli) Digestive gland tissue - liver, pancreas, salivary (SL & SM) Urogenital system - epithelium of urinary bladder and urethra Mesoderm also gives rise to serous membranes (pleura, pericardium, peritoneum). Neuroectoderm also gives rise to the retina. The integumentary system received contributions from both ectoderm and mesoderm. Salivary glands include parotid, sublingual, submandibular, minor salivary glands, including von Ebner glands in circumvallate papillae of the tongue (associated with taste). |
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Describe the process of neurulation.
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Cut away/side view of trilaminar germ disc. Neural groove forms and deepens due to its association with the notochord. The ventral aspect of the neural fold acts like a joint and allows the neural folds to “close” together much like the leaves of a book. The central space enclosed by the neural folds is the neural canal.
-neural groove seperates from overlying ectoderm to form neural tube. -neural crest cells split and migrate |
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Describe the Turing Reaction diffusion model.
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-morphogen generates a pattern structure
>2 substances that can activate their own expression >slowly and rapidly diffusing substances >rapidly diff. sub. can inhibit the slowly diff. subs. -pos. feedback: peaks of slowly diff. sub surrounded by larger islands of rapidly diffusing sub. |
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Describe how protein gradients establish body polarity.
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-maternal effect genes (diffusing in from mother)
-gradient of bicoid mRNA=transcription regulator, DNA-binding regulator; regulates hunchback (levels are parallel with bicoid) -can manipulate sys by increasing/decreasing levels of bicoid, etc. -nanos is the posterior transcriptional regulator (tends to inhibit vs. activate) that produces a gradient -morphogens are proteins that make gradients that regulate the proteins expressed in localized areas |
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What are the GAP genes?
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-hunchback has a similar expression pattern
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Describe how different gene classes control segmentation.
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-whole regions go missing if you knock out Kruppel or knirps
-pair rule genes are dependent on expression of GAP genes and gradient genes -segment polarity genes are dependent on pair rule genes >KO gooseberry results in anterior only expression and patched results in posterior only expression -at this stage, each segment is 1 cell thick |
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What is pleiotropy and name an example from Shaw's lecture?
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-pleiotropic: if you KO 1 gene you get more than one phenotype
-toll-receptor responsible for dorsal/ventral gradient and immunity (example of pleiotropy) |
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How does dorsal regulate mesoderm formation in the embryo?
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-pleiotropic: if you KO 1 gene you get more than one phenotype
-toll-receptor responsible for dorsal/ventral gradient and immunity (example of pleiotropy) |
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How would you induce homeosis in an organism?
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-homeosis: wrong structure in a position on the embryo
-utlrabithorax yields 4 wings instead of balance organs -antennapedia yields legs instead of antennae -map of flies body in genome >homeobox: 60 aa DNA-binding domain >genes in order of positional expression on fly |
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What is one receptor-mediated mechanism in vertebrates involved in developmental cell signalling?
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-TGF beta receptors
>ser/thr kinases >SMAD can bind to DNA <Genes in the SMAD gene family provide instructions for producing proteins that are involved in cell signaling, regulating the activity of particular genes, and cell growth and division (proliferation). The SMAD proteins are part of the transforming growth factor beta (TGF-β) pathway, which transmits signals from the outside of the cell to the nucleus. This type of signaling pathway allows the environment outside the cell to affect how the cell produces other proteins.> |
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Describe how SMAD's are localized to the nucleus.
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-importin mechanism allows nuclear entry
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Describe how cellular signalling via SMAD's can be differentiated to meet multiple needs.
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-2 receptors react in diff. ways
-diff. heterotrimeric complexes can form -differentiate response of cells |
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Describe the patched/hedgehog signalling pathway.
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-Smo related to GPCR
-Ci75 is effector -in absence of Hh, Smo inhibited from going to mem. >Ci75 gets cleaved and transcription activity inhibited -when Hh present, Ci doesn’t get cleaved and can transcribe target genes for development -kinases involved |
Hedgehog = diffusible ligand; 3 in human Sonic Hedgehog, Desert Hedgehog and Indian Hedgehog
Patched = 12 transmembrane protein; 2 in human Smoothened = 7 transmembrane G protein coupled receptor like Costal 2 = Kinesin like microtubule based motor molecule- no human? Fused = ser/thr kinase associated with Costal 2 Cubitus interuptus (Ci) = Zinc finger transcription factor- 3 in human Slimb = supernumery limbs, F box protein targets ubiquitin to Ci PKA = cAMP dependent protein kinase CK1 = casein kinase 1 GSK3 = regulates metabolism (also Su/Fu) = suppressor of fused) Hedgehog signaling associated with cilia… |
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How is hedgehog involved in embryonic neural differentiation?
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-notochord secretes sonic hedgehog and prod. gradient throughout developing spinal cord
>diff. cell types have diff. fates (moto-, sensory neurons, etc.) |
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What signalling molecules are involve in the graded induction of diff. cell types in the neural tube?
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-combo of Shh and BMP IV
>BMP IV secreted from roof plate >sys. interact to define fate of neurons |
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What signalling molecule sets up the dorsal patterning of the neural tube?
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-ectoderm added to tissue expresses neural crest markers as does BMP 4/7
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What is the purpose of establishing signalling gradients?
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-gradients fine-tune expression of particular genes (to define phenotype of neuron)
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What are sensory organ precursors, how do they arise, and what is their fate?
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-define 1 cell in skin as sensory organ precursor
>migrates under skin to prod. 4 cells that have diff. fn. |
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Describe Notch/Delta signaling.
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-all cells express both notch and delta
>they interact and signal >a random cell with more delta signals more and turns off delta in surrounding cells |
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What is the mechanism of notch/delta signalling?
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-mechanism of prev. slide
-gamma-secretase cleaves |
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What is the result of delta/notch binding?
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-results in turning off ac genes (define cells to become hair or neurons)
>cell becomes epi -left: cell does opp. >signals more delta and transcribes ac |
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What is another pathway implicated in embryogenesis that is similar to hedgehog pathway?
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LRP=LDL receptor related protein APC=Adenomatous polyposis coli (APC) GSK-3Glycogen synthase kinase 3
-similar to Hh pathway >in absence of Wnt, complex of kinases are degraded and transcription inhibited >in presence of Wnt, phospho. events occur, beta-catenin migrates to nucleus and ternary complex factor is allowed to initiate transcription |
Components of pathway
Wnt = Wg (wingless) of Drosophila and Int, pronounced “Wint”. About 19 human genes. Frizzled = family of 10 7-transmembrane receptors possibly related to G protein coupled receptors. (Frzb (frisbee), secreted frizzled related proteins (sFRPs), Dkk- competes with Wnt for Frizzled receptor) LRP – Very large protein (~4,500 aa) involved in internalization of beta-amyloid and amyloid precursor protein Dishevelled = three genes making a cytoplasmic phosphoprotein- anchored to C-terminus of Frizzled by a PDZ domain APC = adenomatous polyposis coli, 800+ mutations- associated with predispositions to numerous colon polyps and – tumor suppressor beta-catenin = cytoskeletal protein associated with cadherin GSK3a = regulates metabolism, also a target of PKB/Akt Axin – contains an RGS domain |
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Describe how hair/fur placement is determined.
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-Turing-model similar to notch delta path
-can explain hair pattern on mice (and humans) -DKK is Wnt antagonist -overexpression of inhibitor yields further spaced hairs (nearly bald with high expression) -TG=transgenic over expression of DKK |
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What are the receptor classes and their method of activation of TF's?
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What are the receptors involved in developmental signalling and their mechanism of TF activation?
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What can be concluded from an experiment where cytoplasm from fert. egg injected into unfert. egg sends cell into mitosis?
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There must be diffusible factor that initiates mitosis.
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What is the diffusible factor that initiates mitosis?
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MPF (maturity/mitosis promoting factor)
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What is MPF?
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MPF is a heterodimer containing a catalytic subunit (cyclin-dependent kinase) and a regulatory subunit (cyclin).
MPF = cyclinB/cdc2 complex of cdc2 (cdk1) which is a kinase, and cyclin B (its substrate |
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Compare and contrast the levels of cyclins and cdk's during mitosis.
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What is key to progression through mitosis? What experimental model would you use to prove this?
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Cyclin B degradation is key to progression through mitosis
-can recapitulate mitosis with JUST cyclin b mRNA -mutant form of cyclin b incapable of being degraded--> causes mitotic arrest -cyclin b required and regulation dictates length of mitosis Frog egg extracts-Mitosis in vitro -eggs centrifuged until cytosplasmic (soluble) extract attained >spiked with sperm chromatin and Ca >get repeated cycles of mitosis in cell-free extracts >micrtoubule asters seen |
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What happens when you add mRNA encoding mutant form of non-degradable cyclin B to cell-free frog cell extract?
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Failure to degrade cyclin B results in mitotic arrest
-couldn't exit mitosis -proved degredation of cyclin b didn't cause transition from metaphase to anaphase >required for exit though |
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What is the driving force behind M-phase advance?
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Proteolysis is a driving force behind M-phase advance.
Anaphase-Promoting Complex (APC) directs ubiquitin-mediated degradation -MPF activity required to enter mitosis >activates APC >directs destruction of component that allows metaphase to anaphase to occur >directs desctruction of cyclin b also to allow cells to exit mitosis |
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What intracellular function does MPF have?
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MPF activity drives nuclear envelope breakdown
-lamina on inside of nuc. envelope (IF meshwork) >lamin subunit phosphorylation promoted by MPF (kinase) causes depolymerization |
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What is the cyclin-centric view of the early embryonic cell cycle?
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-syn of cyclin b drives kinase activity of MPF
-synchronous waves of mitosis caused by peaks and valleys of cyclin b |
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What can you deduce about the function of cdc25 and wee1 within the cell cycle?
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-levels of ctrl over cell cycle advance in cells beyond early embryo stage
-temp. sensi mutant yeast -cdc25 mutants were incapable of entering mitosis (very large cells) -wee1 mutant went into precocious mitosis w/o enough growth phase -wee1 inhibits MPF activity and cdc25 promotes it -cdc25 is a phosphatase and wee1 is a kinase -wee1 substrate is Y residue on cdk1/cdc2 >phosphorylation being inhibitive in this rxn |
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How is MPF regulated?
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-MPF activated in numerous ways (via phosphorylation rxn's)
>closely regulated event MPF is only active when it exists in a specific phosphorylation state (T161 phospho but not Y15) |
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How is MPF localization controlled?
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-most of substrates for MPF are nuclear (a few cytoplasmic)
>abundance ctrl'd by CRM1 |
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How is M-phase entry regulated?
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The M-phase entry is regulated by
a variety of mechanisms 1) Cyclin Accumulation (controlled at transcriptional level) -has to be syn. and accumulate to a critical level to activate enough cdc2 2) Cyclin Destruction (triggered by APC-depedent ubiquitination) -allows exit from mitosis 3) Cdc2 catalytic activity (phosphorylation/dephosphorylation) -by wee1, cak, and cdc25 4) MPF localization (CRM1-mediated nuclear export) -if not nuclear, not able to advance cell cycle |
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Summarize cell cycle regulation.
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-commitment point to enter mitosis occurs at S phase
>DNA replication commits a cell to cell division >very few cells are arrested at G2 |
There are 4 families of cyclins currently known to control
cell cycle progression • cyclin D • cyclin E • cyclin A • cyclin B There are 4 cyclin-dependent kinases that are currently known to control cell cycle progression • cdk6 • cdk4 • cdk2 • cdk1 (cdc2) -multiple cyclins and cdk's |
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When are the various cyclin/cdk complexes active?
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-cyclin d important in transitioning cells from G0 to G1
-quiescence=no cyclin activity -cyclin E drives the bus into S-phase, and A activates S phase (doesn't get degraded until prophase, part of mitosis but not as long as B) |
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What are the early and late response genes to growth factors?
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-growth factors initiate division
>causes gene responses due to signal transduction >fos/jun=transcription factors >cyclins are gene targets of fos and jun (early responders) Among the first late response gene products to accumulate are: • cyclin D • cdk2 • cdk6 These will form cyclinD/cdk2 and cyclinD/cdk6 complexes. These kinase complexes set the stage for cell cycle advance by: • inactivating APC • phosphorylate Rb protein -cyclin D can complex with cdk2 and 6 >early events determined by complexes and activity (inactivate APC to allow entry into division) >phospho Rb |
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How is E2F regulated?
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-Rb binds E2F
>Rb hyper-phospho causes it to let go of E2F which then becomes active TF >substrates include E2F itself (pos. feedback), cyclin E (ramped by E2F, also early responders) -Rb=tumor suppressor gene |
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What are CKI's, their classes, and some examples?
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Cyclin-dependent kinase inhibitors (CKIs)
A class of proteins which inhibit cyclindependent kinases by binding t CKI proteins fall into two classes: The INK4 family (Inhibitors of cdk4) are specific for cdk4 and cdk6: p16INK4A p15INK4B p18INK4C p19INK4D The Kip (Kinase inhibitor proteins) family inhibit all G1and S phase cdk enzymes: p21Cip1/WAF1 p27Kip1 p57Kip2 -during G1, molecules that inhibit CKI's -p16=tumor suppresor -inhibit cell cycle advance -none are catalytic in function...structural in function >can be overcome stoichiometrically (titrated) -INKs function by inhibiting cdk4/6 association with cyclin --p16 forms complex with cdk4 to inhibit cdk from complexing with cyclins |
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How do CKI's work?
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-Kips bind to active complexes to inhibit
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How is p27 regulated?
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-p27 must be gotten rid of to adv. thru cell cycle (interphase-->S)
-cyclinE phospho. it to promote p27 ubi and degradation |
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Describe control at the transition point of the cell cycle.
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-sustained input of growth factors needed to express the molecules necessary to drive progression thru cell cycle (sustained signalling to drive cyclin expression)
>overcome stoichiometric expression of p16 and subsequent fx >inactivating p27 -end-product: huge amt. of cyclin E and cdk4 |
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How is cyclin A activated?
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-cyclin activates cyclin A
>phospho. sic1 that forms complex with cyclin A cdk? to activate cyclin A >activates replication -G1 cyclin= cyclin D+CDK -SCF=ubi ligase that poly ubi sic1 to degrade via UPS (also ubi p27 for degradation) |
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What is the result of SPF activation?
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-essential to activate DNA rep. on specific cues
>low level of cyclins causes rep. machinery at origins to only be half assembled (pre-RC) that are also assoc. with inhibitors -cyclin A causes cdc6 to go cytoplasmic (turns it off), recruits cdc45, activates ORC's (Cdc45 is required for the subsequent binding of RPA, the heterotrimeric protein that binds singlestranded DNA generated when the MCM helicase unwinds the parental DNA duplex.) |
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Describe the mitotic spindle at different stages of mitosis. What does taxol cause?
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-accumulation of cyclins and degradation drive cell cycle
>pro. degredation catalyzed by enzymes are driving force so that the rxn cannot go backward (forces cell cycle to go forward only) -interphase to metaphase requires major re-org. of MT's >centrosome replication, migration, org. of mitotic spindle, MT attaching to chromosome and causing to align (metaphase progression) <MT-dependent> -taxol prevents progression to anaphase due to MT "freezing" in polymerized state (loses dynamic characteristic and won't be able to pull apart chromosomes due to inability to depoly.) (doesn't effect MPF so nuc. still breaks down) -reformation of nuc. env. depends on low MPF (anaphase A=chromosomes moving to poles, anaB=poles moving to opp. directions) |
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What are some other important kinases in mitosis besides cdk's?
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-bora=inhibitor of polo-like kinase
-polo-like kinase activates aurora A (activate each other) Currently, five polo-like kinases have been identified in humans (PLK1–5). A common feature of polo-like kinases is the presence of a highly conserved polo-box domain(PBD) in their carboxy-terminal region. The PBD serves as a phospho-dependent interaction motif serving to brings the kinase into close proximity with its substrate. For example, phosphorylation of PLK substrates by either cyclin-dependent kinase 1(CDK1) or PLK itself promotes the interaction between PLK and its substrates Not all PLK family members function in mitosis but: PLK1 promotes the activation of cyclin B–CDC2 (MPF) by inducing the degradation ofWEE1 and by activating the CDK-activating phosphatase CDC25C. Inactivation of Plk1 results in a failure of spindle assembly. During M-phase exit, PLK1 appears to activate APC by directly phosphorylating three subunits of the anaphase-promoting complex. PLK4 has a well-established role in centriole duplication, PLK2 has also beenimplicated in centriole duplication -PBD=phospho binding motifs (like SH domains) >force protein-protein rxn's >brings kinase into close prox. to substrate -PLK1 important in helping activation of MPF (efficient...degrading major inhibitor and activating major activator); modulates formation of cytoskeleton |
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What is the main function of Aurora?
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-aurora localizes at centrioles
>plays a role in spindle assembly and dynamics during chrom movement >oncogene (aur A expression can be increased resulting in misformed spindles and mis-segregation of chromosomes leading to chrom. misbalances (polyploid cells) and subsequent cancer) -some of the MT's interact with kinetochore at centromere of chromosomes |
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Describe how sister chromatids are separated during anaphase.
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-metaphase to anaphase transition is major commitment point to finishing cell division (everything downhill from that point)
-securin is probably first substrate of APC >securin inhibits seperase -once cohesins are degraded the cell undergoes anaphase >allows opposing forces of pulling MT's attached at kinetochores to pull apart the chromosomes |
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Describe the spindle checkpoint.
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-chromosomes on metaphase plate
-cdc20 is regulatory subunit of APC (ubi securin and activates seperase) |
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Contrast mitosis and meiosis.
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In meiosis there is 1 round of DNA replication, 2 rounds of cell division.
-end prod. of meisois is haploid (1n) gametes >shares many bichem. cues as mitosis |
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What occurs during the first phase of meiosis?
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-2 homologous chromosomes (from mom and dad)
>synapsed >1 cell has both copies of mom and 1 has both copies of dad |
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What occurs during the second phase of meiosis?
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-meiosis II is like mitosis
>sis chromatids seperated |
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How are sister chromatids kept attached during anaphase of meiosis I?
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-Rec8 is specialized cohesin
>cannot be degraded by separase at meiosis I >Rec8 can be degraded during meiosis II |
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What significant event occurs during prophase of meiosis I?
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-lepotene stage is entry into synapse formation
The snaptonemal complex forms between homologs that align during prophase I. These homologs are now referred to as synapsed and this facilitates crossing over and the formation of chiasma. Chiasma are visible during diplotene stage. Crossing over actually occurs duringpachytene stage but dissolutionof some of the synaptonemalcomplex is required to seechiasma. -synaptonemal complex physically holds together mom and dad chromosomes to allow recombination -chiasmas=covalent bonds between mom and dad (cross-over structures) |
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