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234 Cards in this Set
- Front
- Back
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Give some basic features of microfilaments |
Smallest of the cytoskeletal features (7-9nm) |
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Give some basic features of intermediate filaments |
medium size (10 nm) |
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Give some basic features of microtubules |
Largest cytoskeletal element (25 nm) alpha beta tubulin dimers make up structure of mitotic spindle |
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What is G actin? |
Globular, monomeric actin |
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What is F actin? |
Filamentous, polymeric actin |
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Give some examples of cellular structures which utilise actin |
microvilli, filopodia, lamellopodia, contractile ring |
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What are the three stages of actin polymerisation? |
1. nucleation (assembly of actin monomers to >3)
2. Elongation (addition of actin monomers to both ends) 3. Steady state (addition of monomers to (+) end and removal from (-) end) |
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What is the rate limiting step in actin polymerisation? |
Nucleation |
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Explain the idea of actin polarity and treadmilling |
Actin is constantly polymerising and depolymerising in the cell. ATP-G actin is always added to (+) end, then ATP is hydrolysed to ADP + P. ADP-G actin is then removed at (-) end causing treadmilling of the actin fibre |
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What other proteins are involved in the treadmilling process and what do they do? |
Profillin: promotes ATP-G actin formation for the (+) end, regulates formins |
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What are the two different classes of actin nucleating proteins? |
Formins = assemble unbranched filaments |
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What are formins and how do they work?
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Proteins that regulate actin assembly. Have an FH2 domain that forms a dimer, which then binds two actin subunits. By rocking back and forth, additional actin monomers are added. Regulated by profillin. Produces unbranched filaments |
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How does WASp and Arp2/3 regulate branched actin assembly? |
Cdc42 activates WASp (takes it out of its autoinhibited state). Active WASp then binds to and activates Arp2/3 by inducing a conformational change. Arp2/3 then forms a weak association with the actin filament. WASp also binds to G actin which binds to and strengthens the Arp2/3-actin connection. Branch nucleation and polymerisation then occurs |
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How is actin used in endocytosis? |
Endocytosis factors (e.g. clathrin) recruit WASp. WASp promotes Arp2/3 assembly of branched actin filaments which help transport the endocytosed vesicle into the cell |
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How is muscular dystrophy linked with actin? |
Dystrophin is a protein that links actin to the cell membrane |
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What are microfilament motors? |
Actin acts as a track for myosin motor motility |
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Where are type I myosins found? |
Found in cell periphery |
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Where are type II myosins found and what do they do? |
Found in muscle and non-muscle cells. required for cytokinesis and focal adhesion |
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What is myosin V? |
A type of myosin required for organelle transport |
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Explain the power stroke? |
ATP bound to mysosin (low energy) --> ATP hydrolysis (high energy state) --> ATP bound myosin binds actin --> Pi dissociates --> ADP dissociates --> power stroke --> ATP binds and starts the process again |
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What determines the rate of myosin movement? |
Length of neck |
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What is the sliding filament model? |
The idea that during sarcomere contraction, there is no actual change in the length of the fibres - just varying levels of overlap |
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Where is myosin II found and what does it do? |
Found in stress fibres and required for cytokinesis |
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What does myosin I do? |
Found in cell periphery |
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Explain the basic process of cell migration |
1. Lamellipodia extension (actin crosslinking) |
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What does Rho do? |
Regulates stress fibre formation |
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What does Rac do? |
Regulates lamellipodia formation |
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What does cdc42 do? |
Regulates filopodia formation |
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In a moving cell, where would Rho, Rac and cdc42 be active? |
Rear: Rho activation --> stress fibre formation and mysoin II (cytokinesis) activation |
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What is chemotaxis and how does it lead to cell movement? |
The sensing and movement towards chemical gradients by motile cells. Chemokines bind to GPCRs which results in PIP3 formation. PIP3 activates Rac to induce actin configuration changes. |
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In cell migration, various receptors, signalling molecules and cytoskeletal elements are moved around. What are some examples of elements that would be found at the rear and the front of the cell? |
Rear = myosin II, lipid phosphatase
Front = lipid kinase, actin, PI(3,4,5)P3 |
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What are some structural features of microtubules? |
Heterodimers of alpha and beta tubulin
Microtubules are made of 13 laterally associated protofilaments to form a tube |
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What is the microtubule organising centre? |
An anchor for the (-) end of microtubules, found near the nucleus, which extends MTs out into the cell |
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How does the MTOC change during mitosis? |
during mitosis, cells completely reassemble their microtubules to form bipolar spindles |
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Explain the idea of microtubule treadmilling |
alpha/beta tubulin dimers bind GTP and add to (+) end of microtubules. |
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What is anterograde transport? |
Transport towards the periphery (+) |
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What is retrograde transport? |
Transport towards the MTOC (-) |
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Explain how kinesin movement occurs |
1. Leading head binds, releases ADP, causing strong leading head connection and weak lagging head connection |
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Kinesin movement is highly processive- what does this mean? |
That it involves catalysing consecutive reactions without releasing the substrate |
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What governs the volocity of kinesin movement? |
ATP hydrolysis |
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How do kinesin and dynein connect to cargo vesicles?
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Both kinesin and dynein require adaptor proteins to join to the cargo vesicles |
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What is kinesin's adaptor protein to connect to cargo vesicles?
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Kinectin |
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What is dynein's adaptor protein to connect to cargo vesicles? |
Dynactin complex |
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What is the basic structure of cilia? |
Consist of a singlet pair of microtubules surrounded by 9 doublets (9 + 2 arrangement) |
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Why do cilia and flagella bend? |
Cilia and flagella bend due to dynein activation- dynein on A subunit of one doublet walks along the B subunit on the adjacent doublet. Because they're connected by nexin, the cilia bends |
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How are microtubules used during mitosis? |
Spindle microtubules attach to the kinetochore and pull chromosomes apart via a combination of sliding and treadmilling |
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The cytoskeleton is a common target for the treatment of what disease? |
Cancer |
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What is vinblastine? How does it work? |
A plant alkaloid used as a cancer treatment |
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What is Paclitaxel (taxol)? How does it work?
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An anti-cancer drug used in ovarian, breast, lung, bladder and other solid tumor cancers |
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Do IFs have polarity? |
No - they are not motors |
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Give some examples of different types of IFs |
Keratins (epithelial cells), vimentin (mesenchymal cells), desmin (muscle cells), lamins (nucleus) |
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What is an example of an intermediate filament disease? |
Epidermylosis bullosa simplex - due to mutation in keratin 14 |
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Why aren't there many microtubule/microfilament diseases? |
Because these tend to be embryonic lethal |
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What is fibronectin, and how does its expression differ between normal cells and cancer cells?
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it is a component of the extracellular matrix |
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Antibodies against fibronectin have what effect if present during development? |
Inhibit branching morphogenesis in salivary glands |
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What occurs if collagen II is deleted? |
Dwarfism |
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How do cells attach to the ECM? |
Receptors for ECM components = integrins. Integrins are heterodimers of different alpha and beta chains (different combinations give different specificity for what they bind to) |
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How do cell-ECM links change as the cell moves? |
Cell-ECM adhesion plaques stay in the same spot as the cell moves over the top of them |
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Explain cell-cell adherens junctions |
Cadherins are the transmembrane proteins, which are linked to actin inside the cell by alpha and beta catenins (alpha joins actin, beta joins cadherin). |
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What do cadherins do? |
They are transmembrane proteins that link cells to other cells via adherens junctions |
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What are podosomes and how are they relevant to cancer? |
Adhesion structures in cells containing Arp2/3. WASp and cortactin. Pososomes, named invadopodia, break down intracellular matrix and direct secretion of matrix metalloproteases |
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What is the fixation step for light microscopy? |
Specimen is fixed in a chemical (e.g. formaldehyde) causing cross linking of protein in the tissue to prevent distortion |
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What is the embedding step for light microscopy? |
Tissue is dehydrated (ethanol) then 'cleared' with solvent (e.g. chloroform), placed into melted wax and left to cool and solidify |
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What is the sectioning step for light microscopy? |
Block of tissue cut into 5 um sections using a knife held in a microtome |
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What is a HE stain and what colours does it produce in what parts of tissue? |
Haematoxylin = basic stain, stains acidic structures (DNA) purple |
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What would a normal cell look like with a H&E stain? |
Purple nucleus, pink cytoplasm |
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What is IHC useful for? |
Analysing spatial expression of target antigens, not so much the quantative expression levels (western blots/ELISA are better) |
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What are the major steps of IHC? |
Fixation and antigen retrieval Visualisation of antibody |
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What is the fixation step of IHC? |
Formaldehyde generates methylene bridges that crosslink proteins in tissue sample to protect morphology. However these can hide antigenic sites so heat or enzymes are used to partially break the bridges |
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How is endogenous target activity blocked during IHC |
Immunodetection that relies of enzymes such as peroxidase needs to have endogenous activity blocked (eg by incubation with hydrogen peroxide) to prevent excessive background staining |
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How is non specific protein binding prevented in IHC? |
Tissue sections are incubated with buffer (skim milk, BSA) which should bind and block proteins to which antibodies may weakly bind, preventing false positives. Detergents are often inhibit non-specific hydrophobic reactions |
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What are the two types of immunodetection in IHC? |
Direct - reporter is conjugated to an antibody (primary)
Indirect - primary antibody against antigen + anti-antibody with reporter attached |
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Does direct or indirect immunodetection have higher sensitivity? |
Indirect |
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How does visualisation of the antibody occur in IHC? |
Through chromogenic or fluorescent means |
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What is counterstaining in IHC? |
A different stain is added to the tissue to contrast the primary IHC stain e.g. haematoxylin to stain nuclei purple |
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What is the primary structure of a protein? |
Amino acid sequence |
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What is the secondary structure of a protein? |
Alpha helix and beta sheets |
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What is the tertiary structure of a protein? |
The protein's 3D structure |
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What is the quaternary structure of a protein? |
Dimerisation, oligomerisation |
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What are structural motifs? |
Combinations of secondary structures e.g. ring finger, zinc finger motifs. For non-covalent interactions (commonly divalent) |
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What are protein domains? |
Distinct regions of tertiary structure which can have its own structural and functional properties (enzymatic etc). |
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What is a Svedberg unit? |
The rate of sedimentation on a centrifuge Related to size, shape and density. Non standard, non-linear (50S might not be twice the size of 25S) |
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What is the benefit of multi subunit complexes vs modular domains in a protein? |
Modular domains can't be swapped out |
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What are post translational modifications? |
covalent modifications that change a protein's structure |
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What is phosphorylation? |
The addition of a phosphate group to a serine, tyrosine or histidine by kinases |
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What is ubiquitination? |
The addition of one or more ubiquitin peptides to lysine residues |
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What is allosteric regulation? |
The change in protein structure/function due to non-covalent binding of a ligand |
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What is calmodulin? |
An example of allosteric regulation where Ca binding changes the tertiary structure allowing it to bind to other proteins |
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What does a GEF do? |
switches GDP out for GTP |
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What does a GAP do? |
switches GTP out for GDP |
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What are chaperonins? |
Barrel shaped folding machine, with a lid homoheptamer, which together fold proteins powered by ATP |
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What are the major features of the nuclear membrane? |
The nucleus has an outer and inner membrane. The inner membrane defines the nucleus and the outer is continuous with the rough ER. Has nuclear pores for key protein exchange
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What is the nuclear lamina? |
A meshwork of intermediate filaments (lamins) which interconnect with nuclear pores - provides structure to the nucleus |
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What is the nucleolus? |
A suborganelle within the nucleus, with no membrane, the site of ribosome biogenesis - hotspot of transcriptional activity |
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What are nuclear bodies? |
Membraneless sub compartments, concentrated regions of protein and RNA transcription and processing. Formation may enhance process efficiency |
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What is chromatin? |
A complex of DNA and histone proteins (nucleosomes) which has a dynamic structure that determines gene expression |
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How it chromatin structure regulated? |
Histone tails extending from a nucleosome can be targets for post translational modification |
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What is chromatin acetylation? |
PTM that makes the chromatin less condensed and more transcriptionally active |
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What is chromatin deacetylation? |
A highly condensed form of chromatin that is less transcriptionally active |
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What is the nuclear pore complex? |
A huge structure in the nuclear membrane made up of ~30 different nucleoporin (Nup) proteins Cytoplasmic and nuclear asymmetry |
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What are nuclear localisation sequences? |
Amino acid sequences that target a protien to the nucleus, generally internal. |
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What are importins and exportins? |
Cargo carrying complexes that overcome the size limit of the NPC, allowing proteins to be brought in/out. They are receptors for FG nups in the NPC, and recognise the nuclear import/export sequences on the cargo |
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How is nuclear import/export controlled? |
Uses a GTPase swtich - RanGTP/GDP |
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What are the steps of nuclear import? |
Importin binds NLE on cargo and moves into the nucleus, along with RanGDP. |
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Show nuclear import with a diagram |
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What drives the movement of the importing complex across the nuclear membrane? |
More GTP inside nucleus (drives GTP out) |
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Explain the nuclear export mechanism |
Exportin and RanGDP diffuse into nucleus |
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What are laminopathies? |
Genetic mutations that affect lamins, nuclear proteins connected to lamins or proteins involved in lamin maturation |
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Give some examples of laminopathies |
Muscular dystrophies and cardiomyopathies |
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What is Hutchinson-Gilford progeria syndrome? |
Accelerated ageing, premature hair loss, muscle wasting, fat loss, reduced bone density |
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What are the morphological differences between the smooth and rough ER? |
Rough = sheetlike cisternae - flattened membrane, covered with ribosomes |
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How does the ER membrane curve? |
reticulons inserted into the membrane are responsible for curvature |
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How is 3 way branching acheived in the ER? |
Comes about via membrane fusion between GTPase alastins in the membrane. When two membranes come close, alastins dimerise and fuse |
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Where is the ER targeting sequence on proteins? |
Signal located at the N terminal of the growing peptide |
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What is cotranslational translocation? |
ER targeting must happen at the same time as protein synthesis |
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Explain the basic process of cotranslational translocation |
Signal recognition particle recognises the ER targeting sequence on the growing peptide in the ribosome. SRP moves to the SRP receptor near the translocon on the ER membrane. GTP hydrolysis in both the SRP and the SRP receptor. Translocon opens and signal sequence is cleaved by signal peptidase. Translation stops, ribosome dissociates and protein folds in the ER. |
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Explain cotranslational translocation with a diagram |
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Show the different types of ER membrane proteins conformations |
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What is a type 1 ER membrane protein? |
A protein which is inserted into the ER membrane, with the C terminal outside and the N terminal inside. Majority of protein is inside. Needs a stop transfer anchor. Has a cleaved terminal ER targeting sequence |
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What is a type 2 ER membrane protein? |
Does not have a cleavable N terminal signal sequence - internal targeting signal anchor sequence instead. |
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What is a type 3 ER membrane protein? |
No cleavable N terminal sequence - signal anchor sequence close to N terminal. |
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How do type 2 and 3 ER membrane proteins have different conformations?
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The location of +ve charged residues --> these prefer to stay on the cytosolic side |
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What kind of modifications occur in the ER? |
Glycosylation |
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What is hereditary spastic paraplegia? |
Progressive stiffness, contraction (spasticity), loss of coordination and lower limb weakness |
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Does the golgi have quality control mechanisms? |
No - unlike the ER. |
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What is the basic structure of the golgi apparatus? |
Parallel stacks of flattened membrane discs connected to form ribbons. Transitions from cis golgi network to cis golgi to medial golgi to trans golgi to trans golgi network |
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What are the cis and trans golgi network? |
Fenestrated tubular netowrks that receive and secreted vesicles respectively |
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What two proteins hold the golgi together? |
GRASPs = golgi reassembly and stacking proteins that dimerise/oligomerise to facilitate stacking |
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What is anterograde golgi transport and what facilitates it? |
Transport from ER to Golgi |
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What is retrograde golgi transport and what facilitates it? |
Transport from golgi to ER |
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How is cargo sorted into vesicles to be transported to the golgi? |
Membrane cargo proteins = sorting signal on the cytoplasmic domain recognised by coat proteins |
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What is retrograde retrieval? Give an example of a protein that has to be recovered in this way? |
Sorting signals are required to maintain residency within a compartment |
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What is cisternal maturation? |
Each membrane compartment over time matures to become the 'next' on e.g. cis golgi matures to become the medial golgi. |
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How are the different golgi compartments funtionally different? |
Each compartment has its own resident enzymes. |
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What are congenital defects of glycosylation? |
A complex and highly heterogenous disease resulting from deficiencies in glycan modifying enzymes in golgi compartments --> reduced branching and glycan extension |
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What is significant about the way that mitochondrial DNA is inherited? |
Inherited cytoplasmically and maternally |
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What does mitochondrial DNA code for? |
Always codes for mitochondrial proteins, but most of the coding is done by the nucleus |
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What does the mitochondrial matrix contain? |
mtDNA and mitochondrial ribosomes |
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What are the different shapes that mitochondria can take on? |
Can be individual spheroids (M phase) to long elongated networks (G1/S) |
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How does mitochondrial fission occur? |
Mitochondrial fission factors recruit G proteins that hydrolyse GTP to constrict membranes and separate mitochondria |
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How does mitochondrial fusion occur? |
Mitofusins are G proteins that hydrolyse GTP to fuse membranes |
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What are the features of the mitochondrial targeting sequence? |
20-50aa N terminal cleaved sequence. |
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Compare the ER, mitochondrial and nucleus targeting sequences |
ER = N terminus, cleaved, short hydrophobic |
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What is significant about the cargo for mitochondrial transport? |
Protein synthesis occurs in the cytoplasm but proteins are kept unfolded by chaperones (cytosolic Hsc70) |
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What translocons are involved in mitochondrial transport? |
To get to the matrix (if that's where they're directed to go) proteins must go through the outer membrane translocon (TOM) and the inner membrane translocon (TIM) SIMULTANEOUSLY |
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Explain the basic process of mitochondrial transport |
Protein synthesis in cytoplasm, protein is kept unfolded by Hsc70 (no ribosome directly involved - no cotranslational translocation) |
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What is oxidative phosphorylation? |
Coupling a series of oxidation/reduction reactions with phosphorylation of ADP to generate ATP |
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What are NAD and FAD? |
High energy electron carriers |
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What is stage one of oxidative phosphorylation? |
Sugar and fat metabolism |
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What is stage two of oxidative phosphorylation? |
Giving up electrons |
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What is stage three of oxidative phosphorylation? |
Pumping protons |
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What is stage four of oxidative phosphorylation? |
Generating ATP |
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How is mitochondrial fission/fusion used for quality control? |
Damaged components are segregated and removed through fission and targeted for degradation
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What is PINK and Parkin and how are they related to Parkinson's disease?
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PINK is a kinase and Parkin is a Ub ligase - they are required to prevent fusion of damaged mitochondria back into the healthy pool |
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How was the secretory pathway first visualised? |
Using a temperature sensitive mutant of vesicular stomatitis virus G |
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What is the basic process of vesicular transport? |
Cargo in donor cell (soluble/membrane bound) |
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What is a class A sec mutant? |
Accumulation of protein in cytosol |
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What is a class B sec mutant?
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Accumulation of protein in rough ER |
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What is a class C sec mutant? |
Accumulation of protein in ER --> golgi vesicles |
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What is a class D sec mutant? |
Accumulation of protein in golgi |
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What is a class E sec mutant? |
Accumulation of protein in secretory vesicles |
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What are the two major types of cargo involved in secretion? |
Passengers to be moved (e.g. newly synthesised proteins) |
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Where does COPII transport go to and what major proteins are involved? |
ER --> cis golgi transport |
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Where does COPI transport go to and what major proteins are involved? |
cis golgi --> ER |
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What provides the selectivity for COP transport? |
Different coat proteins/GTPase provide specificity and selectivity |
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Why will some cargo bind to a receptor in the golgi and not in the ER? |
The capacity for soluble cargo to engage receptors and be transported depends on the binding properties which differ between compartments
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Give an example of a type of soluble protein that undergoes retrograde transport |
ER resident proteins with a KDEL (lys-asp-glu-leu) binds to the KDEL receptor in the lower pH of the golgi and is returned to the ER via a COPI vesicle |
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How is the KKXX signal sorted and transported? |
KKXX is a sorting signal on ER resident membrane proteins |
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How is the X-Arg-Arg-X signal sorted and transported? |
X-Arg-Arg-X is a sorting signal on ER resident membrane proteins |
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Explain the basic process of COPII vesicle formation |
Sar1 in its cytoplasmic GDP form interacts with sec12 (a membrane bound GEF), and is transformed into its GTP, ER-membrane-bound form. Sar1 GTP, in addition to sec23/24 interact with the cytosolic signalling domains of particular cargo (which is sorted to go to the golgi) forming the prebudding complex. Sec 13/31 is then recruited, and polymerisation of these proteins occurs until a bud is formed. The bud then undergoes scission by dynamin to become a free-floating, coated vesicle. The coat is then removed when sar1 hydrolyses GTP (sec23 is the GAP). Coat disassembly produces a free floating, uncoated vesicle which travels via the microtubule system to the golgi |
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Explain the mechanism of vesicle fusion |
Free floating vesicle has VAMPs and RabGTP
RabGTP docks on the Rab effector on the acceptor membrane, GTP hydrolysis stabilises binding. VAMP interacts with SNAP25 and syntaxin to form the SNARE complex (2 x SNAP25, 1 x VAMP, 1 x syntaxin). Fusion occurs NSF/alpha SNAP disassemble SNARE complex by ATP hydrolysis - VAMPs are returned |
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What are the GTPases involved in COPII, COPI and vesicle fusion? |
COPII = Sar1 |
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What are the components of a SNARE complex in vesicle fusion? |
2 x SNAP25 1 x VAMP (vesicle) |
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What is the segregation sorting mechanism? |
Having different proteins in the cytoplasm and lumen that physically will not meet |
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What is an active sorting signal? |
Sorting signals on proteins that will include them in transport vesicles - can be just the protein (membrane bound) or through protein-protein interactions (soluble protein) |
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How does active retention work as a sorting mechanism?
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Protein retained in cell due to interactions e.g. with another complex too big to bud off |
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How does retrieval work as a sorting mechanism? |
A specific sorting mechanism on the protein allows it to be retrieved e.g. KDEL |
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What are the two major types of secretion? |
Constitutive |
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Why does the golgi have different compartments? |
So the various processes (e.g. glycosylation) can be completed correctly and in the correct order (if all the proteins and enzymes were in one big compartment there would be many errors)
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What is the evidence for cisternal maturation? |
- live cell imaging (fluorescent tagging of yeas cell golgi shows transition from cis (green) to trans (red)) |
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What is constitutive secretion? Give an example |
Sorting signal independent - used when protein needs to be constantly secreted (bulk flow). Soluble and transmembrane proteins included - level of secreted protein depends on the level produced by individual cells |
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What is regulated secretion? Give an example |
Sorting signal dependent pathway that involves protein being accumulated in vesicles and released in response to a stimulus. e.g. insulin (seen in its proinsulin form in new secretory vesicles and mature form in vesicles near the membrane) |
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Explain the regulated secretion of insulin |
Munc molecules are key regulators - bind syntaxin and prevent SNARE complex formation |
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What is I cell disease?
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A disease characterised by a deficiency of lysosomal acid hydrolase proteins leading to build up of substances in the cell that can not be degraded as they should (inclusions). A deficiency of a phosphatase in the golgi means that lysosome-bound proteins are secreted normally because they don't have a mannose-6-phosphate signal (to be recognised by the M6P signal and sent to the golgi). This results in growth and physical defects. |
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Explain the mannose-6-phosphate pathway |
The M6PR in the golgi recognises the M6P signal on lysosyme-bound proteins (acid hydrolases). When bound in the TGN, this complex stimualtes clathrin-coated vesicle (via adaptins) transport to the late endosome. Here, lower pH stimulates receptor-ligand uncoupling - receptor is transported via retromer back to the TGN or to the cell surface. Any acid hydrolyase that was randomly secreted via constitutive secretion can be brought back via this surface receptor (endocytosis). As the late endosome matures to become a lysosome, the acid hydrolase becomes active |
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What are tethers? |
Peripheral membrane proteins that reach out into the cytoplasm to sense and bind particular vesicles. They don't have quite the same specificity as SNAREs, and are more about increasing the probability of successful SNARE interaction. They often involve a small GTPase (Rab). |
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What are the major functions of plasma membrane proteins? |
Transport |
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Why does a cell need endocytosis? |
Uptake of nutrients |
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What is LDL? |
Low density lipoprotein - a structure through which cholesterol can be absorbed in our bodies |
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Explain the mechanisms behind LDL binding/uncoupling from its receptor |
LDL-R is on the cell membrane and has an internal cytoplasmic sorting signal. Signal = NPXY (Asn-Pro-Val-Tyr). Adaptin binds NPXY signal and initiates endocytosis by recruiting clathrin. |
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What sorting signal is on LDL? |
NPXY |
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How does LDL bind to its receptor? |
At high pH (extracellular) the ligand binding arm binds apoprotein B |
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How does LDL unbind from its receptor? |
At low pH (endosome) the B propellor domain becomes +vely charged and binds the ligand binding arm, releasing LDL |
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Explain the basic LDL pathway |
LDL-R N terminus binds LDL, C terminus binds adaptin. Adaptin recruits clathrin which stimulates endocytosis bud formation. Bud undergoes scission when fully formed, by dynamin. Clathrin coat disassembly occurs. Uncoated vesicle duses with endosome, where low pH causes receptor and LDL to dissociate. Receptor is brought back to surface by recycling endocytosis, LDL stays in maturing endosome and is eventually broken down into cholesterol/fatty acids etc |
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What is the functional unit of clathrin? |
Triskellion
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What is dynamin? |
A GTPase that creates a ring, hydrolyses GTP causing a conformational change that pinches off a membrane bud |
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How is actin polymerisation used in endocytosis? |
Actin polymerisation (Arp2/3, WASp etc) is used to drive the vesicle away from the membrane (particularly bigger vesicles). |
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What is coincidence detection? |
Recruitment of a protein to a particular point can depend on a number of factors e.g. lipids and proteins that must both be present for binding to occur |
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What is familial hypercholerterolemia? |
excess LDL in blood - recessive single gene mutations in LDL receptor |
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What is macropinocytosis? |
bulk fluid phase endocytosis - not selective for cargo. Regulated by growth factor receptor signalling. Leads to extensive remodelling of plasma membrane. Involved in many processes - immune system, tumor progression. |
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How do pathogens modulate macropinocytosis? |
- induction of cell signalling (act on receptor) |
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What are endosomes? |
A sorting site which sorts proteins away from the lysosome, and a major site of protein degradation. Endosomes become acidified via a proton pump (lowers pH)
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Explain the process of transferrin uptake into a cell
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Ferrotransferrin (with Fe3+) binds to receptor and stimulates clathrin coated endocytosis. Scission and uncoating of vesicle in cytoplasm. Fusion of uncoated vesicle with endosome. Low pH causes release of Fe3+ from ligand but NOT THE RELEASE OF THE LIGAND. Receptor + ligand is recycled back to cell surface. Low pH causes ligand release from the receptor (when there is no Fe3+ attached - apotransferrin) |
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What happens to membrane proteins and intraluminal proteins in the late endosome/lysosome? |
Membrane proteins tend to survive whereas material within the intraluminal vesicles is degraded |
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Explain the basic process of EGF receptor |
An example of receptor/ligand degradation |
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How can the endosome act as a signalling platform? |
Signalling can occur from a receptor + ligand on the cell surface. Signalling can also occur from an internalised receptor + ligand in the endosome. Differential outcomes of signalling cascades due to different initiation points (different signalling cascades from the same endosome/different signalling from early vs late endosome). |
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What are multivesicular bodies? |
Specialised endosomes that transit from early (sorting) endosome to late endosome/lysosome. |
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Explain the process of the internalisation of a vesicle into a MVB.
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Ubiquitination of HRS proteins and cargo proteins on the endosome surface stimulates the formation of a bud. As bud grows, ESCRT proteins bind to ubiquitinated HRS proteins which stimulates the internalisation of the vesicle. ESCRT proteins are still bound to the Ub HRS proteins, and Vsp4 is required to use ATP to separate them. |
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Explain how HIV uses the MVB pathway to exit cells
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HIV gag protein on cell surface is ubiquitinated and bud containing HIV core particle is formed. ESCRT proteins then bind to the Ub Gag, stimualting the release of the vesicle (an enveloped HIV virus). Vsp4 then uses ATP to disassemble the ESCRT proteins |
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What is autophagy? |
The recycling of worn out organelles and degradation of specific cargo. Used in response to nutrient starvation, infection or apoptosis |
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Which would appear more dense during electron microscopy - an endosome or a lysosome? |
A lysosome - full of enzymes and degradative material |
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How do lysosomes lower their pH? |
A proton pump |
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How is the cell protected from the degradative enzymes it synthesises? (Why do they only work in the lysosome?) |
Degradative enzymes may only work at the low pH of the lysosome and/or may be synthesised as precursors which require cleavage before they become active |
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How are nutrients retrieved from the lysosome to the cytoplasm? |
Nutrient pump |
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What are some intracellular tracking pathways that involve recycling back to the plasma membrane? |
LDL |
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Give an example of a receptor pathway that requires retrograde transport? |
M6P pathway - recovered from late endosome to the golgi |
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Give an example of a receptor pathway in which both the ligand and the receptor are degraded |
EGF pathway |
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How is the maturation of the endosome defined? (i.e what differentiates an early from a late endosome?) |
As endosomes mature, the outside components change- maturation is defined by presence of specific Rab molecules and phosphoinositide content
This allows different trafficing machinery to be recruited and different cargo to be sorted |
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How can early/late endosomes have different functions? |
The different Rab/PI content allows different trafficking machinery to be recruited and different cargo to be sorted |
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What are the different Rab effector functions? |
Sorting = Rab can activate a sorting adaptor to sort a receptor into a budding vesicle |
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What is myosin V |
Actin associated motor that moves vesicles around |
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What are Lewy Bodies? |
Intracellular protein aggregates found in parkinson's disease- still debated whether these are a cause of the disease or a symptom |
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What is alpha synuclein? |
The most abundant protein found in lewy bodies, and is directly linked to the activation of apoptotic pathways |
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How is immunofluorescence microscopy prepared? |
Sample prepared on slide |
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How are monoclonal antibodies made? |
Mouse injected with antigen X |
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What is the Stokes shift? |
The difference between the peak absorption and peak emission of the fluorescent protein used in IF microscopy. |
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What is confocal microscopy? |
Relies on elimination of out-of-focus light to create optimal sections |
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What is two photon microscopy? |
Used for imaging live tissue |
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What is electron microscopy? |
A higher resolution/magnification microcopy technique than light microscopy. Samples are always processed. Live imaging is possible however processing artifacts can occur. Samples are stained with heavy metal prior to being imaged. |
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Why is yeast a good model organism? |
- unicellular but a model of multicellular organisms |
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What is VRP1? |
The yeast homolog of human WASp |
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What is the phenotype of a VRP1 mutant? |
Changes in morphology, proliferation rate and temperature sensitivity |
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Explain the basic process of PCR |
Repeated heating cycles to make multiple copies of a target section of DNA |
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What happens if the annealing temperature is too high/too low? |
Too high = primer may not bind |
