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398 Cards in this Set
- Front
- Back
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Cells are small...why? |
-so they can have maximum surface area to volume ratio - important in gas exchange -ensures that cytoplasm volume per nucleus is low - ensures optimal production of mRNA and proteins -membrane is fragile so a smaller cell is better as it requires less support, therefore less tension on membrane |
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Cells are complex |
-made of many different connected parts -cells are ordered and consistent entities |
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Consequence of cells being small and complex |
-cell biology is reductionist (understanding the parts is crucial to understanding the whole) -b/c cells are small & complex, we need special tools to study them |
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What kind of observation has led to our knowledge/understanding of cells? |
Indirect observation |
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Compound microscope |
Allows us to see cell processes |
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Transmission electron microscope |
1st microscope that allowed us to see things |
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Robert Hooke |
-coined the term "cell" -credited w/ discovery of cells -Head of Royal Society |
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Anton van Leeuwenhoek |
-made his own microscopes -created a lens superior to other microscopes -first person to describe many single cell organisms including protists (animalcules), bacteria, sperm -first to observe animalcules (microscopic organisms) -wrote letters to Royal Society describing these microscopic observations |
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Creators of first 2 tenets of cell theory and the 2 tenets |
Matthias Schlieden (botanist), Theodore Schwann (zoologist) - 1800s 1. All organisms composed of 1 or more cells 2. The cell is the structural unit of life -These 2 points proved to be less insightful as they both agreed that cells could arise from non-cellular materials |
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Creator of third tenet of cell theory and the 3rd tenet |
Rudolf Virchow 3. Cells can arise only by division from an existing cell The exception being the very first cell ever (The cell theory asserts the importance of cells) |
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Chemical interactions in the cell |
-all work and interactions happen inside a cell is result of chemical bonds forming and breaking up -at the molecular level, every event in the cell has to trigger the next automatically -chemical interactions are EVERYTHING -movement of molecules is actually not smooth at all -the cell is a very crowded place |
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Basic properties of cells |
1. Cells are complex 2. Cells possess a genetic program and the means to use it 3. Cells can reproduce 4. Cells acquire and utilize energy 5. Cells carry out chemical reactions 6. Cells engage in mechanical activites 7. Cells interact w/ their environment 8. Cells are capable of self-regulation (homeostasis) 9. Cells evolve |
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Cells are complex |
more complex = more parts must be in proper place, less tolerance of error |
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Cells possess a genetic program and the means to use it |
Genes constitute recipes for constructing cell structures, directions for running cellular activities, and a program for making more of themselves Main difference b/w cells and viruses = cells have a recipe and can carry it out |
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Cells acquire and utilize energy |
Energy from sunlight trapped by light absorbing pigments present in membranes of photosynthetic cells, convert into chemical energy and stored in energy rich carbohydrates Energy used in the form of ATP |
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Cells carry out chemical reactions |
Sum of total of chemical reactions in a cell represents its metabolism |
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Cells engage in mechanical activities |
Materials transported from place to place Structures assemble and disassemble Entire cell moves itself Motor proteins carry out these mechanisms |
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Cells interact with their environment |
Cell has receptors that provide pathways through which external stimuli can evoke specific responses in target cells |
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Cells are capable of self-regulation |
Cells are robust b/c they are protected from dangerous fluctuations in composition or behavior |
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Cells evolve |
All living organisms have evolved from a single common ancestral cell (LUCA) that lived more than 3 billion years ago |
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Evolution |
Life - photosynthetic bacteria - cyanobacteria - eukaryotes - algae - invertebrates - vascular plants - mammals - humans Pre-cambrian: Life -- Invertebrates Cambrian: vascular plants -- humans |
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Domains of life |
-Carl Woese made a phylogenetic tree of life to distinguish b/w the 3 domains based on rRNA sequence 1. Bacteria (prokaryote) 2. Archaea (prokaryote) 3. Eucarya (eukaryote) - protists, fungi, plants, animals |
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Why was rRNA used to make phylogenetic tree of life? |
-all domains have ribosomes -all domains need ribosomes to function -ribosomes are less prone to mutation |
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Development of the eukaryote |
-by endosymbiosis -prokaryote engulfed aerobic prokaryote and made use of its ability to make energy (the mitochondria) and also engulfed another prokaryote and made use of its ability to convert light energy into stored chemical energy (chloroplast) -anaerobic heterotrophic prokaryote engulfed an aerobic prokaryte and used the oxygen it provided - prokaryote's metabolism figured cell could survive with this aerobic prokaryote so did not destroy it - aerobic pokaryote becomes mitochondrion - nuclear envelope and ER precursors - turns into eukaryote - host cells profited from the chemical energy the mitochondrion produced while the mitochondrion benefitted from the protected nutrient-rich environment surround it |
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Order of appearance in eukaryote: mitochondria and chloroplast |
Mitochondria were incorporated BEFORE chloroplasts |
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Characteristics common to prokaryotes and eukaryotes |
-similar plasma membrane: lipid bilayer -both use DNA, similar genetic code: made of 4 nucleotides, encodes the recipe for itself, nucleotides form codons which code for amino acids -similar transcription & translation mechanisms: ribosomes necessary for transcription, ribosomes present in all domains -shared metabolic pathways -plants and cyanobacteria: similar photosynthesis |
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Features unique to eukaryotes |
-nucleus -separate chromosomes: DNA inside chromosomes, recipe stored in multiple linear chromosomes in eukaryotes whereas in prokaryotes it's stored in singular circular chromosomes -membranous organelles: organelles form compartments where reactions are kept separate from each other which is beneficial if some reactions are harmful to other organelles/parts of cell -presence of mitochondria (for anaerobic respiration) and chloroplasts (for photosynthesis) -complex cytoskeleton -ability to phagocytose -endocytose (engulf and incorporate) material -diploidy - 2n chromosomes -mitosis (cell division) using a mitotic spindle: microtubules form mitotic spindle, ensures each new cell gets correct number of chromosomes, allows each daughter cell to receive equivalent array of genetic material -sexual reproduction using meiosis |
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4 types of macromolecules |
-carbohydrates (sugars) -proteins -nucleic acids -lipids |
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Common feature between 3 out of 4 of the types of macromolecules |
Common feature b/w carbs, proteins, and nucleic acids: monomers combine to form polymers *Lipids are not monomers that combine to form polymers |
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Common feature among all 4 types of macromolecules |
-they all have a carbon backbone -carbon has 4 valence electrons making it easy to form multiple bonds |
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Silicon has 4 valence electrons...why isn't the backbone made of Si? |
Silicon can only form short chains whereas carbon is packed closer to the nucleus so the bond is stronger and carbon can make longer chains |
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Name of precursor and its respective macromolecule for all 4 types |
Precursor --> macromolecule Monosaccharide --> polysaccharide Fatty acids --> lipid Amino acids --> protein Nucleotides --> nucleic acids |
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Monosaccharide (carbohydrate) |
-one sugar monomer -can be cyclical - (CH2O)n where n is the # of carbons triose n=3 tetrose n=4 pentose n=5 hexone n=6 |
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Disaccharide |
-2 monosaccharides attached by glycosidic (alpha) bonds (covalent bonds) -readily available energy stores Ex: glucose + fructose = sucrose |
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Oligosaccharide |
-small chain, 3-10 monomers -branched -covalently attached to lipids and proteins making them called glycolipids and glycoproteins -forms "ID card" for protein -serves to distinguish one cell from another and help mediate specific interactions of a cell with its surroundings |
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Polysaccharide |
-repetition of mono or disaccharides -glycogen, starch, and cellulose area all glucose polymers |
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Why can't we eat cellulose? |
Because we don't have enzymes to digest beta bonds (cellulose is made from beta bonds) Humans can only digest alpha bonds (units are oriented in the same direction) |
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Why can cows eat cellulose? |
Cows themselves do not have beta bond digestive enzymes but bacteria living in their stomach do |
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Are sugars polar? |
Yes -due to their large # of hydroxyl groups, sugars tend to be highly water soluble and therefore polar -hydrophilic |
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What joins sugars to one another? |
Glycosidic (covalent) bonds |
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Lipids (3 types, common feature) |
3 types: fats, steroids, phospholipids Common features: -dissolve in organic solvents -do not dissolve in water (water insoluble) |
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Fats and their structure |
-fat = triacylglycerol = triglyceride -structure: glycerol moiety attached to 3 fatty acid tails via ester bonds - long unbranched hydrocarbon chains w/ a single carboxyl group at one end - |
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Where are fats stored? |
-stored in adipocytes whose cytoplasm is filled w/ 1 or few large lipid droplets -stored in adipocytes b/c water insoluble -insoluble in water due to lack of polar groups so stored in adipocytes in form of dry lipid droplets |
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Fatty acids |
-amphipathic --> hydrocarbon chain (fatty part) is hydrophobic/non-polar and the carboxyl group (acid part) is hydrophilic/polar -structure: carboxyl group (acid part) is water soluble/fat insoluble and polar and a variable length C chain & methyl group make up the fatty part and is water insoluble/fat soluble |
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Are fats amphipathic? |
A triglyceride (fat) is not amphipathic because there are no water soluble carboxyl groups on the end However, a free fatty acid is amphipathic |
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Effect of double bonds in a fat |
-unsaturated fat -bonds don't always introduce kinks -the more double bonds a fatty acid chain has, the less effectively these long chains can be packed together |
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Cis fatty acid |
-C's on double bond in "cis" conformation -kink in chain -more flexible -liquid (oil) at room temperature -bent |
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Trans fatty acid |
-C's on double bond in "trans" conformation -straight chain -rarely exist in nature -solid at room temperature -straight |
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Steroids (lipid type) |
-built around characteristic 4-ringed hydrocarbon skeleton -steroids are amphipathic -cholesterol is important because it is a pre-cursor to fat-soluble hormones (testosterone, estrogen, etc.) |
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Phospholipids (lipid type) |
-one fatty acid chain in a fat is replaced by a polar head group --> only 2 fatty acid tails -diacylglycerol - hydrophilic polar head group and glycerol backbone and 2 hydrophobic fatty acid tails -phospholipids are amphipathic |
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Proteins |
-made of same 20 amino acids -3 components: amino group, R group, carboxyl group |
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What determines amino acids? |
-R-groups (the side chain) determine the chirality of amino acids - note: amino acids always have L-stereoisomer in proteins |
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Polar charged R-group |
-hydrophilic (b/c polar) -act as acids or bases so fully charged at physiological pH -form ionic bonds -involved in chemical reactions -strong |
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Polar uncharged R-group |
-hydrophilic -partial charge allowing them to participate in chemical reactions -form H bonds -associate with water -slightly weaker |
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Non-polar R-group |
-hydrophobic -made of C and H only -form inner core of soluble proteins -buried away from aqueous medium -associate w/ lipid bilayer of membrane |
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Unique side chain: Glycine |
-no actual side chain - just the H atom - which gives flexibility to the amino acid -can fit into either hydrophobic OR hydrophilic environments -resides at sites where 2 polypeptides come into close contact |
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Unique side chain: Cysteine |
-polar uncharged side chain -still forms strong covalent bond (b/c no charge) with another cysteine to form a disulphide link - these links stabilize the intricate shapes of proteins |
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Unique side chain: Proline |
-hydrophobic side chain -creates cyclic structure thereby forcing protein to bend and creates kinks in polypeptide chain -disrupts ordered secondary structure by introducing kinks -rigid structure |
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Orientation of non-membrane, soluble proteins |
-constructed so that the polar residues are situated at the surface of the molecule where they associate with surrounding water and contribute to protein's solubility in aqueous solution |
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Orientation of membrane, non-soluble proteins |
-non-polar residues situated at inner core of molecule where the hydrophobic residues of protein interior are tightly packed so water molecules are excluded |
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Peptide bonds |
-join the amino acids that makeup polypeptides -formed via condensation reaction (which occurs in the ribosome) between the carboxyl group of one amino acid and the amino group of another amino acid where they get so close that a water molecule is formed & released and a covalent bond is formed -N-terminus: where amino group is on the end of joined amino acids -C-terminus: where carboxyl group on end of joined amino acids |
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Protein size |
-molecular mass measured in Daltons (Da) or kiloDaltons (kDa) -Dalton: unit of mass equal to a single carbon 12 atom divided by 12 (close to molecular mass of hydrogen) |
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Calculating the mass of a real protein |
Mass of 1 mol of protein 13.7 kDa x (6.022 x 10^23) = 13.7 kg |
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Primary protein structure |
-describes linear sequence of amino acids -never branched, always linear -starting end is N-terminus (amino acid end) -single nucleotide mutation can encode for a different amino acid causing a defect in the protein (Ex. Sickle Cell Anemia - one amino acid mutation causes clumped hemoglobin which makes fragile RBCs and can cause clogs in blood vessels) -primary structure determines next levels of organization -all information necessary to produce the secondary, tertiary, quaternary structures is in the primary structure |
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Secondary protein structure |
-describes folding/coiling
-3D arrangement of protein -alpha helix or beta-pleated sheet |
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Secondary structure - alpha helix |
-stabilized by H bonds -structure found in proteins that span the membrane -twisting spiral |
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Secondary structure - beta pleated sheet |
-forms a single plane were amino acids in sequence -R groups alternate pointing up and down -H bonds oriented perpendicular to long axis of polypeptide chain -neighboring strands can run either parallel OR antiparallel -a lot of Beta pleated sheets in a protein increase structure stability and tensile strength |
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Secondary structure - other conformations
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-portions of the polypeptide chain not organized in an alpha helix or beta pleated sheet may consist of hinges, turns, loops or fingerlike extensions -often, the most flexible portions and sites of greatest biological activity |
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Tertiary protein structure |
-describes conformation -determined using x-ray crystallography -structure of a whole protein -highest level of organization for a single polypeptide strand -stabilized by non-covalent bonds -divided into domains (which give protein their function) |
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The non-covalent bonds that stabilize tertiary protein structure |
-Van der Waals forces b/w non polar charged parts -H bonds b/w non polar & polar charged parts -ionic bonds b/w polar uncharged & polar charged parts |
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Protein domain |
-protein domain part of protein structure and sequence that can evolve, function, exist independently of the rest of the protein chain -each domain forms compact 3D structure and can be independent stable and folded -many proteins consists of several structural domains -one domain may appear in a variety of proteins -molecular evolution uses domains as building blocks and these may be recombined in different arrangements to create proteins w/ different functions -domains allow us to predict the functions of a newly identified, unknown protein |
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Quaternary protein structure (and an example from lecture) |
-applies to multimeric proteins only (more than 1 subunit/chain) -named by stoichiometry -- tells us which subunits make up the protein Example: Nicotinic acetylcholine receptor - enables muscle contractions |
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Protein folding experiment |
-conducted by Christian Anfinsen -proved that tertiary structure was held within the primary structure -treated ribonuclease A protein with urea & mercaptoethanol which breaks disulphide bonds and denatures the proteins causing the protein to unfold and its loss of function --> realized that by removing urea and mercaptoethanol, the protein could regain its structure & function as it refolded and it actually did |
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Protein folding |
-secondary structure folds THEN tertiary structure develops |
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Molecular chaperones |
-bind to hydrophobic amino acids that are expose in non-native proteins -function: to prevent interactions b/w proteins that are not supposed to bind together, prevent interaction b/w the protein leaving the ribosome and other proteins in the cell |
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2 types of molecular chaperones |
-Hsp70 - binds and restricts conformation of chain, prevents premature (mis)folding -Chaperonin - has a space in the middle of its structure where nascent protein folds itself into proper conformation, chaperoning made of amino acids, hydrophobic non polar groups point to empty core space (U-shaped) and hydrophilic polar charged or uncharged groups pointing towards outside |
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Molecular chaperone in action |
1. Hsp70 binds to nascent elongating peptide growing from ribosome 2. Once detached from ribosome, the new protein either folds by itself into native conformation OR 2 (alternative). Enters empty space in middle of chaperonin 3. Cap closes/covers chaperonin 4. Signal occurs to switch the chaperoning amino acid arrangement so that the hydrophobic group now points to the outside and the hydrophilic group points inside 5. This new arrangement allows nascent polypeptide to properly fold/coordinate assembly of structure |
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Cell membrane functions |
-compartmentalization - separate sections/organelles for reactions to occur without interference, allows for cellular activities to be regulated independently -scaffold for biochemical activities - attachment site, place for chemical reactions to occur -provides selectively permeable barrier - hydrophobic things less capable of crossing membrane, anything water soluble needs help to cross -transporting solutes - usage of energy by transport proteins -responding to external signals - cell covered with sensors for detection -intercellular (b/w cells) interaction - communication among cells -energy transduction - glucose converted to ATP, energy conversions dependent on membrane |
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Membrane composition |
-lipids (bimolecular layer) -proteins -carbohydrates |
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Membrane lipids |
-include phospholipids, cholesterol, glycolipids -amphipathic -hydrophilic - polar solvent, polar head group in lipid bilayer -hydrophobic - non polar solvent, nonpolar fatty acid in lipid bilayer -has phospholipids - can have different polar head groups -up to 50% of membrane lipids can be cholesterol -basically impermeable to water |
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Contribution of cholesterol to membrane lipids |
Cholesterol fits between hydrophobic phospholipid tails preventing fatty acids from moving too much - this adds rigidity |
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Lipid movement |
Flex - fatty acid tails can move and shake Lateral shift - fatty acid tails can move along same plane Transverse diffusion - enzyme flippase only takes certain phospholipids and flips them from one leaflet to the other (cell exterior to cytosol) *Cholesterol molecules can flip too |
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Consequence of flipping of phospholipids |
Leaflets will not be identical - asymmetrical aspect of phospholipid bilayer |
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Lipid bilayer (3 main points) |
1. Fluid 2. Asymmetric - as caused by flippase flipping phospholipids to produce different leaflets 3. Seal spontaneously - try to reseal if there's a tear -lipid bilayer prevents random movement of water soluble materials in and out of the cell |
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Carbohydrates of cell membrane |
-present in membranes as glycolipids or glycoproteins - sugars covalently linked to lipid or protein in membrane -asymmetric -short oligosaccharides -face away from cytosol -N-linked: always attached to asparagine [N-acetylglucosamine] -O-linked: attached to serine or threonine [N-cetylgalactosamine] |
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Proteins (3 types) |
-integral membrane proteins -peripheral membrane proteins -lipid anchored/GPI anchored proteins |
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Integral membrane proteins |
-amphipathic -asymmetry -penetrate the lipid bilayer - transmembrane proteins -portion spanning the membrane is alpha-helix (hydrophobic) whereas the hydrophilic amino acid groups point to cytosol and cell exterior |
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Peripheral membrane proteins |
-located entirely on outside of the lipid bilayer -can attach to integral membrane proteins or lipids via non-covalent electrostatic bonds -dynamic -asymmetry |
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Lipid anchored/GPI anchored |
GPI: glycosylphosphatidylinositol GPI-anchored protein is a type of lipid anchored protein -located on outside of lipid bilayer -covalently linked to a lipid molecule that's situated within the bilayer -called "myristate" or "farnesyl" (2 types) |
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Fluid Mosaic Model |
-asymmetry - b/c proteins and lipids don't flip-flop, only phospholipids do and require the enzyme flippase to do so -embedded proteins -mosaic arrangement - not uniform, patches of different things -quasi-fluid - different restrictions of movements |
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Restriction of protein movements within lipid bilayer (6, example) |
A: proteins moving randomly B: proteins attached to cytoskeleton right at membrane - not moving C: movement but very directed b/c being transported by motor proteins - unidirectional movement D: crowded by other proteins so unable to move E: movement by only in small sections until jumps to another area to move around in F: proteins that form the extracellular matrix Example: acetylcholine receptor at neuromuscular joint |
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Fluorescence Recovery After Photobleaching (FRAP) |
-technique for studying membrane protein mobility Process 1. Label protein of interest w/ fluorescent dye 2. Photobleach spot w/ laser beam - the light is so intense that the fluorescence spot disappears 3. Recovery - measure time/speed of unbleached fluorescently labelled proteins migrating into bleached area -the faster the recovery, the more mobile the protein is |
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Lipid rafts |
-contributes to mosaic characteristic of membrane/example of how membrane is mosaic -introduces order into random sea of lipids -cholesterol & sphingolipids cluster together for a short time -the "spikes" protruding from these clumps are GPI-anchored proteins - serve as floating platforms that concentrate particular proteins, thereby organizing the membrane into functional compartments |
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Restriction of movement for lipids within bilayer |
-diffusion is limited for lipids -G40-DOPE - lipid labeled w/ gold particle and can be tracked amongst unlabeled lipids (technique to follow movement), allows for observation of mobility of individual lipid molecules |
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Membrane composition summary |
Fluid Mosaic Model: asymmetry, many embedded proteins, mosaic arrangement of components, rotational and lateral diffusion, no flip flop without flippase (just phospholipids) Restriction of movement: molecules do not diffuse freely in the membrane, movement of membrane constituents is restricted in a number of ways Lipid rafts: some lipids pack together and form ordered regions that include some proteins (GPI-anchored) and exclude others, cholesterol & sphingolipids |
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Metabolism
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All the chemical reactions that occur in a cell |
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Metabolic pathway |
Product from one reaction (metabolic intermediate) becomes substrate for another reaction -Enzymes in the same metabolic pathway are physically linked |
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Metabolic reactions: catabolic and anabolic |
Catabolic: releases energy for cell to use by breaking down complex molecules, also make available the raw materials from which other molecules can be made Anabolic: uses energy to build up complex molecules |
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Mitochondria
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-can have different shapes - heavily branched or cylindrical -can divide by fission and fuse by fusion -have their own genome -gets transported around the cell by motor proteins (kinesins) to wherever energy required in the cell -have to import most of their proteins even though the mitochondrial ribosome does make some -ATP made in ATP synthase particles -have double membrane and inter membrane space -cristae - folds within the inner membrane, membranes of cristae (crystal membrane) continuous with the inner membrane but they have a different composition |
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Outer-mitochondrial membrane |
-50% lipid, 50% protein -have embedded channels called "porins" -porins made of Beta barrels (Beta sheets that form tubes) -these tubes are large and let large molecules in -allows small molecules from cytosol to have access to intermembrane space -porins can open and close -porins make outer membrane quite permeable |
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Inner-mitochondrial membrane |
-3:1 protein:lipid ratio -impermeable -protein rich -cardiolipin (lipid present) |
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4 types of proteins in the inner-mitochondrial membrane |
1. Those that carry out oxidation reactions of the ETC/respiratory chain 2. F1/F0 particles that compose ATP synthase which produces ATP in the matrix - cristae covered in F1 particles, F0 particles embedded in membrane 3. Transport proteins that regulate passage of metabolites into and out of the matrix 4. Protein import machinery - proteins to help import other proteins |
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Lipids (Cardiolipin) |
Cardiolipin -unusual lipid in inner membrane -double phospholipid = has 4 fatty acid chains -insulates the membrane -stabilizes activity of proteins in ETC -impermeable to many things like hydrogen |
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The matrix of the mitochondria |
-contains ribosomes -contains high concentration of proteins -site of TCA cycle/Kreb's cycle -contains mitochondrial genome and gene transcription/translation machinery |
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Summary of the mitochondria |
-Vary in shape and are dynamic -Double membrane: outer membrane permeable to small molecules due to porins, inner membrane impermeable, rich in protein and contains rare phospholipid called cardiolipin -Matrix has high protein concentration and houses the TCA cycle |
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Processes of cellular respiration that occur in the mitochondrion |
-TCA cycle -ETC [oxidative phosphorylation] -Proton motive force and F1/F0 particles [oxidative phosphorylation] |
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Glycolysis |
-occurs in cytoplasm -1 glucose (6C) --> 2 pyruvate (3C each) -need investment of energy, 2 ATP, to kickstart glycolysis -4 ATP made - 2 ATP used = net production of 2 ATP -2 NADH (reduced electron carrier) produced |
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Glycolysis starting & ending products, full reaction |
Start: 1 glucose, 2 NAD+, 2 ATP End: 2 pyruvate, 2 NADH, 2 ATP glucose + 2NAD+ + 2ADP + 2Pi --> 2 pyruvate + 2NADH + 2ATP + 2H+ + 2H20 |
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Link reaction |
-NOT part of the TCA cycle -pyruvate dehydrogenase takes pyruvate and combines it with coenzyme A to produce Acetyl-CoA -loss of 1C to production of CO2 -1 NADH produced -remaining 2 C's (from 1 pyruvate) will continue on to be fed into TCA cycle pyruvate + Coenzyme A + NAD+ --> via pyruvate dehydrogenase --> acetyl coenzyme A + CO2 + NADH + H+ |
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Acronym NAD |
Nicotinamide adenine dinucleotide |
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TCA/Krebs/Citric Acid Cycle |
-2 turns of TCA cycle for 1 glucose b/c of 2 pyruvates -aldol condensation - b/w acetyl-CoA (2C - what's remaining of pyruvate) and oxaloacetate (4C) to produce 6C citrate at beginning of cycle -the 2 C's fed into the cycle are broken down to CO2 -succinate dehydrogenase reduces FAD to FADH2 (and also belongs to the ETC) -GTP produced in TCA cycle -NAD+ and FAD reduced to NADH and FADH2 |
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Oxidation of Acetyl group in TCA cycle |
Acetyl-CoA + 2H20 + FAD+ + 2NAD+ + GDP + Pi ------> 1 round of TCA cycle --> 2CO2 + FADH2 + 3NADH + 3H+ + 3GTP + HS-CoA |
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Oxidation of glucose in TCA cycle |
glucose + 2 ADP + 2Pi + 10NAD+ + 2FAD --> 2 rounds of TCA cycle --> 2ATP + 10NADH + 2FADH2 + 6CO2 |
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Acronym for FAD |
Flavin adenine dinucleotide |
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Oxidative phosphorylation |
-occurs in 1) ETC and 2) chemiosmosis -formation of ATP from ADP as driven by transfer of electron to oxygen -process involves proton motive force (electrochemical gradient) across inner-mitochondrial membrane by electron transport followed by the controlled movement of protons back across the membrane to generate ATP |
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Electron transport chain |
-pumps protons against a gradient to the inter membrane space -energy is required for this pumping -NAD, electron carrier, arrives at Complex 1 and gets oxidized and donates 2 electrons becoming NADH -Complex 1 uses this energy from the electrons to pump protons to inter membrane space -Ubiquinone receives e- from NADH at complex 1 and carries them to complex 3 -Oxygen at complex 4 is the ultimate electron acceptor and receives e- from Cytochrome C |
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Ubiquinone |
Receives e- from NADH at Complex 1 in ETC and carries them to Complex 3 |
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Cytochrome C |
Carries e- from Complex 3 to Complex 4 in ETC |
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Succinate dehydrogenase |
-complex 2 - where succinate is transformed to fumarate in the TCA cycle -reduces FAD to FADH2 |
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Which complexes are proton pumps and which aren't? |
Complex 1, 3, 4 are proton pumps Complex 2 is not a proton pump - it is part of TCA Cycle |
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Rank in order of highest to lowest redox potential: cytochrome C, oxygen, Complex 1, Complex 2, Complex 3, Complex 4, ubiquinone |
Complex 1 (NADH) and Complex 2 (FADH2) Ubiquinone Complex 3 Cytochrome C Complex 4 Oxygen |
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Analogy used in class to describe oxidative phosphorylation |
-hydroelectric dam -produces electricity by using the potential energy stored in water to drive a generator -in the mitochondria, a proton gradient drives the F1/F0 particle ATP generator |
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Proton gradient |
-pH gradient (concentration difference in H+) + charge difference (voltage) = establishes electrochemical gradient -energy from this electrochemical gradient establishes a proton motive force (measured in millivolts) |
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What determined that ETC is separate from ATP production in oxidative phosphorylation? |
2,4-dinitrolphenol -an ionophore that allows protons to flow through it |
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Chemiosmosis |
-production of ATP using energy of H+ ion gradient (proton motive force) across membrane to phosphorylate ADP |
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What would happen if we poked holes in the inner membrane that allowed H+ ions to flow freely? |
The proton motive force would be lost |
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ATP synthase |
-what generates ATP -consists of F1 and F0 particle -F0 - embedded within the inner mitochondrial membrane -F1 - outside membrane on cristae but inside the matrix of the mitochondria |
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Rotor (turning) and Stator (stable) subunits of the F1 and F0 particle
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F0 -rotor: 10-14 c subunits -stator: a subunit, 3 b subunits F1 -rotor: gamma, epsilon -stator: 3 alpha units, 3 beta units, 3 delta units |
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ATP synthesis in F0 particle - coupling of proton diffusion to c ring rotation |
-occurring in membrane -protons enter through subunit a, then enter the rotating ring of c subunits -proton binds to aspartic acid in c subunit and travels around the ring until it leaves -the proton's exit drives the rotation of the rotor part/causes turn of gamma subunit (which initiates binding change mechanism) |
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ATP synthesis in F1 particle - Binding Change Mechanism (Paul Boyer) |
-occurs in matrix -beta subunit has multiple conformations (open, loose, tight) -every 1/3 turn of a gamma subunit causes the beta subunit to go through different conformations -rotational catalysis = the turning triggers activation of subunits, rotation driven by movement of protons through membrane via the channel in base of F0 particle |
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Conformational changes of beta subunit in the Binding Change Mechanism |
1. Open - ready to receive ADP+Pi 2. Loose - ADP+Pi on subunit 3. Tight - ADP+Pi pushed together on subunit *Spontaneous ATP formation* 4. Tight - ATP now 5. Open - ATP can leave Beta subunit |
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What is the gamma subunit? |
-a cam -changes input motion, by rotary motion, to a reciprocating motion of the follower -turn of gamma subunit causes conformational change of Beta subunit |
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Experiment w/ actin filament |
-placed actin filament on the gamma subunit of the F1 particle of ATP synthase and saw that the actin filament was swung around - due to rotating motion of gamma subunit |
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Summary of chemiosmosis & binding change mechanism |
-energy released by movement of proton NOT sued to drive phosphorylation BUT used to change binding affinity of active site for ATP product -each active site on the Beta subunit progresses through 3 different conformations -ATP is synthesized by rotational catalysis where one part of ATP synthase rotates relative to the others -chemiosmosis drives the rotation -F1 + F0 = ATP synthase |
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Peroxisomes |
-function in catabolism of very long chain fatty acids -microbodies, organelles -have special enzymes to breakdown acids -cannot synthesize their own proteins so they get protein import after translation -glyoxysomes = plant peroxisomes |
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Eukaryotic cell compartments |
-eukaryotic cell composed of many compartments with membranes -these compartments are NOT independent - they form an integrated functional unit, all dependent on one another |
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Endomembrane system
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Organelles that make up biosynethic pathway (synthesis, sorting & secretion) -endoplasmic reticulum -Golgi complex -endosomes -lysosomes -plasma membrane |
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Smooth endoplasmic reticulum |
Structure: more like tubes/tubular shaped Functions: -synthesis of steroid hormones -detoxification by oxygenases - cytochrome P450 transforms hydrophobic molecules into hydrophilic ones so that they can be excreted in urine -glucose release -sequestering Ca2+ |
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Rough endoplasmic reticulum |
Structure: long and flat, contains ribosomes Functions: -protein secretion -lumen proteins -membrane proteins |
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Cell Fractionation experiment |
-to study cell parts in isolation 1. Homogenize cells (blend cells/tear apart) 2. Centrifuge to separate cell parts - end up with whole cells, nuclei, mitochondria on the bottom & supernatant on top containing ribosomes 3. Centrifuge the supernatant - microsomes (rough ER membrane fragments with attached ribosomes) accumulate at the bottom and the supernatant on top w/ free ribosomes |
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Pulse-chase experiment |
-used to determine the intracellular pathway followed by secretory proteins from their site of synthesis to site of discharge 1. Incubate cells in radioactive amino acids ("pulse") 2. Wash cells free of excess isotope 3. Transfer cells to medium containing only unlabeled proteins ("chase") - period where additional proteins are made using non-radioactive amino acids -the longer the "chase", the farther the radioactive proteins made during "pulse" will have travelled from site of synthesis -can follow movement by observing wave of radioactive material moving through cytoplasmic organelles of cells |
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Pulse-chase + cell fractionation experiment |
-enables use to figure out where proteins travel to 1. Treat cells w/ pulse chase 2. Rinse cells and homogenize them 3. Centrifuge and separate ribosomes & microsomes into two tubes 4. Measure radioactivity in both tubes - should be lots of radioactivity in microsomes 5. Cells secrete "pulse" and radioactivity disappears but radioactivity remains same in ribosomes in supernatant |
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Pulse-chase + autoradiography |
-allows determination of location of radioactively labeled materials within the cell 1. Perform pulse chase 2. Cells containing radioactive isotope covered w/ photographic emulsion (which is exposed to radiation emanating from radioisotopes within the tissues) - sites in cells containing radioactivity revealed under microscope by silver grains in the overlaying emulsion 3. Cells quickly fixed - put chemical on to freeze cells in place 4. Determine locations of proteins that had been synthesized during incubation w/ labelled amino acids (pulse chase part) autoradiography - those labelled proteins appear black *Silver grains are not the actual protein but they get excited in autoradiography which is why you can see them when cells covered in photoemulsion |
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Purpose of pulse-chase experiments in combo with cell fractionation or autoradiography |
-can figure out where proteins travel to -BUT does not tel us the actual machinery that makes the proteins travel |
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2 types of proteins |
1. Those that are being made, travel, and secreted 2. Those that enable those things to happen/enable protein secretion --> secretory proteins |
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What are secretory proteins? |
-not being secreted themselves -help other proteins move from compartments to a resident compartment OR out of the cell |
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Yeast SEC mutants (purpose, how they were discovered) |
-mutations in secretory proteins -looking at mutant phenotypes is beneficial b/c the nature of the deficiency (due to the mutation) provides information on the function of the normal protein How they were discovered: 1. Introduce mutations w/ mutagenic chemicals 2. Pulse chase to screen proteins 3. Perform autoradiography to see where proteins have travelled --- noticed proteins from pulse-chase were stopping at different points along the secretory pathway --- this created 5 classes of mutants |
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Class A secretory protein mutant |
Fate of secreted protein: accumulation in cytosol Defect: transport into ER |
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Class B secretory protein mutant |
Fate of secreted protein: accumulation in the rough ER Defect: budding of vesicles from rough ER |
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Class C secretory protein mutant |
Fate of secreted protein: accumulation in ER to Golgi transport vesicles Defect: fusion of transport vesicles w/ Golgi |
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Class D secretory protein mutant |
Fate of secreted protein: accumulation in Golgi Defect: transport from Golgi to secretory vesicles |
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Class E secretory protein mutant |
Fate of secreted protein: accumulation in secretory vesicles Defect: transport from secretory vesicles to cell surface |
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Why are yeast cells used? |
Yeast cells used because of a similar protein found in humans like yeast Comparable with humans |
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Heat sensitive mutant |
-mutation does not totally kill the protein function -need to raise the temperature to activate mutation |
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Experiment with heat sensitive mutant |
1. Grow temperature sensitive mutant yeast and non-mutant control yeast at room temperature 2. Raise temperature to active mutation (approx 36 degrees C) 3. Pules chase - to screen 4. Autoradiography - to get picture 5. Examine under microscope and determine class of mutants |
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2 biosynthetic pathways |
1. Constitutive secretory pathway 2. Regulated secretory pathway |
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Constitutive secretory pathway |
-always secreting -materials transported in secretory vesicles from sites of synthesis and discharged into the extracellular space in a continual manner -membrane proteins destined to reach the cell's surface via constitutive pathway in secretory vesicles |
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Regulated secretory pathway |
-materials to be secreted are stored in large densely-packed membrane-bound secretory granules and are discharged only in response to an appropriate stimulus |
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Endocytic pathway |
-not a secretory pathway -materials move from outer surface of cell to compartments (such as endosomes or lysosomes) located within the cytoplasm |
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Vesicle transport |
-orientation is always maintained -lumen of one compartment is inside the cell -lumen can be equivalent to the outside of the cell -cytosol always kept separate from lumen of vesicles |
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3 ways for proteins to move between cellular compartments |
-gated transport -transmembrane transport -vesicular transport |
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The Signal Hypothesis (who made it, points) |
-by Gunter Blobel 1. Ribosomes are all the same, whether attached to the ER or not (free or membrane-bound) 2. It's a signal on the protein that tells the protein where to go |
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The Blobel experiment (and Sabatini) |
-key experiment to proving The Signal Hypothesis -deconstructed cell and isolated parts necessary for in-vitro translation -protein produced in-vitro was longer than the same protein made in-vivo b/c in vivo, the protein was cleaved -the extra bit on the in-vitro protein was the signal (signal sequence on N-terminus) that tells the protein where the to go -the signal peptide is the "postal code" of the protein |
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Co-translational translocation mechanism (what it is, steps) |
-protein synthesis on the rough ER, mechanism by which proteins injected into ER, proteins not destined for membrane but rather those inside the ER -goes with the Signal Sequence Hypothesis 1. Translation starts on free ribosome 2. SRP (signal recognition particle) binds to the signal peptide on the nascent polypeptide and temporarily locks translation 3. SRP docks ribosome and nascent polypeptide to SRP receptor on the ER membrane - both SRP and SRP receptor bid to GTP 4. Once docked, SRP detaches (SRP receptor dissociated too) and the polypeptide moves into translocon 5. Translocon plug removed by elongating peptide (as translation had resumed) that entered translocon and the signal peptide is cleared off by a signal peptidase 6. Chaperones bind to elongating peptide to ensure proper folding |
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Start-transfer sequence (what it is, steps) |
-how proteins incorporated into membrane, proteins destined to be part of membrane -hydrophobic amino acids that comprise internal start transfer sequence will span the membrane and form into alpha helices -one end of the sequence will be positive and one will be negative - this determines orientation of sequence in the membrane 1. Start-transfer sequence tells ribosome to go to membrane to continue translation 2. Internal start transfer sequence becomes embedded in translocon 3. Bound GTP to SRP and SRP receptor hydrolyzes to GDP so then SRP leaves ribosome so it can continue synthesizing protein - translation starts at N-terminus and ends at C-terminus 4. Elongating protein - C-terminus grows into ER lumen 5. Mature protein dissociates from translocon and is now embedded in membrane - internal start sequence (the part that's in the ER lumen) becomes anchor for membrane protein -these proteins stay in the membrane forever and the orientation stays the same forever (part of protein in ER lumen will never be on the outside of the membrane and vice-versa) -peptide not cleaved after transfer of protein into ER membrane (start-transfer sequence not cleaved) |
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Orientation of start-transfer sequence |
-the start-transfer sequence can "snap" into the translocon in the other direction (based on + and - end) depending on how the protein is encoded -this ability to incorporate in either direction ensures the protein will be oriented in the correct way |
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Double pass transmembrane protein |
-membrane proteins can have many transmembrane domains but all proteins use the same basic mechanism to get incorporated -2 parts of protein embedded in the membrane 1. When a peptide containing a stop-transfer sequence inserts into the translocon, the translation process stops 2. Translocon releases the polypeptide chain containing the stop-transfer sequence laterally to the lipid bilayer 3. Stop-transfer sequence becomes a transmembrane segment too -the peptide containing stop-transfer sequence is NOT cleaved but rather just remains in membrane |
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Membrane lipid synthesis
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-asymmetrical -2 leaflets represent the 2 layers of the bilayer ---cytosolic leaflet: always facing cytoplasm ---exoplasmic leaflet: facing inside lumen or outside of cell (b/c when vesicles fuse with membrane the leaflet on the inside of the lumen becomes continuous with extracellular space leaflet) -precursors of phospholipids always inserted on cytosolic side -phospholipids inserted into pre-existing membranes in the ER -phospholipid synthesis occurs in cytosolic face -flippases needed to transfer phospholipids to exoplasmic face |
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Why is lipid composition of organelles different?
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Modifying lipids AFTER they've been introduced into the ER membrane -modification by enzymes - changes nature of polar head groups -vesicle formation - can select certain lipids and leave others behind -phospholipid transfer proteins - plucks individual lipids from one membrane and puts it onto another |
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Summary of membrane compositions |
-membranes arise from pre-existing membranes (components added in ER) -asymmetry maintained by flypasts -synthesis of phospholipids occurs on cytosolic face and are transferred to exoplasmic face via flippase -lipids are then carried from ER to Golgi via transport vesicles -the action of enzymes in the lumen, differential inclusion of lipids in vesicles, and phospholipid transfer proteins produce membranes with different compositions in different organelles |
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Post-translational protein modifications |
-disulfide bond formation -folding -proteolytic cleavage - done by proteases -multimer asesmbly - quarternary structure -glycosylation - addition of sugars |
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2 kinds of glycosylation |
N-linked: sugar called N-acetylglucosamine added to N end of asparagine O-linked: sugar called N-acetylgalactosamine added to O end of serine or threonine (less frequently occurring) |
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Glycosylation |
-sugars added to protein 1 monomer at a time -sugars are not just floating around in the cytosol - they get attached to a nucleotide - nucleotides act as carriers so that the nucleotide sugar can get recognized -happens at the ER always |
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Glycosyltransferase |
-genearl name for group of enzymes that adds nucleotide sugars to growing oligosaccharide chain -sequence in which sugars are transferred during oligosaccharide assembly depends on sequence action of glycosyltransferases which in turn depends on location of specific enzymes within various membranes of secretory pathway |
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N-linked glycosylation |
-occurs at the ER -production of core oligosaccharide -entire oligosaccharide made before being added to nascent protein 1. Sugars, including manses, transferred one at at a time to dolichol *Dolichol: special lipid found in ER membrane, acts as a holder for growing carbohydrate chain *Sugars are added by glycosyltransferases *Carbohydrates face cytosol early (exception to the usual case where carbs normally point towards inside of lumen) 2. Flippase flips carb to now face the inside of the lumen 3. Oligosaccharide protein transferase plucks preassembled sugar tree from dolichol and adds it to certain asparagine in the elongating nascent peptide Components: -dolichol: holder of sugars on cytosolic side -mannose: added too -N-acetylglucosamine: total sugar added to asparagine of growing polypeptide |
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Protein quality control |
-ALWAYS occurs -ensures proteins are properly folded -protein was made by ribosome, injected through the translocon and is now in the lumen 1. Glucosidase 1 and 2 cleave off a few terminal glucoses residues on the oligosaccharide 2. Calnexin or calreticulin (molecular chaperones) bind to oligosaccharide - giving protein time to fold 3. Protein should be finished folding and removal of final glucose by Glucosidase 2 leads to release of glycoprotein from chaperone *If protein is properly folded, then the protein can leave and travel to its destined 4. If protein mid folded, its hydrophobic ends stick out and it will linger in the lumen 5. Monitoring enzyme UGGT recognizes hydrophobic amino acids pointing out so will add a glucose onto mannose residues at exposed end 6. Now the "tagged" glycoprotein is recognized by same chaperones which give protein another chance to fold properly |
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Reverse translocation |
-if protein continues to misfold and stays in the ER for a long time, some enzymes will cleave the mannose residues off -if mannose is gone, then UGGT cannot add glucoses onto it and the protein can no longer be recycled through cycle -thus, the protein gets sent back to the cytosol to a protease which destroys proteins |
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Proteasome
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-big protein complex -Beta subunits are the active subunits -where proteins are fed into and get destructed to their single amino acid fragments |
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Ubiquitin
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-example of proteasome -small protein tag added to other proteins to mark them for destruction by a proteasome -3 enzymes necessary for adding ubiquitins: E1, E2, and E3 (aka ubiquitin ligase) -ubiquitins stick to a cap of a proteasome while the protein is then fed through the proteasome - ATP is used in this process |
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Unfolded protein response |
-occurs under stress that leads to the accumulation of misfiled proteins (example: raising temperature leads to unfolding and denaturation of proteins) -the ER contains protein sensors that monitor the concentration of unfolded or misfiled proteins in the ER lumen -under no stress, sensors kept in the inactive state by the molecular chaperone BiP which is bound weakly to the lumen side of the sensors 1. Proteins start to unravel and get denatured 2. BiP chaperones will recognize hydrophobic amino acids sticking out - will need tons of BiP to go stick to unraveling protein, BiP keeping sensors inactive when bound 3. When BiP leaves sensors to go to the misfiled protein, sensors undergo dimerization and become dimers and are now active *1st set of sensors: they are kinases - they phosphorylate translation factor eIF2alpha, phosphorylated translation factor eIF2alpha binds to small ribosome unit causing decrease in translation *2nd set of sensors: gets transported into nucleus where it'll trigger transcription of mRNA which gets translated into proteins capable of alleviating stress |
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What family does BiP belong to? |
-belongs to heat shock family because BiP concentration increases when cell feels heat -BiP actually discovered when cell experienced heat shock |
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What happens to the protein if nothing else works? |
Apoptosis |
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Summary of protein synthesis/response |
-in the protein quality control process, the calnexin or calreticulin chaperone uses glucose as a marker for folding while UGGT detects misfiled proteins and adds a glucose if another round of folding is needed -if the misfolded state persists, the protein is sent to the cytosol by reverse translocation (through the translocon) where the protein is polyubiquinated and degraded by the proteasome -if the proteins can't be sent to the cytosol quickly enough, the unfolded protein response comes into play and gene expression of chaperones (especially BiP), coat proteins and quality control proteins is increased while overall translation is decreased -if this does not work still, the cell commits suicide (apoptosis) |
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Camillo Golgi |
-invented silver based histological stains allowing for visualization of many new sub cellular structures -discovered a darkly stained reticular network which was later identified in other cell types which he named the Golgi complex |
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Golgi complex |
-defined stacks of membranes (<8 stacks) that form cup shape -Cis golgi network, cis/medial/trans cisternae (membranous stacks), Trans golgi network -the ER forms a single continuous compartment that traverse the Golgi at multiple points, extending in opposite directions beyond the cis-most and trans-most cisternae |
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Cis and trans golgi network |
Cis golgi network -tubular membranes -bottom of the "cup" -entry face closest to the ER -sorting station that distinguishes b/w proteins that should be sent back to the ER and those that are allowed to proceed to the next Golgi stack Trans Golgi network -tubular membranes -top of "cup" -exit face at end of stack -sorting station where proteins segregated into vesicles heading either to plasma membrane or to various intracellular destinations |
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ERGIC |
Endoplasmic Reticulum Golgi Intermediate Compartment -after proteins bud from the ER membrane, transport vesicles fuse with one another to form larger vesicles & interconnected tubules in the region b/w the ER and Golgi -this region is the ERGIC -VTC's are present in this area |
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VTC
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Vesicular Tubular Clusters -pre-Golgi areas -form in the ERGIC region as transport vesicles fuse together to form larger vesicles -go directly from ER to Golgi |
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N-linked glycosylation in the Golgi |
Recall that N-linked glycosylation is the modification of a protein's carbohydrates 1. Core oligosaccharide begins attached to cis-Golgi (recall that glucose residues were removed) 2. Oligosaccharide moves through cis to medial to trans cistern - mannose residues are removed during this 3. Various glycosyltransferases within different membranes add sugars - different enzymes trigger different changes to the oligosaccharide *It is important for these enzyme reactions/adding of sugars to occur in the correct sequence, which is made possible by the arrangement of the Golgi membrane stacks |
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Vesicular transport model |
-probably wrong model -cisternae remain in place -cargo (secretory, lysosomal, membrane proteins) shuttled through the Golgi stack in vesicles - proteins leave Golgi & enter Golgi & travel b/w Golgi stacks in vesicles -vesicles travel in anterograde direction: cis to trans |
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Anterograde vs Retrograde
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Anterograde: cis to trans, forward Retrograde: trans to cis, backward |
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Cisternal maturation model |
-each cisterna "matures" into the next cistern along the stack -major elements of the cistern continually being formed at the cis face, moving through the stacks & dispersed at trans face -proteins NOT being secreted in vesicles -new proteins to be secreted remain in cistern -vesicles containing enzymes that make changes to the proteins in cistern travel through the stacks |
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Evidence supporting why cisternal maturation model is more correct |
-only vesicles seen travel in retrograde direction and contain Golgi-resident enzymes -the Golgi resident enzymes are continuously sent back to the previous stack so that they can continue to perform their function at the correct stage in sequence *Vesicles with enzymes in trans citernae sent to medial cisternal whose vesicles are sent to cis cisternae |
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Role of protein coats in vesicle transport
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-protein coats form from soluble proteins that assemble on the cytosolic side of donor membrane at sites where budding takes place -each coated protein has their own area where they're found 1. Mechanical device - shapes the cell membrane by causing membrane to curve and form budding vesicle 2. Selects components to be carried - cargo/proteins |
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3 types of coated vesicles |
1. COPII-coated: moves materials from the ER in the anterograde direction/forward to the ERGIC and Golgi 2. COPI-coated: moves materials in the retrograde direction from (1) the ERGIC & Golgi to the ER and (2) from trans-Golgi backward to cis-Golgi 3. Clathrin-coated: moves materials from (1) trans-Golgi network to endosomes & lysosomes and (2) from plasma membrane to cytoplasmic compartments |
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COPII-coated vesicle assembly |
Recall that COPII-coated vesicles transport cargo from the ER to the Golgi complex 1. Sar1 recruited to ER membrane and activated by exchange of bound GDP for GTP 2. Sar1-GTP molecule inserts itself into ER bilayer beginning conversation of flattened membrane into spherical vesicle 3. Sar1-GTP recruits Sec24 protein to further induce curative of membrane in the formation of a vesicle 4. Cargo proteins bound to cargo receptors associate with Sec24 polypeptide of COPII-coat 5. Once entire COPII coat is assembled, the coated bud is separated from ER in form of COPII-coated vesicle -ER resident proteins excluded from coated vesicles -before the COPII-coated vesicle can fuse with a target membrane, the protein coat must be disassembled and its components released into the cytosol |
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Disassembly of COPII-coated vesicle (why does this occur, steps) |
Disassembly necessary so that the COPII-coated vesicle can fuse with a target membrane and release its components -triggered by hydrolysis of GTP (on Sar1) to GDP -Sar1-GDP has decreased affinity for vesicle membrane so it leaves the vesicle membrane -then, other COPII subunits are released and vesicle falls apart |
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COPI-coated vesicle |
-transporting escaped proteins back to the ER -coat contains membrane-bending, GTP-binding protein Arf1 whose membrane-bound GTP must be hydrolyzed before the coat can disassemble -retrograde transport -trans to cis direction in Golgi, ERGIC & Golgi to ER |
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Retrieval signals |
-ER resident proteins contain retrieval signals ensuring their return to the ER should they accidentally be carried forward to the ERGIC and Golgi -KDEL: soluble protein, KDEL sequence on soluble ER resident proteins recognized by the KDEL receptor (integral membrane protein that shuttles b/w cis Golgi & ER) allowing it to be sorted into the COPI-coated vesicle -KKXX: sequence for ER membrane proteins, sequence on KDEL receptors which binds to COPI coat *ER resident proteins bearing KDEL binds to KDEL receptor and the KKXX on the KDEL receptor binds to COPI-coated vesicle allowing for the ER resident protein to be incorporated into vesicle and returned to ER *if protein with KDEL sequence in it accidentally gets sent to the Golgi, it will be transported back to the ER |
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Targeting of ER synthesized proteins |
-acid hydrolases (type of lysosomal enzyme) are what is sent to lysosomes 1. ER synthesized lysosomal proteins sent to cis Golgi 2. Once in Golgi cisternae, enzymes recognize these lysosomal proteins and realize phosphate must be added to the mannoses 3. N-Acetylglucosamine phosphotransferase adds phosphates to the mannose 4. Phosphodiester glycosidase trims off N-acetylglucosamine so there is only phosphates and mannoses 5. Lysosomal enzymes make way to trans Golgi 6. Lysosomal enzyme attaches to mannose 6-phosphate receptor (receptor spans Golgi membrane sticking into TGN lumen) 7. GGA adaptor (binds to MPR) has 3 parts/domains -one domain binds to clathrin coat holding clathrin scaffolding onto surface of vesicle -one domain binds to Arf1-GTP (active state) which acts in conjunction to bind clathrin & initiate formation of budding vesicle -one domain binds to mannose 6-phosphate receptors (MPRs) that carries the lysosomal enzymes w/ mannose 6-phosphate signal 8. Once clathrin coat is formed and vesicle has budded from TGN, the clathrin coat is lost (by hydrolysis of GTP to GDP on Arf1) so vesicles are able to release the lysosomal enzymes to the lysosome 9. Rab proteins (specifies vesicle destination) recognizes cytosolic tails on vesicle surface and knows where it should go based on the tail - Rab proteins bind to GTP to become active then binds to vesicle AND also Rabs that bind to the target membrane 10. Tethering proteins (2 of them) act as links b/w the Rab on the transport vesicle and the Rab on the target membrane *Rabs recruit the tethering proteins 11. GTP on Rab hydrolyzes to GDP and then Rab leaves 12. During the docking stage leading up to membrane fusion, the V snare on transport vesicle interacts with t-snares on target membrane causing the snares to coil which brings membranes in close contact with each other and they fuse spontaneously 13. After membranes fuse, dissociation of the snare complex is achieved by protein NSF (shaped like a donut) that uncoils snares os they can be reused *When uncoiled, SNAREs store potential energy which is later used for when they partake in fusion again |
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SNAREs |
Transport vesicle has: v-snare (transmembrane) Target membrane has: t-snare (transmembrane) and t-snare (peripheral) -all snares contain snare motif capable of forming complex with another snare motif -snares already have enough energy to do so -lumen of transport vesicle becomes consistent with the lumen of the lysosome |
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Rabs |
-specify vesicle destination -targeting specificity -one associates with the vesicle membrane and the other with the target membrane, connected by tethering proteins -bring vesicle and target membrane in close contact just prior to fusion |
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Summary of targeting of lysosomal enzymes
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-vesicle formation: coat proteins -coat shedding: depends on hydrolysis of GTP on Sar and Arf proteins -targeting & tethering: Rabs and tethering proteins -docking and fusion: SNAREs -SNARE dissociation: NSF |
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Example of lysosome function: Autophagy |
-regulated destruction of the cell's own organelles/process of organelle turnover -old mitochondria produces toxins - need to get rid of them 1. Double membrane of ER forms around organelle - forms autophagic vacuole/autophagosome 2. Lysosome fuses with ER and autophagosome - forms autophagolysosome 3. Inner membrane of autophagosome and enclosed contents are degraded 4. Once digestive process in autophagolysosome complete, the organelle forms a residual body 5. Contents of the residual body are then exocytosed OR the remnants may be retained within the cytoplasm as a lipofuscin granule *Lipofuscin granules increase in number as individual becomes older *As you age, more organelles produce toxins and autophagy is required |
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Types of endocytosis
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-receptor-mediated: uptake of receptors and any material bound to them -phagocytosis: uptake of particulate matter, cell eating -pinocytosis: uptake of solutes, cell drinking *Autophagy (process of organelle turnover) is not really a type of endocytosis but it does use a similar process to phagocytosis |
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Endocytosis |
-mannose 6-phosphate receptors also partake in endocytosis -endosome intermediate between normal vesicle and lysosome -when endosome fuses with lysosome it becomes even more acidic -recruited proton pumps pump H+ into early and late endosomes that induce acidity higher than in normal vesicles |
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Clathrin triskelion
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-6 subunits: 3 heavy chains, 3 light chains |
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Receptor mediated endocytosis |
AKA: clathrin mediated endocytosis 1. At plasma membrane, AP2 adapt (multimeric) is NOT a GTPase and recognizes receptors w/ their cargo and also recruits clathrin 2. Coated pit forms and continues to sink inward to form coated but 3. Dynamin bound to GTP forms ring around neck of clathrin bud and is responsible for pinching off neck of vesicles to release them 4. GTP hydrolysis causes dynamin to undergo conformational change thus releasing the vesicle to leave cell surface 5. GTP-gammaS - looks like GTP but cannot be hydrolyzed - causes multiple dynamin rings to form one on top of another leading to a very long neck *GTP-gammaS does NOT undergo conformational change since it doesn't get hydrolyzed |
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Housekeeping receptors |
-transferrin and low density lipoprotein (LDL) -responsible for uptake of materials that cell uses/needs -acidity in the peripheral early endosomal carrier breaks connection b/w housekeeping receptor and cargo -material transport continues in endosomal carrier vesicle while housekeeping receptor sent back to plasma membrane in recycling compartment for additional rounds of uptake |
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Transferrin |
-a housekeeping receptor -brings in iron |
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LDL |
-low density lipoprotein -a housekeeping receptor -brings in cholesterol |
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Signalling receptors
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-hormones and growth factors -produces receptor-down regulation - instead of getting recycled (like housekeeping receptors do), they get carried along with the cargo from housekeeping receptors which decreases the number of receptors on the cell surface (i.e. less receptors to recognize cargo so less cargo being brought in) |
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Early endosomes vs. Late endosomes |
Early endosomes: acidic, located near peripheral region of the cell Late endosomes: more acidic, located closer to cell's nucleus |
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Post-translational protein uptake at the mitochondria |
-requires 2 energy sources: ATP and proton motive force 1. Targeting sequence recognized by molecular chaperones which then bind to it - mitochondrial matrix proteins contain sequence with positively charged residues on end 2. Proteins pass through TOM complex on outer membrane 3. Proteins destined for the matrix must pass through TIM23 complex (on inner mitochondrial membrane) where they are pulled through the complex by molecular chaperones already in the matrix -- use energy from ATP hydrolysis to pull down on proteins, molecular chaperone performs biased diffusion and acts as a Brownian ratchet (random diffusion, allows movement in only one direction) 4. Proteins destined to be incorporated in the inner mitochondrial membrane must enter TIM22 complex where they are then embedded into the inner mitochondrial membrane |
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Types of cytoskeleton |
*Each type is a polymer of protein subunits held together by weak non-covalent bonds which allows them to break apart & link up easily -microtubules - made of tubulin -microfilaments - made of actin -intermediate filaments - made of several related proteins |
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Cytoskeleton functions
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-maintains/determines cell shape -positions organelles inside the cell - microtubules -directs movement of materials and organelles in the cell -moves cell from place to place - grows cytoskeleton to some areas to travel there -mitosis and cell division - spindle made of microtubules |
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Microtubule (structure, protofilament, heterodimer, alpha & beta) |
-long, hollow unbranched tubes composed of tubular subunits -big - a lot thicker than plasma membrane -protofilaments - form the wall of the tube, made of dimers of alpha and beta subunits, each protofilament assembled from dimeric building blocks consisting of 1 alpha-tubulin subunit and 1 beta-tubulin subunit (heterodimer - non-identical because 1 alpha and 1 beta) -there is a seam in the tube where subunits are not aligned properly - beta next to an alpha -alpha has GTP bound all the time -beta has both GDP and GTP - can convert -plus end: end of tube with Beta subunits -minus end: end of tube with Alpha subunits -have polarity |
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Microtubule functions
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-mechanical support to cells - cell shape -internal organization -movement of intracellular components - w/ molecular motors -movement of cells - sturdy frame, microfilaments grow on the frame -mitosis and meiosis - movement of chromosomes |
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Microtubule associated proteins (MAPs) (2 types) |
Structural MAPs -stabilize microtubules and promote their assembly -without structural MAPs = grow but fall apart -microtubules have dynamic stabilization -Example: MAP2 - clamps to tube, keeps subunits in place -MAP1, MAP2, MAP4, tau Dynamic MAPs -motor proteins -move through the cell |
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Axonal transport
(anterograde vs retrograde) |
-use motor proteins -signal b/w nerve cells - travels down axon -vesicles - move materials to axon, mediated by microtubules (both directions) -vesicles attached to microtubules by cross-linking proteins including motor proteins such as kinesin & dynein -microtubules always arranged same way: plus end to axon, minus end to cell body -anterograde: away from cell body to axon -retrograde: towards cell body |
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Kinesin and dynein
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Kinesin - bring material to plus end on microtubule, anterograde travel Dynein - bring material to minus end on microtubule, retrograde travel |
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Motor protein movement (2 cycles)
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Mechanical cycle - as protein moves along, it undergoes a series of conformational changes Chemical cycle -provides energy necessary to fuel motor's activity -steps include binding of ATP molecule to the motor, the hydrolysis of ATP, the release of products (ADP and Pi) from motor, and binding of new molecule of ATP |
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Kinesin
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-45 different kinds -all have similar structure: head, neck, stalk, tail -has 2 heads: attach to microtubules, act like feet that walk on the microtubule, has a catalytic core that uses/hydrolyzes ATP -stalk: has flexible hinge -tail: where the variety is, has "light chain" which recognizes cargo to transport -heavy chain: composed of head & stalk, two chains -light chain: composed of tail, two chains, variable region |
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Kinesin based movement
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-moves material toward plus end on microtubule -in axons, kinesin transports vesicles/other cargo toward the synaptic vesicles 1. Tail binds to cargo 2. Heads walk on microtubule to move - each step has a different binding to ATP, each step produces conformational change *When one head binds to microtubule, the resulting conformational change in adjacent neck region of motor protein causes the other head to move forward to next binding site on the protofilament 3. Binding and hydrolysis of ATP to ADP on the head -drives a power stroke that moves the motor a precise # of nanometers along its track -neck moves/rotates (power stroke) causing head to rotate -after head rotates, head w/ ATP binds to microtubule 4. Other head ejects ADP and docks onto filament 5. Hydrolysis of ATP results in head detaching |
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Chemical cycle and mechanical cycle steps of kinesin based movement
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Chemical cycle --> mechanical cycle Binding of ATP --> causes neck region to move, power stroke Ejection of ADP --> docking of head onto microtubule ATP hydrolysis --> detachment of trailing head |
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Kinesin-1
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-one member of a superfamily of related proteins called Kinesin-like proteins (KLPs) |
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Cytoplasmic dynein (and dynactin) |
-walks toward minus end of microtubule -walks towards cell body -uses ATP -2 heavy chains - 2 heads -several light chains -does not interact directly with membrane-bounded cargo but requires an intervening adaptor - dynactin -dynactin - associates w/ light chain, intermediate b/w light chain and cargo, also re-regulates dynein activity by increasing processivity of dynein |
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Kinesin & dynein and tails |
-kinesin and dynein both recognize cytosolic tails -both can bind to same molecule - more of one decides what direction it'll travel in -more tails for kinesin - will travel anterograde -more tails for dynein - will travel retrograde |
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Kinesin vs dynein movement
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-kinesin walks towards plus end, toward axon terminus, anterograde -dynein walks toward minus end, toward cell body, retrograde |
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Summary of dynamic MAPs |
-motor proteins move unidirectionally along the microtubule in a stepwise manner from one binding site to the next -mechanical cycle: conformational changes -chemical cycle: binding & hydrolysis of ATP -kinesin movement is anterograde (minus to plus end) -dynein movement is retrograde (plus to minus end) |
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Microtubule organizing center (MTOC) |
-the centrosome - found in all different cells -located close to the nucleus -made of tubulin - 9 groups of 1 complete tubule and 2 half tubules of tubulin = 9 groups of 3 sub tubules (A, B, C) -protein material joining the microtubules -a centrosome is 2 centrioles oriented at 90 degrees to each other -surrounded by cloud of electron dense "pericentriolar material" (PCM) - centrioles recruit PCM to form a new centrosome -centrioles grow out of another centriole -can reproduce BEFORE the cell can divide -microtubules do not contact centrosome directly - contact made in PCM cloud |
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Gamma tubulin and Y-TuRC
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-placed on PCM (pericentriolar material)
-can only associate with alpha tubulin -Y-TuRC (gamma tubulin ring complex) determines diameter and orientation of microtubule -critical component to microtubule nucleation |
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Microtubule dynamics |
-microtubules in axons need to be stabilized by structural MAPs -in cell division, microtubules must be dynamic to grow and search for chromosomes and also be able to fall apart |
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Dynamic instability
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-microtubule's ability to undergo rapid growth or rapid shrinkage -explains the observation (1) that growing and shrinking microtubules can coexist in the same region of a cell and (2) that a given microtubule can switch back & forth unpredictably (stochastically) b/w growing and shortening phases-GTP dimers are non-exchangeable on alpha subunit -GTP is exchangeable on beta subunit so there are two shapes of a Beta subunit: one when GTP is bound and one when GTP hydrolyzed to GDP -if GDP is deep inside microtubules, it will stay inside |
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Dynamic instability process |
1. Tip consists of open sheet containing tubulin-GTP subunits (GTP Cap) 2. Tube has begun to close, forcing hydrolysis of GTP on the beta subunit 3. Tube has closed to its end, leaving only tubulin-GDP subunits - GDP-tubulin subunits have curved conformation which makes them less able to fit into straight protofilament 4. Strain resulting from presence of GDP-tubulin subunits at plus end of microtubule is released as the protofilaments curl outward from the tubule and undergo catastrophic shrinkage/catastrophe |
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Catastrophe vs. Rescue
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Catastrophe: GDP dimers falling apart b/c unstable, caused by GTP hydrolysis occurring faster than new tubulin addition Rescue: allows microtubule to incorporate enough GTP dimers to grow back again, added at the plus end |
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True or false: concentration of GTP dimers varies all around the cell |
True |
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Intermediate filaments |
-smaller in diameter than microtubules -strong, flexible, rope-like fibres that provide mechanical strength to cells subjected to physical stress |
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What is plectin?
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-crosslinking protein between microtubule and intermediate filament |
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Intermediate filament distribution
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-Epithelial cells -Keratinocytes - highly specialized epithelial cells that produce keratin -Keratin -Desmosomes -Hemidesmosomes |
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Keratin |
-keratin filaments constitute the primary structural proteins of epithelial cells -very stretchy -keratin-containing IFs radiate through cytoplasm tethered to nuclear envelope and anchored to outer edge of cell by connections of cytoplasmic plaques of desmosomes and hemidesmosomes -keratins interact with desmosomes & hemidesmosomes |
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Desmosomes |
-cell to cell adhesion -structure by which two adjacent cells are attached-formed by 2 protein plaques in cell membrane linked by filaments -contain cadherins (proteins) that link 2 cells across a narrow extracellular gap -IFs are linked to cytoplasmic domains of desmosomal cadherins by additional proteins |
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Hemidesmosomes
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-cell to ECM adhesion -half of a desmosome-similar to a desmosome BUT the attachment is between the cell and extracellular matrix -involve only one cytoplasmic plaque |
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Intermediate filament protein structure |
-monomer structure -alpha helices made up of a repetition of 7 amino acids = Heptad repeats -Heptad repeats form coils which make the proteins stretchy, add stability and stretchiness -alpha helix can form coiled coil arrangements with other monomers |
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Intermediate filament structure build-up (polarity vs non-polarity) |
1. One polar monomer
2. Polar dimer = two polar monomers 3. Non-polar tetramer = two polar dimers 4. Non-polar filament = 8 non-polar tetramers 5. Non-polar intermediate filament = filaments *Monomer & dimer are polar b/c you can tell one end from the other *Cannot detect polarity in tetramer b/c cannot distinguish one end from the other (2 dimers facing each other) *Filament and IFs also non-polar b/c cannot distinguish one end from the other |
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Disassembly of intermediate filaments |
-phosphorylation by a kinase -in the case of lamins (lamin network lines the inside of the nucleus in a cell), disassembly required for cell division so that the nucleus can fall apart (Ex. Cdc2 kinase) |
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Assembly of intermediate filaments
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-dephosphorylation by a phosphatase |
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Mutation in intermediate filaments
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Ex. Epidermolysis bullosa simplex - caused by loss of epidermal cell-to-cell adhesion so get blistering effect -if you have a mutation in keratin, the IFs have no stretch so desmosomes have nothing to pull against causing the skin to blister |
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Microfilaments |
-monomers made up of actin (actin is a central component of muscles) -actin is responsible for whole cell movement |
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Actin isoforms
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-similar in size -similar in amino acid sequences -copolymerize -common ancestor gene |
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Two types of actin |
G-actin (globular) -just actin monomers -single subunit -forms 2 lobes on each slide of ATP binding cleft -ATP-binding cleft & magnesium atom -2 domains on each lobe -actin is an ATPase F-actin (filamentous) -actin part of microfilament -assembles facing same way foreign helix so these filaments have polarity |
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Decoration experiment |
-isolated myosin -F-actin + S1 fragment of myosin in presence of ATP results in a decorated filament where myosin assembles on F-actin -when S1 fragments are bound, one end of the actin filament appears pointed like an arrowhead (pointed end) while other looks like a barbed end (barbed end) -plus end (barbed end) polymerizes 10x faster than the minus (pointed) end -this experiment showed which end polymerizes Basically, the F-actin was decorated with myosin heads and it was found that the barbed end (plus end) polymerized faster than the pointed (minus) end - this showed which end of F-actin polymerized |
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Microfilament assembly/disassembly |
Before incorporated into a filament, an actin monomer binds to an ATP - actin is an ATPase 1. Form seed - 4 subunits assembled together - need 4 G-actin subunits in F-actin filament for growth to occur 2. Add high conc. of actin - both ends of filament grow, lots of growth at plus/barbed end and little growth at minus/pointed end 3. If actin conc. drops, probability of subunits being added and detaching are equal at the minus end but subunits are still being added to plus end -addition of monomers continues at plus end - critical conc. of ATP-actin lower at plus end -addition of monomers stops at minus end - critical conc. of ATP-actin higher at minus end 4. If conc. of actin drops further, tendency for subunits to leave minus end is greater but growth still occurring at plus end -monomers continue to be added to plus end but a net loss of subunits occurs at minus end 5. If concentration remains constant, subunit added to plus end and leaves at minus end = tread milling -subunits being added to plus end and removed from minus end of each filament at steady state -the relative position of individual subunits within each filament is continually moving = tread milling Actin subunit needs to be recharged before binding ATP and reintegrating into filament |
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Microfilament assembly/disassembly review |
-starts with pool of ATP bound G-actin monomers which aggregate until a stable nucleus is formed = seed -seed rapidly elongates by addition of monomers to both of its ends -plus end elongates faster than the minus end -F-actin filament ends in steady state equilibrium with G-actin monomers -- equilibrium concentration of pool of unassembled units is called the "critical concentration" -adding more G-actin raises monomer concentration above CC favouring further polymerization -at monomer concentrations below CC, F-actin will depolymerize |
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Critical concentration
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-the plus and minus ends require different minimal concentrations of ATP-actin monomers in order to elongate -critical conc. of plus end is much lower than the minus end meaning that the plus end can continue to elongate at lower ATP-actin concentrations than the minus/pointed end can |
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Myosin (what is it, types, commonality between the different types) |
-molecular motor -uses ATP for energy -chemical cycle (ATP hydrolysis) coupled to mechanical cycle (how the head interacts w/ actin) -two types: conventional Type II found in muscles and unconventional myosin found everywhere else -both types have two heads |
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Type II (Conventional) myosin |
-primary motors for muscle contraction -uses ATP -moves toward the PLUS end of actin filament -light chains associated w/ neck, regulate neck movement -assembles into bipolar filament Structure -SI fragment (isolated myosin head) -pair of globular heads -pair of necks (each consisting of a single alpha helix & 2 associated light chains) -single tail (formed by intertwining of long alpha helical sections of 2 heavy chains) |
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Type II myosin into a bipolar filament |
-assembles into a bipolar filament -assemble so that the ends of the tails point toward centre of the filament and the globular heads point away from the centre -because they are bipolar, the myosin heads at the opposite ends of a myosin filament have the ability to pull actin filaments toward one another |
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Unconventional myosin - myosin V |
-involved in vesicle transport -moves toward PLUS end -cooperates with kinesins -interacts indirectly with Rabs |
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Myosin exists in 3 conformations |
1. An ATP pre-hydrolysis state with myosin NOT bound to actin 2. An ADP+Pi state with myosin bound to actin weakly 3. A post power-stroke state (rigor conformation) - Pi has left, neck moves causing head to move, affinity for actin increases |
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What happens when there's no ATP around? |
-filament cannot move -myosin stays stuck to actin -conformation is kept the same -muscles won't relax because myosin is stuck to actin in rigor conformation -- rigor mortis |
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Myosin as a molecular motor in action
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-2 myosin heads (dimer) act independently and are attached to a lever arm -ATP binding to head region causes detachment (because ATP bound myosin has weak affinity for actin) -ATP hydrolyzes which resets head position and allows for attachment between myosin head & actin -Pi leaves causing power stroke - myosin head pulls on actin filament -release of ADP leads to rigor conformation and binding of new ATP will release myosin from actin and cycle repeats |
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What does the power stroke do?
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causes the muscle to contract |
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Microfilament assembly & organization |
Two arrangements seen in non-muscle cells: (1) Actin network - loose arrangement (2) Actin bundle - parallel, tight arrangement -actin network not in muscle cells found in cortex - right underneath the plasma membrane -loose arrangement allows for constant reorganization of actin as cell moves |
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Acting-binding proteins
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Goal: to polymerize network where it needs to be formed or break apart where it needs to break apart -these proteins typically seen in cortex -starts with monomers -8 different types |
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Monomer nucleating (acting-binding protein) |
-forms base at minus end which has pseudo monomers where these monomers can form without seeds or nucleus -base facilitates formation of new actin filament -similar to MTOC in microtubules Ex. Arp 2/3 complex |
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Monomer sequestering (acting-binding protein)
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-binds to actin ATP-monomers and prevents polymerization Ex. Thymosin B4 |
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End-blocking/capping proteins (acting-binding protein)
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-responsible for capping filaments -capping proteins at either the plus or minus end -effect of capping: at the critical concentration where the whole filament is at a steady state and if we cap plus end, the filament will shrink (because monomers will leave at minus end and no growth will occur at plus end) and likewise if we cap the minus end then the filament will grow -capping one end will STOP tread milling -can control growth or shrinking w/ these proteins Ex. CapZ (plus end), Tropomodulin (minus end) |
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Monomer polymerizing (acting-binding protein)
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-exchange factor -recharges the monomers -promotes exchange of ADP for ATP so able to become part of filament Ex. Profilin |
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Depolymerizing (acting-binding protein)
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-binds to actin-ADP at minus end causing depolymerization Ex. Cofilin |
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Bundling (acting-binding protein) |
-proteins that interact w/ network of actin to form either loose network or tight bundle -both called bundling proteins Ex. Filamin - long flexible rod, results in loose network Ex. Villin - small globular, results in tight parallel bundle |
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Filament severing (acting-binding protein) |
-breaks filament into 2 in presence of lots of G-actin -when you cut filament in 2, it can form seeds which would promote growth of new filament Ex. Gelsolin |
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Membrane binding (acting-binding protein)
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-attaches microfilaments to membrane Ex. Vinculin - found in lots of cells Ex. Dystrophin - muscle cells, role in muscular dystrophy (progressing weakening & wasting of muscles) |
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Summary of microfilaments
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-actin binds ATP -forms rigid gels, networks & linear bundles -regulated assembly from a large number of locations -highly dynamic -polar -tracks for myosin (molecular motors) -contractile machinery & network at the cell cortex |
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Summary of microtubules
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-alpha & beta tubulin binds GTP -rigid and not easily bent -regulated assembly from a small number of locations -highly dynamic -polar -tracks for kinesins and dyneins -organization and long-range transport of organelles |
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Summary of intermediate filaments
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-intermediate filament subunits don't bind to a nucleotide -great tensile strength -assembled onto pre-existing filaments -less dynamic -polar -no molecular motors -functions in cell tissue & integrity |
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Cell locomotion |
-not something the whole cell necessarily does -important during development -sometimes we have cell migration that is bad (ex. cancer cell migration) -cell has a leading edge, tail, and cell body w/ nucleus -myosin motors mediate movement of cell body over actin skeleton -traction forces -actin polymerization is responsible for pushing the leading edge of cell outward whereas myosin is responsible for pulling the remainder of the cell forward Ex. Fish keratocytes - good for studying locomotion because their rapid gliding movement depends on formation of very broad, thin lamellipodium |
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Cell locomotion steps
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1. Extension phase -cell pushes lamellipod forward, driven by rapid polymerization of actin filaments at leading edge -leading edge extends by growing its actin skeleton inside the leading edge -pushes lamellipod forward -movement initiated by protrusion of part of cell surface in direction in which cell is to move 2. Adhesion stage -has to attach to substrate - lamellipod adheres to underlying surface using specialized membrane adhesion sites -portion of lower surface of protrusion attaches to substrate forming temporary sites of anchorage -firmly anchored cell is now ready to generate the force to move the cell body forward, new adhesion site formed 3. Translocation of cell body -cell body translocates to part where leading edge was before -bulk of cell is pulled forward over adhesive contacts -accomplished by contraction of actin and myosin bundles at the lamellipod and cell body border 4. De-adhesion -after cell body translocates, the back of the cell releases from adhesion sites & moves forward -has to unattach from temporary attachment sites on substrate -cell breaks rear contacts with substrate causing retraction of trailing edge/tail |
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Leading edge (of a cell during cell locomotion)
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-lamellipodium: thin veil, broad, flattened, veil like protrusion -sometimes lamellipodium has filopodia - thin extensions, tend to be present when cell is not moving fast and when cell is in decision-making process of where to go, full of receptors for chemical attractants, if it picks up an attractant it sends a signal telling actin skeleton where to start forming/signals cell movement direction |
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Protrusion of lamellipodium leading edge |
1.Chemical attractant receptor picks up chemical attractant that tells cell what direction to move in 2. Receptor releases WASP protein (WASP = Wiskott-Aldrich Syndrome Protein, rare genetic diseas, occurs b/c protein mutated cells don't travel to where they're needed, poor immune response) 3. When WASP not mutated, it activates Arp 2/3 complex 4. Arp 2/3 complex serves as nucleating site for formation of new actin filaments w/ actin monomers that have profilin on so that filaments can grow out quickly 5. Once filaments have formed, Arp 2/3 complexes attach to sides of filaments which stimulates their nucleating activity and as a result, the bound Arp 2/3 complex initiates side branches that extend outward (new filament grows from it) 6. Plus end of actin filament push against plasma membrane causing cell to push out (as side branches polymerize, this pushes plasma membrane outward resulting in extension of leading edge of lamellipodium) 7. Barbed/plus end of previously formed filaments become capped by capping protein which prevents further growth 8. Minus end begins to depolymerize releasing ADP-actin monomers - promoted by cofilin which binds to ADP-actin monomers within filament & stimulates dissociation of subunits from minus end *The purpose of depolymerization at the back is to provide monomers for polymerization at the front 9. Profilin recharges (actin-ADP to actin-ATP) actin monomers that had fallen off from the filament - this allows them to be used to build onto new filament/ready to engage in actin polymerization |
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Heterochromatin vs Euchromatin |
Heterochromatin - tightly packed DNA, not actively read by cell, attached to edge of nucleus Euchromatin - loosely packed, less electron dense, actively read/transcribed by cell |
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What is one mean of gene regulation?
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Chromatin arrangement
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Functions of the nucleus
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-DNA storage -DNA replication -DNA transcription |
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Nuclear structural components |
-nucleoli - ribosome factory -nuclear envelope - double membrane -nuclear pores in the membrane -nuclear matrix - proteins that hold chromatin together -nucleoplasm |
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Nuclear membrane
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-double membrane -continuous with the ER -has own distinct complement proteins -embedded proteins made at the ER -inner and outer nuclear membranes fused at sites forming circular pores that contain complex assemblies of proteins -outer membrane is continuous w/ the rough ER -inner surface of nuclear envelope is bound by integral membrane proteins to a thin filamentous meshwork called "nuclear lamina" |
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Nuclear lamina
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-provides mechanical support to nuclear envelope -serves as site for chromatin fibres at nuclear periphery |
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Nuclear Pore Complex (NPCs)
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-massive structure extending across nuclear envelope forming gateway that regulates flow of macromolecules b/w cell nucleus & cytoplasm -nuclear basket sticking into nucleoplasm -filaments stick out to cell cytoplasm -8-fold symmetry -cytoplasmic filaments -proximal filaments -central transporter - bundle of protein fibres -spoke ring assembly - holds everything together -nuclear basket - nucleoporins: all proteins associated w/ nuclear basket |
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Nucleoporins
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Proteins that makeup the NPC (nuclear pore complex) |
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Nuclear Localization Signals (NLSs) |
-proteins to be imported to the nucleus all have signal: lysine (w/ positive charge), arginine (w/ positive charge) and proline [these amino acids form the signal] -the amino acids can be separate within the sequence (not right next to each other) and still emit the signal - the way the complex folds together makes it so the amino acids line up beside each other to display the signal -sequence can be separated by up to 10 amino acids but the protein folds in a way so that the sequence comes together and can be recognized -need a positive charge for nuclear import |
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Transport receptors |
-float around in the cytoplasm -importins and exportins |
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Importin (transport receptor)
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-have alpha & beta subunit
-soluble NLS protein getting imported binds to alpha subunit |
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Protein nuclear import
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1. Detection of NLS on the protein to be imported - alpha importin recognizes NLS protein and binds to it and then the beta subunit binds to the alpha subunit *importins are also known as transport receptor 2. Receptor-cargo complex docks to cytoplasmic filament of the nuclear pore complex (NPC) 3. Receptor-cargo complex moves through NPC into nucleoplasm 4. Once receptor-cargo complex enters nucleus, the GTPase "ran" assembles with beta subunit to cause the dissociation of the receptor from the cargo 5. Importin beta in association with ran-GTP transported back to the cytoplasm via the NPC and then GTP hydrolysis occurs and ran-GDP dissociates from beta importin 6. Importin alpha is transported back to cytoplasm with help of an exportin |
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GTPase activity |
On = GTP bound Off = GDP bound GTP hydrolysis: on to off GDP-GTP exchange: off to on, required to bring protein back to on state GTP-ase activity for most proteins is the rate of GTP hydrolysis - which is usually low *Need something to adjust for the time between GDP and GTP |
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GAP vs. GEF
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GAP: GTPase accelerating protein -increases GTP hydrolysis -without GAP, protein would stay in the "on" state for too long GEF: guanine exchange factor -removes GDP so it can be replaced by GTP |
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If a protein has a lot of GTPase activity, what state would it spend more time in? |
The off state
GTPase activity is referred to activity of GTP hydrolysis (conversion of GTP to GDP) |
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Proteins involved in the RAN cycle
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RCC1 - a GEF -in the nucleus -converts ran-GDP to ran-GTP -maintains high concentration of ran-GTP RANGAP1 - a GAP -in cytoplasm -converts ran-GTP to ran-GDP -maintains low concentration of ran-GTP *These conversions provide the only energy required *GTP hydrolysis maintains the ran-GTP gradient across the membrane - this gradient drives nuclear transport, NO other energy sources required |
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Nuclear lamina |
-lattice of proteins right inside nuclear plasma membrane -intermediate cytoskeleton proteins = lamins -lamins A, B, and C -lamin B, an isoprenyl group - the lamin actually attached to membrane by isoprenyl group -all cells have these 3 lamins that maintain nucleus shape and provide mechanical support to nuclear envelope -lamins have the ability to depolymerize |
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Nuclear membrane disassembly
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-phosphorylation - addition of a phosphate Lamin kinase (eg. Cdc2 kinase) Maturation promotion factor (MPF) - what triggers mitosis |
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Nuclear membrane reassembly
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-dephosphorylation - removal of a phosphate
Protein phosphatase - reassembly occurs in telophase |
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Ribosomes |
-some ribosome from the LUCA is origin of all ribosomes -ribosomes make all of our proteins -rRNA is the major component of the ribosome -ribosomes assembled in a way to produce proteins -ribosomes are conserved over time -ribosomes have a short lifespan so new ones need to be made all the time -ribosomal proteins all together form a large and small subunit which form the whole ribosome -full ribosome size: 24nm, 80S |
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S (Svedburg) value
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-indicates how particles migrate after centrifugation
-value dependent on molecular weight and surface area -produces sedimentation coefficient Ex. Sucrose centrifugation -put macromolecule on top of sucrose gradient -centrifuge -particles suspended in layer relative to size & surface area within sucrose gradient 40S + 60S is not 100S but rather 80S - not additive |
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Nucleolus |
-ribosome factory -no membrane, just an area 3 parts -granular component: granules that are nascent ribosomes in various states of maturation -fibrillar center: centre of ring -dense fibrillar component: where rRNA made, outside of ring |
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Nucleolar rRNA genes in action |
-sequences of rDNA back to back in "tandem arrangement" -rDNA centre of unit -rRNA are branches off the unit = nascent rRNA transcript -rRNA genes separated by non-transcribed spacer -transcription begins at the top of the unit -each bead/molecule along the tree is a protein of RNA polymerase I -each branch is a rRNA precursor -each bead reads the rDNA -rRNA polymerase beads read rDNA from 5' to 3' -5S made on separate gene elsewhere in the nucleus - doesn't get chopped up -45S is a full transcript - gets chopped up into smaller subunits that'll later be part of ribosome -18S, 28S, 5.8S - final products made from initial transcript and will be incorporated into ribosome subunits |
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Chromatin |
-chromatin = chromosomes composed of DNA and associated proteins, DNA + histones -orderly packaging depends on histones - small proteins that package the DNA Heterochromatin - tight packing of DNA Euchromatin - loose packing of DNA |
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Histones |
-ratio of DNA to histones is fixed (9 histones per 200 DNA base pairs) -5 types of histones: H2A, H2B, H3, H4, H1 -small highly basic proteins -amino acid sequences highly conserved -octomers - 2 copies of each histone per 200 base pairs of DNA --- the case for all histones EXCEPT for H1 |
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Histone H1 |
-only one H1 per 200 DNA base pairs
-twice as big as the other histones -less conserved -more loosely associated w/ DNA |
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Nucleosomes |
-repeating subunits of DNA and histones -histone core of each nucleosome consists of 2 copies of histones H2A, H2B, H3 and H4 assembled into an octamer while the remaining H1 resides outside the nucleosome core particle -chromatin = beads on a string -140 bp DNA |
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Levels of chromatin organization
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-naked DNA - 2nm -nucleosome filament/beads on a strong - 10nm -30nm fibers -looped domains - loops of 30nm fibers |
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True or false: the tighter DNA is wound, the less transcriptionally active it'll be
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True |
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General features of signalling pathways |
-start with a ligand (1st messenger) that was produced by another cell -ligand binds to transmembrane receptor Two different paths to follow: (1) Effector bound to transmembrane receptor on inside of cell stimulates 2nd messenger - 2nd messenger binds to other things - triggers cascade to occur (commonly involving kinases) (2) Instead of the receptor triggering the effector to produce 2nd messenger, receptor gets phosphorylated which then becomes a recruiter for other molecules promoting their interaction - allows cascade to occur -at the end of the cascade, there is a target molecule/protein which becomes activated and carries out a specific function (transcription, survival, protein synthesis, movement, cell death, metabolic damage) |
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G-protein coupled receptors |
-process coupled to activation of an effector -receptors all have 7 transmembrane domains (aka: 7 transmembrane receptors) -N-terminus of receptor is always on outside of cell, C-terminus is always on inside of cell -3 loops on the outside (form ligand binding pocket) and 3 loops on inside (form binding sites for intracellular signalling proteins) -each receptor has specific binding for a certain ligand - specificity -G-protein coupled w/ receptor - heterotrimeric receptor -alpha, beta, gamma subunits form G-protein -only the alpha subunit on the G-protein is a GTPase -trimeric receptor activates effector -G-protein anchored to membrane by lipid anchor - just the alpha & gamma subunits (the beta is attached to both the alpha and gamma) |
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Receptor mediated activation of effectors by heterotrimeric G-proteins
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(1) Ligand binds to G-protein coupled receptor -causes conformational change in extracellular ligand binding site (this change in conformation is transferred across plasma membrane) -alpha subunit binds to receptor (2) Conformational change in cytoplasmic loops of the receptor -increase in affinity of the receptor for G proteins -ligand-bound receptor forms a receptor-G protein complex -conformational change in alpha subunit of G protein -release of GDP followed by binding of GTP to alpha subunit (activated receptor acts as a GEF) -G-protein now activated *NOTE: in activated state, a single receptor can activate a number of G-proteins [signal amplification] (3) Once GTP bound, active alpha subunit dissociates from beta & gamma subunits and goes to activate effector (4) Effector enzyme (ex. adenylyl cyclase) produces 2nd messenger (ex. cAMP) -uses ATP to produce cAMP -adenylyl cyclase has 12 transmembrane proteins -other 2nd messengers: Ca2+, phosphoinositides, diacylglycerol, cyclic GMP, nitric oxide (5) GTP hydrolyzed to GDP -by RGS (Regulators of G-protein signalling): a specific GAP for trimeric G-protein, turns off signal causing detachment of GTP (6) alpha subunit of G-protein dissociates from effector -alpha then goes back to bind with beta and gamma subunits to reform inactive trimeric complex (7) Process of desensitization -blocks active receptors from turning on additional G proteins, turning off the signal at the level of the receptor -GRK (G-protein coupled receptor kinase) phosphorylates the G-protein coupled receptor - negative feedback onto receptor to turn it off -if there's a low signal: few phosphates prevent receptor from binding to alpha subunit -if there's a strong signal: will cause reduction in number of receptors by exocytosis (8) Phosphorylated receptor binds arrestin (arrestin competes with G-protein) -arrestin can bind to AP2 adaptor molecules situated in clathrin-coated pits - clathrin molecules assembled around arrestin-bound receptor -uptake of phosphorylated G-protein coupled receptors by endocytosis and return them to the surface to continue triggering cascades or degradation in lysosomes |
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Producing cyclic AMP |
-adenylyl cyclase has 12 transmembrane domains with the catalytic domain on the inside -synthesis of cAMP follows binding of a 1st messenger to a receptor at outer surface of cell and as a result, 2nd messengers enable cells to mount a large-scale coordinated response following stimulation by single extracellular fluid -cAMP produced at the active catalytic site inside adenylyl cyclase which is a site that is connected to the 12 transmembrane domains -uses ATP to produce cAMP |
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Protein Kinase A (PKA) activation |
Inactive PKA: some bound catalytic domains and regulatory domains with 2 nucleotide binding sites each, the regulatory subunits bind to active site of catalytic domains to inhibit it Process of activation: cAMP fills nucleotide binding sites on regulatory domains --> catalytic domains dissociate --> now active PKA Now, the catalytic domains can go on to activate other proteins by phosphorylating them -example of allosteric regulation - cAMP binding to regulatory protein at other sites away from the active site but still causes conformational change and stimulation/activation |
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Allosteric regulation
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Regulation of a protein by binding an effector molecule at a site other than the enzyme's active site The site to which the effector binds to is the allosteric site |
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Lipid derived 2nd messengers |
Phospholipase - lipid splitting enzymes -cleave ester bonds on phospholipids -different phospholipases cut at different points -Phospholipase C Beta cuts between the phosphate & glycerol molecule on a phospholipid Phospholipid kinases - lipid phosphorylating enzymes Phospholipid phosphatases - lipid dephosphorylating enzymes |
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Phosphatidylinositol derived 2nd messengers
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Phosphatidylinositol -is a phospholipid -the inositol ring has lots of OH groups on it & can be phosphorylated at different places -when phosphate groups are added, it is called a "phosphoinositide" -there are not a lot of phosphoinositides in membranes BUT there are more of them on the cytosolic face than the exoplasmic face |
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Phosphatidylinositol (PI) derived 2nd messengers process |
1 & 2. Kinases phosphorylate the inositol ring in positions 4 and 5 to form PIP2 -PIP2 sits in the membrane (membrane phospholipid) and waits for signal to get triggered -PIP2 forms anchor point for proteins -a pH domain is a recognition domain for PIP2 and so any protein w/ pH domain will bind to PIP2 in membrane and phospholipase C beta has a pH domain 3. Ligand binds to G-protein coupled receptor (GPCR) causing GDP to GTP exchange on alpha subunit of G-protein 4. Alpha subunit of G-protein stimulates Phospholipase C beta (PLCB) -PLCB was situated at inner surface of membrane bound there by interaction b/w its pH domain & a phosphoinositide embedded in bilayer 5. PLCB catalyzes reaction in which PIP2 is cleaved into the inositodol ring (IP3) and a diacylglycerol (DAG) -IP3 floats around in cytosol, DAG stays in membrane 6. DAG recruits protein kinase C (PKC) to membrane & PKC attaches to DAG at membrane but is in its inactive state (needs Ca2+ to activate) 7. IP3 diffuses into cytosol 8. IP3 binds to IP3 tetrameric receptor on smooth ER (which is a storage of Ca2+) which causes channel opening on receptor allowing influx of Ca2+ into cytosol thereby activating PKC 9. PKC now active & goes on to phosphorylate other things |
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Signal amplification |
-binding of a single hormone molecule at cell surface can activate a number of G-proteins, each of which can activate an effector that can then produce lots of 2nd messengers in short period of time -production of a 2nd messenger provides a mechanism to greatly amplify the signal generated from original message |
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Calcium as a 2nd messenger |
-calcium stored in smooth ER -IP3 receptors stimulated to trigger release of calcium from these stores -1000x higher concentration of Ca2+ in these stores than in the cytosol -ATP-driven Ca2+ transport system (pumps) - pumps calcium out of cell or into stores to keep Ca2+ out of the cytoplasm -voltage gated Ca2+ channels - as voltage difference occurs, Ca2+ channels open resulting in Ca2+ influx from outside of cell in a quick brief manner -easier to control conc. of Ca2+ if taken from the intracellular stores than when cell decides to take it from the outside of the cell -if too much Ca2+ inside cytosol for too long, the mitochondria releases substance that triggers apoptosis of the cell b/c too much Ca2+ makes the cell think the plasma membrane is breached |
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Calmodulin
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-adaptor (small protein) used by calcium to act on things
-very abundant, everywhere -almost entirely made of alpha helices -contains 4 Ca2+ binding sites -when calcium conc. increases, calmodulin activated & has low affinity for calcium so that it only activates when calcium conc. very high -all 4 binding sites must be calcium bound for calmodulin to be active -when calmodulin is activated it is folded up -when calmodulin is inactive it unfolds, opens up, and hydrophobic amino acids are exposed -activity of calcium depends on proteins and enzymes that respond to calmodulin Ex. Troponin C - calmodulin for muscle, allows myosin to walk on actin |
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Receptor tyrosine kinases (RTKs)
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-act to recruit proteins to the cell surface -activated by dimerization - 2 RTKs need to get together -2 ways of activation (1) ligand mediated dimerization - ligand with 2 binding sites that bind to each receptor so receptors come together and dimerize (2) receptor mediated dimerization - more than 1 ligand binds to receptors which causes conformational change that increases affinity for one another and receptors come together & dimerize *example of allosteric regulation -receptors initially have low kinase activity when separate but when they come together through dimerization they participate in trans-autophosphorylation -after phosphorylated, becomes fully active & recruits proteins to cell membrane |
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Trans-autophosphorylation
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-done by RTKs once they come together & dimerize -one receptor phosphorylates the other and vice versa at multiple sites so lots of kinase activity upon dimerization -enzymatic activity is tyrosine kinase - tyrosine is what's getting phosphorylated |
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Autophosphorylation sites
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-binding sites for cytoplasmic signalling molecules -these sites are recruiting sites for proteins with 1 of these domains: --Src-homology 2 (SH2) domain --Phosphotyrosine-binding (PTB) domain |
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RTK-regulated signaling proteins |
-adaptor proteins - bind to phosphate and recruit (ex. Grb2: SH2 domain, 2 SH3 domains) -docking proteins - potential to extend how many proteins you can recruit, gets phosphorylated by kinase activity of receptor, increase # of phosphorylated tyrosines -transcription factors - STAT: phosphorylated tyrosines by kinase activity of receptors, dimerizes when phosphorylated tyrosine on one STAT interacts with SH2 domain on another STAT protein and becomes active, has SH2 and tyrosine, dimers get sent to nucleus to initiate transcription *transcription factors belonging to STAT family play important role in function of immune system -signalling enzymes - 3 possible enzyme activation mechanisms (1) Activated as a result of translocation to the membrane which places them in close proximity to their substrates (2) Activated through allosteric mechanism - binding to phosphorylated tyrosines results in conformational change in SH2 domain which causes conformational change in catalytic site resulting in change in catalytic activity (3) Phosphorylated tyrosines directly regulates catalytic activity |
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GDI: guanine nucleotide dissociation inhibitor
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-accessory protein for GTPase -when GDI binds to small G protein, it prevents the dissociation of GDP therefore causes molecule to remain in the "off" state |
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G-protein cycle
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1. GDI bound to G-protein keeping it in off state/bound to GDP
2. GEF binds to replace the GDP with GTP, activating the G-protein 3. G protein can now go activate other target proteins 4. GAP binds to G-protein causing GTP to switch back to GDP, turning G-protein back into "off" state 5. Inactive G-protein also means it dissociated from target protein thus the target protein is inactive too |
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Components of Ras-MAP kinase pathway
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-Ras is a small protein attached to inner part of cell membrane by lipid anchor and it is a GTPase which is activated by exchange of GDP for GTP -Grb2 is an adaptor protein and recruits SOS - Grb2's SH2 domain attaches to the phosphate of phosphorylated receptor and the two SH3 domains attaches to SOS -SOS is the GEF for Ras - activates Ras by exchanging GDP for GTP -Ras is important b/c it is mutated in a lot of cancers -Cells are on the path to cell division when Ras is activated |
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Ras-MAP kinase pathway |
1. Growth factor (ligand) binds to RTK 2. RTKs possess phosphorylated tyrosines that act as docking sites for Grb 2 adaptor protein 3. Grb2 recruits SOS (SOS is a GEF for Ras) 4. SOS activates Ras turning it from Ras-GDP to Ras-GTP 5. Ras-GTP causes phosphorylation of Raf protein that becomes membrane bound 6. Raf is a kinase (MAPKKK) that phosphorylates another kinase MEK (MAPKK) which then phosphorylates another kinase ERK (MAPK) 7. ERK (MAPK) which moves inside the nucleus to phosphorylate transcription factors 8. These phosphorylated transcription factors cause initiation of transcription of genes (FOS and JUN) that promote cell division 9. Eventually the pathway leads to activation of genes in cell proliferation including cyclins |
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Dual specificity kinase |
-MEK (MAPKK) is a dual specificity kinase -phosphorylates tyrosines, serines, and threonines -safeguard because only kinase to activate ERK therefore only activates ERK to cause cell division if it really needs to |
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Genes to be produced after transcription intiated |
-FOS, JUN, and eventually cyclins -one gene produced is MKP-1 - a phosphatase that removes phosphate from ERK, introducing negative feedback mechanism to inactivate ERK so that transcription of these genes stops & cell division is not totally continuous |
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Mutation in Ras genes
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-mutation in one of the Ras genes that leads to tumor formation prevent the protein from hydrolyzing the bound GTP back to GDP form and as a result, the mutant version of Ras remains in the "on" position sending a continuous message downstream along cascade keeping the cell in proliferative mode
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Oncogenes |
-genes that produce proteins that produce cancer when they're no longer normally responsive to regulation
-all the genes/proteins in the Ras-MAP kinase cascade would be considered oncogenes if they had a mutation -if proteins in the cascade were not responsive to the protein above it, this would result in uncontrollable cell division -problem arises if oncogenes are present and apoptosis isn't working |
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Apoptosis (what are phagosomes)
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-final safeguard for genes dividing uncontrollably -controlled cell death so we don't have harmful substances spill out & cause inflammation -cells undergoing apoptosis show cell blebbing -phagosomes: remnants of cells from cells that have died by apoptosis, engulfed by macrophages through phagocytosis *Note that our cells die by necrosis |
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CED-3 gene |
-mutation in this gene of worm means that cells won't die -when CED-3 gene in C. Elegans is mutated, the cells that are supposed to die don't -CED-3 gene is key player in apoptosis -homologs of CED-3 gene in both humans & animals are called caspases and are necessary for apoptosis |
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Caspases
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-cysteine proteases -caspase-3 is an executioner caspase - cleave proteins that are important for the cell to live -cleave proteins like... (1) Protein kinases (FAK, PKC, Raf) - to block cell division pathway (2) Lamins - lamins are intermediate filaments attached to inside of nuclear envelope and hold the nucleus shape - if lamins are destroyed, nuclear membrane falls apart (3) Cytoskeleton - causes blabbing -caspases activate endonucleases - a "caspase activated DNAse" (CAD) --CAD will cut DNA at specific lengths/same lengths --testing for the result of DNA getting cleaved by DNA: extract genomic DNA from cells undergoing apoptosis - run through gel electrophoresis - long DNA won't spread but lysed cells will show DNA laddering b/c DNA cut in same size sections (multiples of same amount of DNA) |
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Necrosis
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-trauma causes necrosis to occur -necrosis is the death of cells in organs/tissue due to disease, injury, or failure of blood supply |
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Apotosis: extrinsic vs. intrinsic pathway |
Extrinsic pathway -triggered by something from outside the cell -mediated by a transmembrane receptor that reacts to an extracellular signal -ligand: tumor necrosis factor Intrinsic pathway -genetic damage, oxidative stress, high cytosolic calcium, high temperature inside the cell, lack of growth factor (usually necessary for cell to survive) -these all trigger apoptosis from inside the cell |
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Extrinsic pathway for apoptosis |
-stimulus for apoptosis carried by extracellular messenger protein tumor necrosis factor -tumor necrosis factor (TNF) (ligand) is a trimer - part of group of ligands called cyclokines -the cell is ready to receive the signal for apoptosis to occur -tumor necrosis factor receptors (death receptors) present as pre-assembled trimers -cytoplasmic domain of each TNF receptor contains a death domain -caspases in cells are called procaspases until they are activated |
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Apoptotic extrinsic pathway process |
1. When TNF binds to TNF receptor, a conformational change in the receptor's death domain occurs 2. Adaptor proteins TRADD and FADD interact with one another by their death domains (the DD in the names stands for death domain) 3. Procaspase-8's (full length caspase) (bound to TRADD and FADD) are brought into close proximity to one another so they recognize each other and cleave one another to make activated caspase-8 4. Initiator caspase-8 has full proteolytic activity and will cleave executioner caspase, caspase-3, to activate it 5. Caspase-3 will cleave proteins and cleave endocnucleases and apoptosis will occur |
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Intrinsic pathway of apoptosis |
-responds to many different things -mitochondria are key to this process - they act as buffers for calcium and as sensors for cellular environment 1. Sensors detect something wrong going on in the cell 2. Activation of Bcl-2 proteins occurs and cytochrome-C is released from mitochondria 3. Cluster of cytoplasmic factors (ex. Apaf-1), procaspase 9 and cytochrome C come together, and procaspase-9 is activated to caspase-9 and formation of initiator caspase-9 complex (apoptosome) *note that caspase-9 does not need proteolytic cleavage to become activated but rather just joining the multi protein complex, apoptosome, activates it 4. The apoptosome activates executioner procaspases into active executioner caspases which then leads to apoptosis |
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What happens to the mitochondria during the intrinsic pathway if survival factor is present or no internal damage has occurred? |
-the protein "Bad" gets phosphorylated -"Bad" cannot bind Bcl-2 or Bcl-XL so then Bcl-2 Bcl-XL inhibit the opening of channel called Bax -no cytochrome C is released and so no apoptosis occurs |
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What happens to the mitochondria during the intrinsic pathway if survival factor is NOT present or internal damage has occurred? |
-no phosphorylation of "Bad" protein -Bad binds to Bcl-2 and Bcl-XL so Bax channel opens -cytochrome C is released and thus apoptosis occurs *the release of cytochrome C is an event that irreversibly commits the cell to apoptosis |
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Significance of BH domain |
-there is a BH domain in all the proteins (Bad, Bcl-2, Bcl-XL, Bax) involved in the intrinsic pathway of apoptosis activation -all said to be members of the Bcl-2 family -Bad and Bax are pro-apoptotic Bcl-2 family proteins - promote apoptosis -Bcl-2 and Bcl-XL are anti-apoptotic Bcl-2 family proteins - protect cells from apoptosis |
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Initial knowledge of the eukaryotic cell cycle |
-M phase and Interphase -Interphase composed of G1, S, G2 -G1: cell grows, carries out normal metabolism, organelles duplicate -S: DNA replication, chromosome duplication -G2: cell grows & prepares for mitosis -Go phase: exit of the cell cycle -decision for cell to copy itself is made at the end of G1 -G1 and G2 are gap phases -G2 prepares the cell for mitosis -cell undergoes mitosis followed by cytokinesis in most cases but there are cases where cells replicate nuclei & do not divide |
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Quiescent cells
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-terminally differentiated -in Go phase -have exited the cell cycle -quiescence is a state of inactivity |
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Experiment that first allowed us to view cells in the M phase or Interphase (using a microscope) |
Experiment 1. Treat an asynchronous cell culture with hydrogen 3 thymidine for 30 minutes -- hydrogen 3 thymidine only incorporates into replicating DNA 2. Autoradiography Observations -hydrogen 3 thymidine never incorporates into cells during mitosis (dark silver grains never appeared in cell undergoing mitosis) - this tells us that DNA is not being replicated during mitosis -only a fraction of the cells in interphase are labelled - this tells us that there are portions of interphase where DNA replication is not happening -conclude that there is a definite period of time between the end of DNA synthesis & beginning of M phase (G2 phase) |
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Fusion experiments: fusing a cell in G1 + cell in S phase |
-the DNA from the cell in G1 starts to replicate right away -this tells us that because of these diffusible signals, G1 entered the S phase Conclusion: diffusible factor initiates S phase |
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Fusion experiments: fusing a cell in G1 + a cell in M phase |
-nucleus in G1 cell skips G2 and S phase because the nucleus thinks it's in the middle of mitosis -results in premature chromosomal compaction of the chromatin at the G1 phase nucleus -diffusible factor tells nucleus it should be in mitosis -- condenses DNA, nucleus begins to fall apart |
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Maturation Promoting Factor (MPF) |
-key diffusible factor for initiating the M phase -MPF = kinase + cyclin -kinase: cyclin-dependent kinase (Cdk) - only active when there is a sufficient amount of cyclin around -cyclin: regulatory component, increases & decreases throughout the cycle -high MPF amount triggers mitosis -if MPF activity decreases, mitosis stops -high cyclin = high MPF activity -when cyclin conc. is low, the kinase lacks cyclin subunit and is inactive -when cyclin conc. rises, the kinase is activated (thus the MPF is activated) causing cell to enter M phase |
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How was the MPF identified? |
-yeast was used -it was exposed to a mutagen -temperature sensitive mutants were identified where proteins work normally but then they lost their function if temp. is increased -when mutated, a gene would cause growth of cells at elevated temperature to stop at certain points in the cell cycle -the product of this gene was cdc2 in fission yeast and CDC28 in budding yeast -cdc2 was eventually found to be homologous to the catalytic subunit of MPF |
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Regulation of the cell cycle in S. pombe (fission yeast) |
-different checkpoints where the cell decides to proceed with phase -checkpoints are dependent on different cyclins -"START" present after G1: cdc2 kinase initiates S phase when combined with G1 cyclins, once a cell passes START it is committed to replicating its DNA and ultimately completing cell cycle -2nd checkpoint at end of G2: cdc2 kinase initiates M phase when combined w/ mitotic cyclins -3rd checkpoint in middle of mitosis: where cell can exit the cell cycle and enter Go phase OR it can re-enter G1 or the next cell -MPF in yeast = cdc2 kinase + mitotic cyclins |
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Step 1 of Cdk regulation by phosphorylation |
-cell is in G2 -mitotic cyclins NOT high enough -2 sites on cdc2 kinase that get phosphorylated and regulate activity -as cell gets closer to beginning mitosis, the amount of mitotic cyclins increases -cdc2 kinase associates with cyclin -CAK (Cdk-activating enzyme) phosphorylates threonin 161 on the cdc2 kinase -Wee1 phosphorylates tyrosine 15 on the cdc2 kinase ---- this gives the cell a break where the DNA integrity is checked |
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Step 2 of Cdk regulation by phosphorylation |
-phosphatase cdc25 removes phosphate from tyrosine 15 allowing the cell the enter mitosis -cdc2 kinase + cyclin now an active MPF -cell undergoes mitosis |
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Step 3 of Cdk regulation by phosphorylation |
-phosphate from threonine 161 is removed -cyclin degrades -Cdc2 kinase and cyclin dissociate - now inactive MPF |
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Overall process of Cdk regulation by phosphorylation |
1. During G2, enzyme Wee1 phosphorylates tyrosine 15 on the cdc2 kinase and a separate kinase CAK phosphorylates threonine 151 2. During G2, the cdc2 kinase interacts with mitotic cyclin but remains inactive due to the phosphorylation of tyrosine 15 (i.e. effect of Wee1 overrides the effect of CAK, keeping the Cdk in inactive state) 3. When cell reaches critical size, enzyme Cdc25 phosphatase is activated and removes the inhibitory phosphate on the tyrosine 15 4. Resulting activation of cdc2 kinase drives cell into mitosis 5. At end of mitosis, stimulatory phosphate group is removed from threonine 161 by another phosphatase 6. Free cyclin is degraded, MPF inactivate, and cell begins another cycle |
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Mutants |
Mutant Wee1 gene: -mutants cannot maintain the Cdk in inactive state so divide at an early stage in the cell cycle producing very small cells -if kinase Wee 1 is mutated, tyrosine 15 does not get phosphorylated so the break in the cycle and the phosphatase cdc25 step is skipped -phenotype: 2 smaller cells Normal (wild-type) cells: -Wee1 keeps Cdk inactive until end of G2 and then at end of G2, inhibitory phosphate at tyrosine 15 is removed by phosphatase cdc25 -removal of phosphate switches the stored cyclin-Cdk molecules into active state -cyclin-Cdk now phosphorylates key substrates and drives cell into mitosis -phenotype: 2 regular cells w/ equal divisions Mutant cdc25 gene: -mutants cannot remove inhibitory phosphate from Cdk and so cells cannot enter mitosis -phenotype: cell does not divide but continues to grow so result is a bigger cell than what started with |
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G2 checkpoint (what happens if there is more or less Wee1 than Cdc25) |
More Wee1, Less cdc25: -more phosphorylation of tyrosine 15 -no mitosis -time for cell growth & to correct mistakes in DNA replication Less Wee1, more cdc25: -MPF activation -proceed with mitosis |
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What would happen if a single yeast strain had mutations in both Wee1 and cdc25? |
Resulting fission yeast cells would be smaller than normal |
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Other regulators of Cdk activity |
-additional kinases and phosphatases -controlled proteolysis - ubiquitin proteasome pathway, APC -subcellular localization - cyclin B1 accumulates in the nucleus |
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Anaphase Promoting Complex (APC) as regulatory of Cdk activity |
-ubiquitin ligases -exit of mitosis to G1 -complexes recognize proteins to be degraded and link these proteins to a polyubiquitin chain ensuring their destruction in a proteasome -APC complex acts in mitosis and degrades may key mitotic proteins |
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Subcellular localization as regulator of Cdk activity |
-process where cell cycle regulators are moved into different compartments at different stages -cyclin B1 (mitotic cyclin) accumulates in the nucleus -cyclin B1 shuttles between the nucleus and cytoplasm until G2 when it accumulates in the nucleus just prior to the onset of mitosis |
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Where did we get our identification of checkpoints from? |
-arrived due to study of disease called AT (ataxia-telangiectasia) -people with this ease get tumors more easily -doctors noticed that radiation would reduce tutors in normal patients BUT in AT-diagnosees the tumors would increase due to radiation -the gene responsible for AT encodes a protein kinase that is activated by certain DNA lesions (double-stranded breaks) |
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Proteins that determine if DNA was fixed properly during the cell cycle |
-ATR and ATM -both proteins are capable of binding to chromatin that contains damaged DNA -once they are bound, they can phosphorylate proteins that participate in cell cycle checkpoints and repair DNA |
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ATR |
-in G2 -activated when a cell preparing to enter mitosis is subjected to UV radiation -ensures completion of S phase and that DNA replication was complete -ensures no mistakes are present in the base-pairing -when ATR is active and is repairing things, it activates a kinase called Chk1 which phosphorylates Cdc25 -when Cdc25 is phosphorylated, it gets caught outside the cell by adaptor protein (b/c the Cdc25 was shuttling back and forth across the membrane due to import & export signals) -the Cdc25 getting caught by adaptor protein inhibits Cdc25 phosphatase's activity and prevents it from being reimported into the nucleus -absence of Cdc25 from nucleus leaves the Cdk in an inactive state and the cell arrested in G2 (Cdc25 also removes the phosphate from tyrosine 15 to active MPF and start mitosis, so its absence would mean mitosis doesn't occur) |
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ATM |
-in G1 -repairs DNA damage -identifies the double-strand break due to ionizing radiation -activates a kinase called Chk2 which phosphorylates p53 stabilizing it -when p53 is stable, it acts as a transcription factor to make p21 gene -p21 gene sticks to Cdk which inactivates it and puts the cell cycle in arrest (p21 is a Cdk inhibitor) *note that approximately 50% of all tumors show evidence of mutations in the gene that encodes p53 |
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Mitosis |
-continuous process -during M phase, all the cell's energy is devoted to mitosis -there is NO transcription or translation -all the proteins needed for mitosis are made BEFORE mitosis actually begins -there is NO response to external stimuli |
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Prophase |
-early stage of formation of mitotic chromosomes -chromosome compaction into sister chromatids -duplicated chromosomes are prepared for segregation and mitotic machinery is assembled |
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Condensin |
-condensin is a protein that is activated by MPF phosphorylating it -located in the centre of looped domains of chromosomes -allows chromosomes to maintain their X shape |
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Cohesin |
-prior to replication, the DNA of each interphase chromosome becomes associated with cohesin and following replication, cohesin holds the 2 sister chromatids together through G2 and into mitosis when they are ultimately separated -protein that keeps the 2 copies of homologous chromosomes loosely together -responsible for primary constriction at the centromere -after prophase, cohesin left at the site of primary constriction and is the centromere |
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Centromere |
-constricted region on chromosome -contains cohesin protein -provides sites for kinetochores -do not bind the microtubules |
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Kinetochore |
-2 kinetochores for every centromere - 1 for each of the sister chromatids -kinetochore is a protein complex that assembles at the centromere -attachment for mitotic spindle -located at the outer surface of the centromere of each chromatid -3 components: inner plate, interzone (space), and outer plate -fibrous corona - fibers projecting from the outerplate -outer kinetochore: microtubule binding, microtubule motor activity, signal transduction -inner kinetochore: centromere replication, chromatin interface, kinetochore formation |
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Fibrous corona on kinetochore |
-fibrous corona fibres have protein attachments that interact with microtubule: (1) motor proteins for microtubules: CENP-E/Kinesin 7 (moves to plus end), dynein (moves to minus end) (2) depolymerase: functions in depolymerization of the microtubule |
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Centrosome cycle (Cyclin E-Cdk2, asters) |
-during prophase, cytoskeleton getting completely rearranged -during G1, centrosome contains a single pair of centrioles that are not as tightly associated as they were in mitosis -right after G1 and just before S phase, duplication & organization of centrosome occurs -each centriole has its own centriole come out at 90 degrees (2 centrioles forms a centrosome) -each centrosome migrates to opposite poles of the cell -when centrosomes begin to migrate to opposite poles, asters form around it (asters = early mitotic spindle) Cyclin E-Cdk2 (G1 cyclin) - responsible for G1 regulation, initiation of centrosome duplication at the G1 to S phase transition |
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Summary of prophase |
-first stage of mitosis -duplicated chromosomes are prepared for segregation and mitotic machinery is assembled -chromosomes condense with help of activated condensin (aka chromatin compaction) -cytoskeleton is disassembled and the mitotic spindle is assembled -nuclear envelope disperses and the ER and Golgi fragment |
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Prometaphase |
-microtubules attach to the kinetochores -congression occurs - where chromosomes move to be oriented at the cell equator |
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Congression (in prometaphase)
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-kinetochore makes initial contact w/ sidewall of microtubule and chromosomes move along microtubule powered by motor proteins located on corona fibrous fibres -mono-oriented: under no tension, chromosome attaches to kinetochore that is attached to microtubule form only one spindle pole -bi-oriented: under tension, both sides of chromosome attached to kinetochore attached to microtubule from both sides -tension applied to microtubules allows chromosomes to separate -syntelic attachment: no tension, microtubules fro same centrosome, no tension so cell not supposed to divide |
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Metaphase |
-cell waiting for chromosomes to get nicely aligned -chromosomes do align along the metaphase plate attached by microtubules to both poles |
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Mitotic spindle components (3) |
Astral fibers - radiate outward from centrosome, help position spindle apparatus in cell, help determine plane of cytokinesis Chromosomal spindle fibers - extend b/w the centrosome and kinetochores, exert force, interact directly with chromosomes Polar spindle fibers - extend from centrosome past chromosomes, determine cell shape, allow chromosomes to get pulled apart *all spindle microtubules have their minus end pointing towards the centrosome |
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Anaphase |
-rapid, synchronized pulling apart of all the chromosomes -cohesin gets destroyed, centromeres split, chromatids separate (during metaphase, the chromosomes are under tension but when cohesin gets dissolved, the chromosomes are able to separate quickly) -chromosomes move to opposite poles -spindle poles move further apart |
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Anaphase Promoting Complex (APC) |
-APC is an E3 ubiquitin ligase - adds ubiquitins to mark protein for destruction |
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APC + Cdc20 |
-APC must be combined with adaptor protein Cdc20 to trigger anaphase -APC + Cdc20 adds ubiquitin ligases to securin to be destroyed by proteasome (labels securin for destruction) -separase (which is usually inhibited by securin) can now destroy cohesin -allows anaphase to happen - cleavage of cohesin triggers the separation of sister chromatids to mark the onset of anaphase |
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APC + Cdh1
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-near the end of mitosis, Cdc20 is inactivated and APC associates with Cdh1 -when APC combined with adaptor protein Cdh1, it adds ubiquitin ligases to mitotic cyclins so they are destroyed -when mitotic cyclins are destroyed, Cdk cannot function anymore so all of the proteins that are usually phosphorylated by MPF will now be dephosphorylated and the cell will exit to G1 - enter telophase -proteins will be dephosphorylated -cell will go back to G1 -APC-Cdh activity during early G1 helps maintain the low cyclin-Cdk activity |
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Spindle assembly checkpoint |
-the unaligned chromosome contains Mad2 protein -during mitotic entry, inactive Mad2 is recruited to kinetochores and activates - while active it is able to bind Cdc20 -when Cdc20 bound to Mad2, APC complexes are unable to ubiquinate securin thus keeping all of the sister chromatids attached to one another by cohesin -Mad2 signals to the APC + Cdc20 when all chromosomes are correctly attached -chromosomes must be bi-oriented - under tension, both kinetochores attached to spindle from both sides -Mad2 sequesters Cdc20 adaptor protein meaning APC cannot combine with Cdc20 so chromosomes will not be lined up at metaphase plate |
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3 checkpoints that precede 3 commitment points |
(1) G1 checkpoint - ATM ---> START - G1 to S transition, G1 cyclins + cdc2 kinase (2) G2 checkpoint - ATR ---> Entry into mitosis - MPF (3) Spindle assembly checkpoint - Mad2 ---> Anaphase - APC + Cdc20 |
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Anaphase A and B |
-occur at the same time -cause things to happen simultaneously -cell really elongates so that new cells have enough plasma membrane -Ndc80 complex attaches chromosomal spindle fibres to kinetochore - acts as a tension sensor -Mad2 interacts with Ndc80 to sense tension of chromosomes - if there is tension then Mad2 is inactivated which releases Cdc20 to associate with APC and therefore trigger anaphase |
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Anaphase A |
-tubulin subunits removed from the plus end and minus end from the chromosomal fibres -chromosomes move apart -dynein walks toward minus end (minus end is facing centrosome) |
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Anaphase B |
-tubulin subunits are added at the plus end (at a high rate) and removed from the minus end from the polar fibres -poles move apart -results in cell elongation -net addition of tubulin subunits to plus end of polar microtubules -the fibres overlapping interact with each other -kinesin 5 (double kinesin: has feet on both sides so it could walk either direction) causes fibres to separate & cell elongation -addition of subunits so spindle gets longer while kinesin walks along |
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Telophase |
-chromosomes cluster at opposite spindle poles -chromosomes disperse (they decondense) -nuclear envelope reforms - because lamina B not phosphorylated - reforms around chromosome clusters -Golgi and ER reform -daughter cells return to interphase condition -daughter cells formed by cytokinesis -mitotic spindle disassembles |
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Mitotic movements |
Microtubule motors: Kinesin like proteins (+ end motor) -CENP-E/kinesin 7: involved in congression, walks along chromosomal fibres -kinesin 5: involved in anaphase B, walks along polar fibres Minus end motor -dynein: involved in anaphase A KINESIN-LIKE PROTEIN (DISASSEMBLY) -kinesin 13: depolymerase, disassembles microtubules at the minus end causing loss of subunits at the minus end, at the kinetochore, depolymerizes chromosomal spindle while dynein walks toward the minus end |
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Summary of polar microtubules |
-contribute to keeping the poles apart -elongate the spindle (and whole cell) during anaphase B |
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Summary of chromosomal microtubules |
-important during congression (pro metaphase) -maintain chromosomes at the metaphase plate (metaphase) -separate chromosomes (anaphase) |
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Prometaphase movement |
-structural MAPs not active/associated with microtubules so microtubules consistently build and then fall apart -kinetochore binds to side of microtubule and then orients to be 90 degrees -kinesin 5 walks towards plus end - chromosome movements away from poles mediated by kinesins -dynein walks toward minus end and kinesin 7 walks toward plus end so movement in 2 direction until microtubules positioned 90 degrees to the kinetochore - poleward chromosome movement mediated by dynein -chromosomes moved to the centre of the spindle |
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Metaphase movement |
-2 halves of spindle maintain their separation as a result of continued plus end directed motor activity associated with polar microtubules -chromosome is bi-oriented -chromosomes maintained at metaphase plate by balanced activity of plus end & minus end directed proteins residing at the kinetochore -congression occurs -subunits aded to plus ends of microtubules during metaphase |
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Anaphase movement |
-kinesin 5 enables anaphase B (elongation of cell) -dynein and kinesin 13 (depolymerase) enable anaphase A -subunits removed from plus ends during anaphase resulting in shortening of chromosomal fibres -chromosomal spindle fibers undergo depolymerization at both minus & plus ends -depolymerization at minus end transports chromosomes towards the poles and depolymerization at the plus end causes tubule to roll up while still towing the chromosomes -movement of chromosomes toward poles thought to require activity of kinesin depolymerases that catalyze depolymerization at both the plus and minus ends of microtubules -separation of poles (anaphase B) due to continuing activity of plus end directed motors of polar microtubules |
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Cytokinesis |
-formation of cytokinetic/cleavage furrow where the cell ultimately divides |
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Contractile ring theory in cytokinesis |
-thin band of actin that forms at equator between 2 poles -actin filaments become assembled in a ring at the cell equator -myosin II underneath the actin -contraction of the ring, requiring the action of myosin, causes formation of furrow that splits the cell into two -cortex: space right beneath the plasma membrane -tons of actin right at the cleavage furrow -force exerted so cells get separated - force generated in a thin band of contractile cytoplasm located in the cortex just beneath the plasma membrane of the furrow |
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Abscission |
-final step in cell separation -dependent on the midbody -midbody: dense structure present in intercellular bridge between two daughter cells -midbody used to be thought of as a remnant of cytokinesis to be discarded/junk but it is actually very important for the actual separation of cells |
