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Final

Biology 2382B Final: Cell bio final study guide (all lectures)

35 Pages
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Department
Biology
Course Code
Biology 2382B
Professor
Robert Cumming

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(Midterm 1 Material) Lecture 1: Cell Cultures • Technique used to grow cells under strictly controlled conditions—fundamental technique for understanding biology • Sticky proteins stick cells together—when you take it out of an organism, must mechanically or metabolically break it up o Breaking down cell-cell and cell-matrix interactions (mechanical fragmentation, trypsine, EDTA) • Growth factors help them grow (amino acids, vitamins, salts etc) • Primary cell culture: Contact inhibition (monolayer “carpet when grown”) o refers to cells taken directly from an organism, these cells usually divide a limited number of times o Cell line: refers to cells that are transformed and can grow indefinitely (less contact inhibition) Ex. HeLa cells: could passage indefinetly—imortilized. Most cell lines come from cancers • Morphology of normal vs. transformed fibroblasts (3T3 mouse fibroblasts transformed with rous sarcoma virus) o Normal: ▪ -Elongated, aligned, orderly packed, grow in parallel arrays, contact inhibition o Transformed: ▪ -Rounded, hairlike processes, disorganized, grow on atop the other, loss of contact inhibition Symmetric and Asymmetric Cell Division • Occurs during development and in adult cells • Symmetric Division ▪ Mother cell will divide and create 2 identical daughter cells (same type of cell) ▪ simple eukaryotes (yeast) • Asymmetric ▪ Mother cell divides and creates 2 phenotypically different daughter cells ▪ Progenerator cell ▪ Cell type foes not contribute to function of tissue. Gives rise to cells that have very specific functions ▪ *Cell fate: potential of cell to divide. Embryonic Stem Cells • Pluripotent (capable of giving rise to different cells) and can be indefinitely in culture under appropriate conditions (invetro) • Can also be induced to differentiate into precursors for various cell types o after a repeated number of cell divisions a blastocyst forms which is then plated with ibroblast feeder cells Adult Stem Cells • Most tissues contain adult stem cells, they are required to repair tissue and are capable of generating limited number • of different cells • Located in a stem-cell-niche (adjacent cells which provide signals to self renew or differentiate • Adult stem cells are more limited (ex. Intestinal epithelial cells) • self-renew and divide into 2 identical stem cells (symmetric division) or can participate in asymmetric division • Must preserve balance between renewal and degradation—aging compromises ability to self renew Induced Pluripotent Stem Cells (iPS cells) • reprogramming of fibroblasts by introducing Yamanaka (transcription) factors into differentiated cells: Oct4, Sox2 and Klf4 and one • frequently expressed in cancer cells (c-Myc) • Could recreate stem cells if they are able to differentiate/reverse them back to a tissue that can replace dead tissue • Safety concerns: might be giving a person cancer because of the use of a retrovirus • potential future of medicine—no genetic intervention • Medical Applications o Usually when people have inherent forms of a disease, there is more that one gene associated with the disease o With the transformed skin biopsy—Disease specific drugs created or creation of healthy cells to transplant o Take a cell from person with Parkinson’s and recreate the neurons using iPS, allowing for tests to be conducted and medications to be trialed o Specifically correct the mutations and place them back into the individual ▪ Patient specific treatment—less likelihood of rejections since the cells are coming from the same person ▪ Possible but safety concerns are thee Isolation and Analysis of Cell Organelles and Molecules Labelling live cells with fluorescent antibodies or stains • Antibodies made against specific cell surface proteins can be linked to flurophores • Used in fixed and live cells (don’t go inside cell, but can bind to anything the recognize on the extracellular domain) • Membrane permeable fluorescent dies can be used to label intracellular strain binds ex. Hoeschst strain binds in DNA nucleus FACS (Fluorescent activated cell sorting) • Solution of cells are transported through tubes that get progressively narrower until they can go through one at a time • Lazer illuminated the sample that can then be detected by a light. Filters filter out unwanted light • Cells with different electric charges are separated by an electric field and collected • Can quantify cells—more fluorescence = more proteins • Cell Cycle analysis o Cells that have their replicated but not divided (G2) will have twice (2x) the Hoechst stain fluorescence intensity than non dividing cells o Addition of an agent that stops s-phase would result in 1 hump in the graph rather than 2 Isolating cell organelles 1. Disruption of cell plasma membrane a. Mechanical Homogenizatio: squishes cells to the side of tube b. Sonication: Break apart cells (not gently) c. Pressure: Rupture as forced down tube d. Detergents: Completely break apart membrane and create holes e. Hypotonic solution: high salt concentration, ruptures as it swells bc of osmosis 2. Centrifugation of cell homogenate a. Differential i. Spining homogenate yields pellet and supernatant. Increasing centreifical force at each stage to isolate organelles based on mass. Pour out as you go b. Equilibrium density-gradient i. Seperation based on density. Homogenate is applied to a gradient of sucrose. High speed/several hours, the organelles migrate to the sucrose layer equal to their own density. Seperating proteins from organelle • Non ionic detergents: Good at disrupting membrane, but will disrupt the cell not the structure of the protein • Ionic: disrupts entire cell and denatures protein (negative charge) SDS-PAGE • Electrophoretic separation of proteins—Polyacrylamide gel electrophoresis • Carried out in the presence of negatively charged detergent • SDS is denaturing and binds to and destabilizes the hydrophobic side of chains within the core of proteins. • Polypeptide chains are forced into extended negatively charged conformations with similar charge to mass ratio ▪ Small protein: faster ▪ Large protein: slower Western Blotting (immunoblotting) • Laterally transfer gel onto a membrane • Apply electric current which allows for the negatively charged gel to transfer • Quantify by exposing blot to radiation of imaging device with camera that takes digital image of blot. o Antibody detection—chromogenic detection • Dark blots—inverted image from what is seen from digital image • More hybridization= darkened band/more protein • Loading control: probing blot with antibody that doesn’t vary Protein Synthesis and Transport Targeting/ Sorting • Targeting: proteins with specific destination (organelle) o During or after synthesis • Sorting: Direct proteins to secretory pathway (ER, golgi, lysosomes) o Inside to out, must go through stages • (Non Secretory pathway) synthesised by cytosolic ribosomes (remain in the cytosol) and are targeted to intracellular organelles (specific signal sequence) • (Secretory pathway) synthesized by ribosomes which are attached to the ER (making it the rough ER) Basic Mechanisms of protein targeting • Signal sequence – receptor for signal sequence—translocon channel—energy source ER structure • Uninterrupted membranus tubules with vesicles sepeated from cytoplasm • RER has ribosomes on the tubules (cisterna which are stacked) • ER extend from nuclear membrane Secretory Proteins Enter ER lumen • Sythetic way of reproducing the ER: secreted proteins must go through ER first o Split sample into 2, ½ detergent punches holes, other ½ protease o SDS page= saw bands, membrane (no detergent) would not allow protease in and proteins were protected ER functions • Secreted and membrane proteins are sorted through RER • Sugars/carbs are added to the polypeptide and disulfide bonds are formed • Proteins are folded by chaperones Translocation • Occurs simulatiously with translation • Protein cant be synthesize and folded in the cytoplasm and then placed in the ER . • Translocation occurs simultaneously. • In the cytosol translation begins—signal sequence is translated which sends the protein to the translocon. Ribosome inserts inself and continues to translate the protein inside the ER. o Amino terminal signal sequences of newly initiated polypeptide (nascent proteins) ▪ SRP’s (signal recognition particle) o SRP receptor embedded in er membrane (translocon: protein channel) o Cleavage site where signal sequence is cut by a signal peptidase • SRP binds to ribosome, then binds to SRP receptor and phosphorylates, opening the translocon. SRP releases from receptor and hydrolysizes to GDP. Signal peptidase cleaves the signal sequence, and protein continues to synthesize inside the ER lumen and fold. The ribosome is then cleaved. Glycosylation in ER • Enzymatic transfer of 14—residue oligosaccharide precursor onto a doichol charrier which is then transferred to ASPARAGINE (Asn) • Proteins with attached carbohydrates are known as glycoproteins • Glycosolation functions: protein folding, confer protein stability, cell adhesion (make sticky) Protein Folding • When a protein is folded: outside is hydrophobic and inside is hydrophilic • Several proteins contribute to proper folding in the ER: o BiP, Calnexin and Calreticuli and PDI • Binding of these proteins is bellied to prevent misfolding or aggregation of nascent proteins. • Only folded proteins are transported from ER to Golgi Import into Mitochondrial Matrix • Mitochondrial targeting sequence—20-50 amino acids at n terminus • Precursor protein is energized, cytosolic Hsp70 sends it to the matrix of the mitochondria. o Binds to Tom 20/22 receptor and is enters intermembrane space through Tom40. Tom44, sends the protein from the IMS to the mitochondrial matrix. o Matrix Hsp phosphorylases. MPP cleaves target sequence Vesicular Traffic of Proteins After ER • Proteins go to the golgi apparatus (intermediate structure between ER and plasma membrane) Golgi Complex • Consists of flattened disk like cisternae with no ribosomes. • Vesicles at the end of cisternae fuse or pinch off • Cis (faces ER) Media (middle) Trans (Opposite RER, middle of bowl) Transport Vesicles: Budding and fusion • Part that sticks out will be recognized by coat and GTP binding proteins—important for vesicle formation • Causes protein to bend and pinch to form vesicle • Snare proteins help fuse the two membranes together (snare protein on vesicle and target protein, they will interact together) • COP1 Vesicles—RER to Golgi (anteriograde transport) • COPII Vesicles—Golgi to RER (Retrograde transport) • Clathrin Vesicles – Transgolgi to late endosomes GTP Binding proteins control assembly and disassembly • Protein that we want to transport from er to golgi—Coat protein on then off • Membrane associated GTP binding proteins promote the association of COPII coat proteins on ER membrane • Once COPII are released from donor membrane, hydrolysis of GTP occurs which triggers disassembly of coat proteins RER to cis-Golgi Transport • COPII mediate anterograde transport. o GTP binding proteins control assembly and disassembly of coat proteins as well as docking vesicles to target membrane • Certain cargo membrane proteins use DXE sorting signal which is recognized by COPII proteins • ATP hydrolysis is required for disassociation of SNARE complexes • COPI mediates retrograde transport (Golgi to RER) Cystic Fibrosis • Recessive genetic disease • Characterized by abnormal transport of chloride and sodium across epithelium leading to thick viscous secretions • Caused by mutation in the gene for the protein CFTR. Mutation causes signals to not be expressed and CFTR to be retained in the ER. COPII proteins cannot bind to the CFTR and it is degraded in the ER • If It can get to the apical cell membrane—chloride channels work perfectly—therapy is attempting to get signals to membrane Cis-Golgi to RER Transport • KDEL sorting signal (receptor is pH sensitive) Lower pH is optimal for binding of the KDEL receptor. • Golgi reroutes soluble ER-resident enzymes back to ER. Proteins bare a C-terminal KDEL sequence (signal) • KDEL receptor has a C-terminal KKXX sequence ( cytosol facing) which bind to COPI coat proteins. • Luminal Proteins including chaperons and lectins have KDEL sequences. Protein Glycosylation in Golgi • Some proteins will be secreted which are important for cell adhesion (sticky) • Golgi sub-compartments differ in the enzymes they contain o Some are Glycosidases (remove) and some are glycosyltransferases (adding) Trafficking from trans-Golgi network (TGN) • Directly to lysosome—or—late endosome to lysosome • COPI Vesicles (retrograde) o From Trans golgi network to trans golgi • Adapter Protein o From TGN to lysosome • Clathrin Coated Vesicle o From TGN to late endosome o Forms structure called triskelion—which is important for forming clathrin coated vesicles TGN to Lysosomes • M6P sorting signal o M69 Sorting signal – carbohydrate residue o Targets soluble proteins to the lysosome, targeting requires M6P receptor o M6P is added to lysosomal enzymes in the cis-golgi (must be a phosphate group on the sugar which is added through phosphorylation) o Receptor binding occurs at pH 6.5 • At low pH M6P detaches from receptor • M6P returns to golgi or plasma membrane • (most acidic is lysosome followed by late endosome) • Recycled either to PM or to trans golgi network • Clathrin coated vesicles • Receptor mediated Endocytosis: from PM M6P receptor on membrane, coat added and removed before reaching late endosome. • Constitutive secretion to PM also occurs Lysosomal Storage Diseases • Absence of one or more lysosomal enzymes resulting in accumulation of undegraded material in lysosomes • Inclusion-cell (I-Cell) disease o Absence of GlNAc Phosphotransferase which is what adds the phosphate to the precursor protein which later becomes MP6 – IE no MP6 Signal o Lysosome enzymes are secreted rather than sorted to lysosomes o Undigested glycolipids degraded by lysosomal enzymes (which should be sorted to the lysosome) accumulate within the lysosome • Clinical onset at birth—fatal Receptor Mediated Endocytosis Internalizing extra cellular materials • Phagocytosis: cells from outside ends up on inside • Pinocytosis: small example, invagination of small molecules—not selective • Receptor mediated endocytosis o Selective internalization of specific extracellular molecules (ligands) o LDL, transferrin and hormones ▪ LDL: 88% cholesteryl esters and mediates cholesterol transport. Receptors on plasma membrane (localized in clathrin coated pits) ▪ Lipids are transported in large water soluble complexes—Lipoproteins Electron Microscopy • Receptors in plasma membrane recognize the LDL • Clathrin coated pit helps internalize the ligand Clathrin/AP coated vesicles • AP1 (Trans golgi network), AP2(plasma membrane) • AP complexes recognize sorting signals of cargo proteins or receptors • Clathrin coated vesicles pinch off using dynamin and GTP hydrolysis pH-dependent binding of LDL particles to LDL receptor • LDLR has 3 domains o Short c-terminal cytosolic segment with sorting signal o Long N-terminal exoplasmic seqment with ligand binding domain and beta-propeller domain ▪ Nomal pH= ligand bidning arm binds tightly to ApoB ▪ At acidic pH (LDL is released), Histidine residues in beta propeller domain become protonated and bind with high affinity to negatively charged residues in the ligand binding arm. • ApoB mediates binding to LDL receptor o Specific sorting signal (NPXY) in the cytoplasmic domain of receptors binds to the AP2 complex that lines the pit (surrounded by clathrin) Acidification • V-class proton pumps transport H+ across the membrane via ATP dependent mechanism • Cl- channels are also present on the lysosome and endosome membranes • Anions passively follow the pumped protons resulting in acidification of the lumen • This allows for the disassociation of LDL from its receptor after is enters the late endosome Endocytic pathway for internalizing LDL 1. LDL receptors are concentrated around the clathrin coated pits and bind to ApoB 2. Binding to ApoB allows for the NPXY signal to recruit AP2 Complexes which allows for budding 3. Dynamin polymerizes and helps pinch off the bud from the membrane 4. The vesicle coat is shed (clathrin and AP2 complex) –turning into early endosome 5. pH in late endosome is lower which causes the LDL particle to separate from the beta-propeller domain and receptor. Receptor/protein is recycled back to the plasma membrane 6. Late endosome (with LDL particle) fuses to the lysosome and the LDL particles are broken down. Why Internalize LDL? • High cholesterol – LDLR mutations (No LDL receptor, receptor binds poorly or receptor can internalize LD) • Results in build up of cholesterol in arteries (plaque) – stroke/heartattack Transferrin Cycle • Ferrotransferrin is internalized and Fe3+ ions are released from apoprotein in late endosomes – receptor ligand complex doesn’t disassociate in late endosome like it does in the LDL cycle • Ferrotransferrin is recognized by transferrin receptor and is engulfed in a clathrin and AP2 coated pit. Dynamin GTP hydrolysis helps pinch of the bud • Coat is shed as it approaches the late endosome, and low pH causes release of Fe3+ from ligand • Complex (receptor and ligand) now apotransferrin is recycled to cell membrane Autophagy • Lysosomal/vacular degradative pathway this is conserved in eukaryotic organisms • Mediates the turnover of long-lived proteins and eccess or aberrant organelle o Important role in aging and disease • Autophagic Pathway o Cytosolic proteins and organelles are degraded by their enclosure in a double membrane vesicle (autophagosome) which fuses to lysosomes o Proteins are enclosed by atg12 atg5 and atg15. Atg8 lengthens the double membrane by adding lipids and closing the double membrane so that protein aggregates can be engulfed in a mature autophagasom. The double membrane then fuses with the lysosome • Loss of atg8 means that mature autophagasoms cant be formed, this leads to an accumulation of protein aggregates (clusters of misfolded proteins) • Over expression means that there is a consistent degradation of protein aggregates and consistent formation of autophagasoms • Atg5 is responsible for the initiation of the autophagic membrane—nucleation o Without it, mature autophagasoms would not be able to form o Overexpression, similar to Atg8, allows for increase in life span because there is a consistant availability of nucleated autophasomes Mitosis and Cell Cycle Control Phases of the cell cycle • G1: generalize growth and metabolism. Most cells arrest when not dividing (11hrs) • S: DNA replication (6-8 hrs) • G2: Preparation for chromosome segregation (4hrs) • M:Chromatin condensation, nuclear envelope condensation, sister chromatids attach to mitotic spindle etc. (1hr) • Cell cycle times vary between species Cell cycle regulation • Kinases • Phosphatases • Cyclins Functional Complementation • Screens for wild type gene that restores function of a defective gene in particular mutant o Temp sensitive o CDK is important for mitosis Control of G2 • Loss of Cdc2 (recessive) prevents S. pombe from entering m phase (continues to grow without dividing) • Cdc2 is transcribed and translated throughout cell cycle and is also a CDK • Cdc2 is homologous to Cdc28 • Mitotic Cyclin and CDK--- MPF mitosis promoting factor • Cdc2 in MPF is inactive for most of cell cycle, activity is low in s phase and peaks as M begins but then drops again • Cdc2 levels are equal during the cell cycle but activity fluctuates, at late G2 Cdc2 binds to cyclin and forms/activates MPF Wee1 and Cdc25 regulate MPF activity via phosphorylation • Wee1 is a kinase that phosphorylates CDK leading into is inactivation o Don’t want premature activation of MPF • Cdc25 is a phosphatase that removes inhibitory phosphate group from Cdk and promotes its activation, allowing for progression into cell cycle • Too much Wee1 = no mitosis (elongated cell) • Too little Wee1= premature mitosis (short) • Regulatin in S.pombe Relys on the phosphorylation of two residues in the catalytic CDK subunit – CAK (activating kinase) and Cdc25 (activating phosphatase) Active MPF • Chromosome Condensation (Histone H1 Condensin complexes) • Disassembly of nuclear envelope (Lamins, nucleoporis, nuclear envelope proteins) • Interphase microtubule disassembly and the mitotic spindle formation (MAP) • Remodeling golgi, ER which blocks vesicular traffic Inactivation of MPF • Inactivated at anaphase • Rapid mitotic cyclin degradation following polyubiquination – APC/c (anaphase promoting complex/cuclosome) – ubiquitin ligase • Cyclin contains a destruction box • Active APC/c recognizes destruction box and induces polyubiquitination, ubiquitin marks M- phase cyclins for proteolytic degradation in proteasomes • MPF activity drops and cell returns to interphase. (Midterm 2 Material) Section 1: The Cytoskeleton/ Microtubules The Cytoskeleton • 3 types of cytoskeleton proteins o Actin (smallest) o Intermediate Filaments (Ifs) o Microtubules (microtubulin) • Movement in cells is achieved through cellular trafficking of organelles and cell migration Microtubules • In a cytoskeleton are simpler than actin – largest component of cytoskeleton in terms of diameter • Made up of tubulin, dimers will polymerize and form microtubules • Can be longer/bigger than a cell—make up structures such as cila and flagella • Microtubules are made of tubulin o Alpha and beta tubulin – when they transfer the monomer it forms an alpha beta dimer o Almost never depolymerizes the dimer into monomers o End to end structure-- protofilament o A-b-a-b (Alpha is the minus end and beta is the plus end) – growth not charge • 13 protofilaments come together to form a hollow tube structure • Dimeric tubulin subunit o Very stable. Alpha subunit permanently binds to GTP while beta can hydrolyze GTP to GDP – as the polymer grows, GTP is hydrolyzed • Arrangment o Singlet microtubule : 13 protofilaments come together to form a ▪ found in most cells o Doublet Microtubule: 13 +10 ▪ Stable, do not polymerize/depolymerize o Triplet: 13+10+10 ▪ Stable, stay the same size ▪ Basal bodies and centrioles • Make up most of the cytoplasm (cytoplasmic) and can also extend out of the cell (axonemal) Microtubule Organization • MTOC: many different types, microtubules grow out of them, ex. Centrioles and basal bodies • Minus (-) end is always inside the MTOC, they are growing plus end away o Caps the minus end – cant grow or shrink • Exception: dendrites grow in a random fashion which makes transport more difficult • Centrosome o Two centroles for every centrosome. o Singlet microtubules extend from this o Have a role in the duplication of the centrosome o Plants don’t have centrioles o Made out of triplet microtubules (number of them in a circle) ▪ Stable, but divides o Centrioles are present but not in direct contact with the polymerizing microtubules • Gama tubulin: Forms a gamma tubulin ring complex o Gama and augmin help polymerize things from the MTOC o Provides nucleating sides for microtubules Polarity of Tubulin Polymerization • Flagellar nucleus—in context not an actual nucleus ( ▪ Nucleating agent: something that starts something else • Flagella are made up of doublet microtubules that are stable o Above critical concentration = polymerization occurs faster than depolymerization at the minus end o Minus end is still trapped by the MTOC Microtubule Formation • In a solution there are still dimers (constant amount) all new dimers are going into growing microtubules • A nucleus helps get to polymerized microtubule faster • Assembly and disassembly is important to their function –Cc and temp are also important. o o Microtubules disassemble when chilled to 4 C Dynamics • Meant to oscillate their lengths and change positions • By putting dimers in areas, rescue can occur, if the microtubules are in an area without dimers= catastrophy or disassembly • Polymerizing is tight, instability depends on the presents of a GTP beta tubulin cap o Once away from the plus end, the beta tubulin is in GTP form and cant grow o Adding subunits in GDP form= constant growth (alpha subunit is bound to GTP) o GTP hydrolysis to GDP weakens cohesion between protofilaments (fraying) o Hydrolysis is slower than growth. (GTP is hydrolyzed slower than GDP) • Smooth ends fray during depolymerization MT Disrupting Drugs • Colchincine: depolymerizes microtubules, normally in interphase they should be all throughout the cell, but when depolymerizes there is nothing • Taxol: Stabilizes the microtubules which stops growth. Also must depolymerize MT for mitosis to occur—cant divide = cell death MAPS (MT associated proteins) • Can o Alter MT stability o Bundle MT o Be regulated – phosphorylated o Promote disassembly (CDK in cell cycle) o Some MAPS contain MT binding domain and a projection domain • MAP2 and TAU- coat the microtubules and don’t allow for depolymerization/polymerixation unless the coats are removed o Required because some situations call for stable MTs • Binding Domain: part of the protein binds and some projects out o If bundled together there will be different space between them because TAU and MAP2 have different sized binding domains (projections are diff sizes, laterally MAP2 would leave a bigger space) • Remove MAP2 in order to continue into mitosis • TIPS o Stabilize the plus (+) end. Sometimes it doesn’t want to polymerize, so TIPS stops growth. o EB1 is a tip that binds—reduces catastrophe and responsible for rescue MT Binding and Severing Proteins • Kinesin-13 (ATP required) – removes terminal dimers (pulls them off). Can work on the minus end if it is exposed. Only works if depolymerisation is already occuring • Stathmin—binds to tubulin dimers in a curve and promotes GTP hydrolysis – can be inactivated by phosphorylation o Helps with fraying and doesn’t require energy Section 2: MT’s Continued Tracks for transport • Vesicle transport: Bi-directional • Motor Proteins – require energy (ATP) Axonal Transport • Squid axons are a good model because they have 1 large axon • Labelling the proteins – found anterograde transport? Kinesin MT’s (+) end directed motor protein • Many types (14) o 2 heavy chains (head, flexible neck and stalk) o 2 light chains (variable) o Heavy chains have ATPase activity and MT binding ability ▪ Head moves and binds to microtubule when atp is hydrolyzed o Light chains recognize cargo • Kinesin 1: conventional, involved in orgabelle transport • Kinesin 2: heterotrimeric, heavy chains are different from one another. Do most anterograde transport—bind to cargo and take to plus end • Kinsen 5: no light chains, just 4 heavy chains. No polarity. Can bind to 2 microtubules—sliding • Kinesin 13: Depolymerisation, motor protein in structure but its function is to depolymerize MT’s. Works on the plus end Movement (Anterograde) • ATP hydrolysis causes conformational changes in kinesin o Leading head binds to ATP o Binding induces conformational change causing the linker to swing forward and dock onto MT o Leading head releases ADP and coordinately the trailing had hydrolyzes ATP to ADP +P releasing the linker. • ATP is hydrolysed as each head moves. 16nm • Kinesin 1 is inactive and folded when not bound to cargo, and is recognized by the light chains. o Waste of energy for empty motor proteins to move around Cytoplasmic Dynein MT’s (-) end motor protein • Most dynein is in the cytoplasm, heavy chain heads have ATPase activity and stalk. Linker and stem turn to interact with dynactin complex to recognize and bind cargo o ATP hydrolysis shape changes that drives movement • No light chains, the heavy chains of dynein bind to MT. Stem domain binds to dynactin. o DYNACTIN COMPLEX: links dynein cargo and regulates movement ▪ Association is regulated by dynamitin (inappropriate levels lead to dynein exploding) ▪ P150glued binds dynein to MT but is not a motor ▪ Head domains are ATPase are consistent Motor Proteins • Kinesin (antero) and Dynein (retro) work together, often motor proteins themselves are cargo. • Posttranslational modification of tubulin affect both membrane stability and transport o Aceytlyation of a lysine residue of alpha tubulin stabalizes the MT and promotes Kinesin 1 movement ▪ Different ways to modify dimers—add different groups Cila and Flagella • Flagella: Propel cells, Cilia: sweep material across tissue • Axoneme: Underlying Structure of Cilia and Flagella o Bending= moves cell o 9(doublets)+2(singlets) array of microtubules. Outter doublet consists of A and b tubules. o Nexin—holds the doublets together, found in a ladder like structure o Axonemal dynein is different than cytoplasmic o Basal body—axoneme is continuous and attaches to the basal body ▪ Basal body is made of triplet MT’s and acts as a centriole • A B pass through transition zone which is why the Axoneme is doublet, while 3 tubule does not. • Axoneme Bending o Generated by sliding of microtubules against each other ▪ Inbetween doublets is nexin connecting the MT’s in certain places ▪ Axonemal dynein permanently bound in other places, with head domain reaching for the B tubule (A tubule is permanently bound to axonemal dynein) ▪ Bending is generated with the axonemal dynein moving and sliding • If there is no nexin—allows the MT’s to slide ▪ Regional bending – has to be localized other B tubule will break apart • Intraflageller movement o Up and down—not related to bending. Cytoplasmic dynein in addition to axonemal dynein o Can secrete things from cilia • Many Interphase Cells contin a non-motile primary cilium o No axonemal dynein but important roles in cell-cell signalling ▪ Can cause issues in non mitotic cells if mutated ▪ Look similar to axoneme but simpler (made of MTs) • Acetylated alpha subunit makes it stable Cell Division • Karyokinesis and cytokinesis o Karyokinesis: separation of the chromosomes (microtubules) o Cytokinesis: separation of cytoplasm (actin) • No specific stage it is a progression, some animals don’t break down nuclear envelope it varied • Interphase: singlet MTs, endocytosis and exocytosis occur during interphase—comes from centrosome. • When enter Mitosis must depolymerize singlet MTs—centrosome duplicates instead of polymerizing (doesn’t need to grow MTs) o Centrosome becomes mitotic apparatus (poles) and must capture chromosomes and line them up at the metaphase plate and separate • Difference between mitotic apparatus and centrsosome polymerized MTs o In interphase: half life is longer o In mitosis: polymerizes very quickly o In interphase: microtubules are involved in traffic: not that much of a hurry, but in mitosis MTs have a job: must capture kinetochores and separate chromosomes o Centrosome makes stable MTs while Mitotic apparatus makes dynamic MTs Mitotic Apparatus • Polymerizes unstable microtubules (during mitosis) • Kinesin 13 depolymerizes things, MAPS is also present • 2 MTOC, mitotic poles both have 2 centrioles and eventually become the centrosome in daughter cells • Spindle MTs o Polar and kinetochore make spidle o Astral point awat from MTs • Centromere: attachment site for microtubules o Chromosomes have kinetochores with different proteins—always hits plus end of MTs. Spindle Formation • All chromosomes must be captured and aligned during metaphase (from both sides) o Move chromosomes by polymerizing and depolymerizing MT’s o Dyenin and kinesin ▪ As MT shortens, chromosome will move with it and can also add dynein to pull it. ▪ Redundant functions Tension Assures Bi-orientation • Chromosome is attached to both spindle poles. Kinetechore MT will only capture for a short time unless it feels tention from the same chromosome being captured from the other side o If no tention: phosphorylation of Ncd80 proteins at kinetochore results in weak MT interactions with the kinetochore Anaphase • Anaphase A: Movement of chromosome closer to poles o Shortening of kinetochore MTs, motor proteins assist but are not necessary for the shortening to occur o Depolymerization occurs at the minus end as well • Anaphase B: Pole separation (requires motors) o Polar MTs overlap from opposite poles where there is overlap kinesin 5 is present – causing sliding o Gets rid of over lap as the polar MPs push appart making the cell longer o Shortening of the astral MTs bound to dynein and anchored to the cell membrane pull the poles towards it Section 3: Actin Filaments- Microfilaments Actin • More complex than MTs • Cortical (near outside) right underneath the plasma membrane but can also extend into the cytoplasm. • Involved in cell shape and movement • Facilitates muscle movement aswell • Responds to extracellular signals that changes the cytoskeleton and dramatically impacts the actin cytoskeleton • Forms bundles and networks while tubulin can only form linear tubes Structure • Different types have different function o Alpha: muscles, beta: cortex, found in all cells, Gamma: Stress fibres • When translated it forms a globular structure (g-actin), comprised of 4 domains with an opening on one side (binding cleft) – has polarity because of the cleft o Because its polar, it is always polymerizing in a way that the ATP cleft is in the same orientation (pointing to the minus end) • Myosin 21 decorates actin and creates arrow heads which indicates polarity of actin o Arrow heads are always pointing to the minus end Polymerization • When decorated with myosin s1 – stabilize the actin and use it as a nuclease in which you can look at polymerization rates o Add monomers and allow to polymerize – polymerizes faster at the plus end than the minus end • As you increase mass—hit critical concentration at which the MF is polymerized ▪ Above cc = polymerization ▪ Must me in ATP form to polymerize – hydrolized after polymerization ▪ Below cc= depolymerization Assembly • Plus and minus end have different critical concentrations o Plus end: 0.12 micromolar o Minus end: 0.60 micromolar • Tredmilling: depolymerizing at one end and polymerizing at the other – looks like it is moving but it isn’t Actin Regulation • A lot of g-actin in a cell, technically should always be polymerizing but these g actin are not all bound to ATP • Thymosin: removes g-actin from cc amount and provides a resoivoir • Profilin: Promotes actin polymerization by energizing G-actin and moving it from unfunctional ADP form to functional ATP forms • Cofilin: Enhances depolymerisation Capping Proteins: block assembly and disassembly • Plus end cap: CapZ o Binds to the plus end and wont depolymerize/polymerize—use this to figure out cc at minus end • Minus end cap: Tropomodulin o Binds to the minus end and stops polymerizing and depolymerizing Actin Disrupting Drugs • Cytochalasin: depolymerizing actin filaments • Phallodin: stabilizes actin filaments—can tag actin with this because it fluoresces Assemble and branching • Actin is found in high levels, so the plus end is highly regulated o Formins: helps nucleating proteins, and acts as a nucleating agent in which other monomers can be added to. Always sits at the plus end. ▪ Formin is also regulated and needs to be active to work ▪ Only regulates speed, must still be above cc ▪ Regulated by rhoGTP. RhoGDP will cap the plus end permanently until active • Branching is mediated by active Arp2/3 o Looks like 2 monomers stuck together, but when stuck on a MF, allows growth in any direction (like a tree branch) o Still requires a nucleation promoting factor ▪ WASp: activated by Cdc42 ▪ WAVE: activated by Rac o Can occur when not wanted—works fast ▪ Listeria uses actin network to push into the cell o Endocytosis and phagocytosis – actin pushes on the membrane or pulls Actin-Binding Proteins and Cellular Structures • Proteins that bundle form networks and link actin to the plasma membrane o Fimbrin and alpha actin • Proteins that take actin and form networks o Actin can already form networks, but these proteins allow for even more complex bridging ▪ Spectrin ▪ Filamin • Linking to the plasma membrane o Dystrophine links actin cytoskeleton to a membrane protein • Red blood cells o Actin cross linked to network with spectrin o Link network to PM with band 4.1 (links actin to transmembrane proteins) o Ankryn binds spectrin to the PM which anchors actin to the cytoskeleton • Microvilli o Ezrin has the ability to be bound in one state and unbound in another ▪ Must be phosphorylated to be active • Muscles o Dystrophin is important in mucle cells o If it doesn’t work: trying to link actin cytoskeleton to extracellular matrix, actin will not connect which means the muscle contractions don’t result in movement – continuous attempts with no resolve results is muscle death (muscle dystrophy) Myosin (actins motor protein) • Plus end • Similar to kinesin o 2 heavy chains with head and neck domain o head domain is binding site with ATPase activity o neck domain bends with ATP hydrolysis ▪ tail domain binds to cargo • Classes o Myosin 1: atypical, short monomer with short tail. Binds to membrane and plays a role in endocytosis ▪ Allows for actin network to pull on membrane and move plasma membrane if needed o Myosin 5: similar to kinesin 2, allows for organelle transport o Myosin 2: involved in muscle contraction, forms thick bipolar filament • Shorter the neck of the MP, the smaller the step size and the slower it moved • Myosin 5 stems are 72nm long Conformational changes of Mysosin • Start in rigor state (tense) o Actin filament with thick filament (myosin 2) above with head domain connecting them o Binds to ATP, causing conformational change and myosin releases from actin o ATP is hydrolyzed causing another change in shape, causing the head domain to move to the plus end and bind somewhere down the plus end. o When phosphate is released, causes another change in shape and a power stroke – straightens the thick filament and actin • Rigor mortis: after death, myosin is bound to actin but no ATP to release it: muscles become stiff Skeletal muscles • Actin needs to be capped in muscles cells o CapZ caps the plus end by the Z disks o Tropopudulin caps the minus end by the M band in the middle • Myosin 2 doesn’t float in the sarcomere, it is held in place by titin • Nebulin binds to actin to stabilize it as well at the minus end Regulation by Calcium • Sarcoplasmic reticulum holds calcium • Calcium is transient in the cytoplasm because when it comes together with phosphate it precipitates which is bad inside of the cell. • Nerve signals trigger calcium release into the cytoplasm (concentration gradient) , as it is released it is quickly pumped back into the SR thorugh ATPase pumps which stops muscle contraction Tropomyosin and Troponin • Tropomyosin coats the actin which covers the myosin binding sites—preventing the MPs from binding to actin when the muscle isn’t contacting • Troponin, which is linked to tropomyosin and can move it, changes conformation when bound to calcium. o Change in conformation removed tropomyosin from the binding sites, allowing MPs to bind Actin and Myosin in Non-skeletal Muscle cells • Contractile ring during cytokinesis which cleaves the cell as it depolymerizes • In smooth muscle o No sarcomeres because they are not controlled by nerve impulses ▪ Slower and more persisten
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