Cytoskeleton - structural framework of the cytosol, filaments made from small protein subunits!- Microtubules, hollow cylinders!- Actin filaments (microfilaments), two stranded helical polymers of actin subunits!- Intermediate filaments, rope like fibers of heterogen. intermed. filament proteins!- Functions:!- Bones and muscles of cells: provides strength and supports the pm, determines cell shape and lets cells change shape, cell polarity, lets cells interact with the envi and adjacent cells, enables cells and their components to move!- Stable and dynamic!- can undergo rapid changes during cell division, fibers in ciliated cells, rapid turnover of subunits!!Microtubules - Extend out to cell from centrosome (in interphase)!- cellular highways= tracks for intracellular transport, organelles can anchor on it!- cell shape and polarity!- form mitotic spindle!- motile structures: cilia and flagella!- polar, hollow cylinders formed from tubulin subunits (alpha [has fixed GTP bound] and beta [can exchange its GTP])!- polar protofilaments and polar hollow cyclinders (circle)!- stiff, difficult to bend!- Dynamic Instability - grow faster at + end than - end (which is usually anchored)!- + end undergoes dynamic instability !- GTP cap= stable, growing!- Loss of GTP cap= unstable, shrinking!- typical MT switches between growth and shrinkage every few min. Back and forth between growth and rescue.!- cells use a lot of energy to maintain dynamic instability--> this flux is necesarry to cells can respond to signals!!- catastrophe= change from rapid growth--> rapid shrinkage!- hydrolysis of GTP leads to a conformational change that weakens the bonds and leads to the peeling off of protofilaments!- shrinking MTs can be rescued!- regain of GTP cap!- shrinking MTs may completely disappear or may be rescued and continue to grow!- high GTP-dimer concentrations promote growth, low GTP-dimer concentrations promote shrinkage!- plant toxins can alter MT polymerization!- by disrupting the MT polymerization, they either bind to free subunits or filaments-->drive the rxn in the direction that favors the form to which the toxin is bound!Ex. colchicine, vinblastine, and ncodazole bind to subunits and prevent them from being assembled into a MT--> not MTs will be formed as they all come off and are bound!Ex. taxol binds to and stabilizes MTs--> they can't depolymerize--> no MT structure changes can happen since subunits can't come off!- use taxol and vinblastine to treat cancer because they preferentially kill dividing cells and cancer cells divide the most!- unwanted side effects on normal human cells that divide a lot (bone marrow, GI lining cells, hair follices)!!- Nucleation= the rate limiting step for formation of MTs!- short oligomers assemble, but are unstable and quickly disassemble!- need a stable nucleus to form a new filament!- nucleation gen. occurs at MTOCs= mitrotubule organizing centers!- y-TuRCs= y tubulin ring complexes= nucleus for MTs!- remain bound to - end and stabilize that end (no subunit addition or loss)!- Animal cells: MTOCs= centrosomes - centrosomes have a fibrous centrosome matrix (pericentriolar material) that has 50 or more y-TuRCs, other proteins, and 2 centrioles - centrioles are a component of centrosomes and spindle poles!- they are short cylinders made of modified MTs and proteins, 9 groups of triplets in cartwheel formation!- 2 centrioles in each centrosome!- centrosomes duplicate during S phase (2 centrioles in G1, 4 centrioles in G2)!- these pairs move to the opp. ends of the nucleus and form the 2 spindle poles!- MTs radiate out from the centrosome--orient the cell!!- Accessory proteins regulate length, stability, number and geometry of MTs normally by binding to free subunits or to MT filaments!- Stathmin (relay) - the concentration of tubulin monomers in a cell is usually much higher than the critical concentration!- to maintain these, tubulin subunits are sequestered by the binding to stathmin protein!- binds the 2 subunits, prevents them from adding to the MT--> more likely that the MT will undergo depolymerization!- if phosphory.--> can't bind subunits!- Katanin (sword) - severes MTs near origin in centrosome-->promotes depolymerization!- Microtubule associated proteins (MAPs) stabilize MTs by binding on their sides!- some MAPs that bind to MT and to another cellular location--lead to diff. MT spacing (ex. axons, nerves)!- MAP2= in dendrites, long extended domain!- Tau= in axons, short, projecting domain-->close MTs!- in Alzheimers people have too large aggregates of these called neurofibrillary tangles- phosphory can determine activity!- Capping proteins - proteins that bind along the sides and edges of MTs and affect their dynamic behavior!- catastrophe factors= increase the catastrophe rate of MTs, bind to + end and pry protofils apart Ex. kinesin 13!- XMAP215= stabilizes the + end, promotes assembly/growing, activity inhibited by phosphorylation, phosporylation= critical for assembling the mitotic spindle!- +TIPs (plus end tracking proteins)= link + ends of MTs to target locations in the cell!!Molecular Motors - made of heavy and light/intermediate chains!- move cargo along filaments via ATP hydrolysis (cyclic binding and release of cytoskeletal filament)!- kinesins= + end directed!- large superfamily, common element= motor domain, most are dimers and bind cargo with their tails!- exception= kinesin 14--moves toward - end of the filament!- move via hand over hand--highly processive= move long distances w/o disassociating !- dyneins= - end directed!- made of 2/3 heavy chain and variable # of light and intermediate chains, faster than kinesins!- cytoplasmic dynein= move membrane vesicles along MTs - axonemal dynein= found in cilia and flagella, drive their beating!- organelle location is determined by association with MT via motor proteins !- centrifugal movement= towards cell periphery (ex. ER) med. by kinesin!- centripetal movements= towards cell center (ex. Golgi) med. by dyneins!- motor proteins transport vesicles and orgallenes along nerve axons (+ end = axon terminus)!- anterograde= move from cell body to axon terminal where synapses have to be maintained, carried by kinesins!- retrograde= from + axon terminal to - cell body, old components brought back to cell body for degradation, by dyneins!!Mitotic Spindle !!- minus ends of MTs are embedded in the spindle poles, + ends radiate out from the poles!- kinetochore MTs attach to sister
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