MCB 150 Lecture 32 Cytoplasmic microtubules maintain axons polarized shapes govern orientation of cellulose microfibrils deposited during the growth of cell walls form mitotic and meiotic spindles for movement of chromosomes special disposition and directional movement of vesicles o Singlet MTs with 13 protofilaments Axonemal microtubules highly organized stable microtubules found in cilia flagella and basal bodies o Doublet or triplet MTs one complete MT w 13 pf a tubule and one or two additional with 10 or 11 pf Doublets in cilia and flagella triplets in basal bodies and centrioles Describe the structure of tubulin and how it is polymerized into microtubules o Straight hollow cylinders inner diameter of 15nm and outer of 25 nm o Consist of linear polymers protofilaments 13 o Basic subunit of protofilament is a heterodimer of tubulin with a tubulin and b tubulin forms AB heterodimer that does not dissociate under normal conditions GTP binding domain at N terminus where colchicine can bind to and block MT assembly All a tubulin face same end each end differs structurally and chemically MT is polar Tubulin isoforms 5 a and b in brain acetylated tubulin forms more stable MTs o Steps for MT formation Sufficient concentration of tubulin dimers and GTP tubulin dimers make oligomers Nucleation oligomers serve as nuclei from which mts can grow Slow lag phase Elongation addition of subunits on either end Faster than nucleation Plateau phase concentration of free tubulin becomes limiting and MT assembly with MT disassembly Critical concentration above growth below disassembly Rapidly grows at plus end and disassembly at minus endtreadmilling Compare and contrast the uses of microtubules during interphase and M phase Explain the concept of a GTP cap in microtubule polymerization and depolymerization and how this leads to dynamic instability o Each tubulin heterodimer binds to two GTP molecules association of GDP bound tubulin too weak to support polymerization hydrolysis of GTP not necessary so use nonhydrolyzable analogue of GTP o Dynamic instability model one growing in length by continued polymerization at their plus ends and the other shrinking in length by depolymerization Growing MTs have GTP bound and shrinking have GDP instead o At a low concentration of GTP tubulin rate of hydrolysis of GTP on b tubulin subunits exceeds the rate of addition of GTP bound tubulin GTP cap disappears loss of GDP subunits from tip favored o Catastrophe complete disassembly rescue abrupt growth phase o Dynamic instability is more pronounced at plus end of MT Explain the action of laboratory and medical chemicals in affecting the state of a cell s microtubules o Colchicine binds to tubulin monomers inhibiting their assembly into microtubules and fostering disassembly of existing ones o Vinblastine and vincristine cause tubulin to aggregate inside cell o Nocodazole inhibits MT assembly but effects more reversible than colchicine o Taxol binds tightly to mts and stabilizes them causing all free tubulin to assemble into MTs and arrests dividing cells in mitosis Describe the formation of new microtubules and identify where this happens o Originate from microtubule organizing center MTOC o Centrosome for animals with two centrioles surrounded by a diffuse granular material known as pericentriolar material they originate from pericentriolar material o Centrioles maybe recruit pcm to centrosome o Large ring shaped protein complexes contain g tubulin with gamma tubulin ring proteins o Tubulin ring complexs nucleate assembly of new MTs o Minus ends anchored to MTOC and plus ends toward cell membrane o Dynamic growth and shirkage of MTs at plus ends tends to happen at periphery of cells o MTOC have limited of nucleation and anchorage sites that control how many MTs can form Lecture 33 Vesicle transport is ATP dependent Kinesin molecule that generates vesicle movement motor protein converts chemical energy in ATP into mechanical energy when ATP is hydrolysized it moves towards plus end o A head section with two globular pieces and a tail with small polypeptides with a stalk that connects head and tail o Head binds to microtubule and tail binds to transport vesicle o 375 steps for second Flagella eukaryotes move by whipping back and forth surrounded by plasma membrane organelle like cilia o Nine microtubule doublets surrounding two central microtubulesaxoneme o Originates from basal body identicle to centriole o Spoke proteins connected each doublet to central pair and molecular links to one another o Have a more propagated bending motion than cilia may even be helical Cilia oarlike pattern of beating with a power stroke perpendicular to the cilium thereby generating a force parallel to the cell surface active power stroke followed by recovery stroke Dynein motor protein moves towards minus end o Because of spokes on axoneme sliding motion by dynein walking is constrained if one doublet slides it transmits force to the rest of the axoneme o If dynein arms on just one side of axoneme are activated then the localized movement results in bending swimming motion Anterograde axonal transport kinesins mediate transport from cell ody down the axon to nerve ending Retrograde axonal transport opposite direction towards minus ends Kinesins one of thw two globular heads moves forward to make an attachment to a new region of the MT coupled to the hydrolysis of ATP 60 to 70 efficient Describe the structure of an animal cell centrosome comparing centrioles with other microtubule based structures Explain how microtubules are used to provide shape polarity and transport in cells Compare and contrast kinesins and dyneins Describe axoneme structure and how it leads to movement of cilia and flagella Basal body has nine sets of tubular structures around circumference each set is a triplet As a cilium or flagella forms a centriole migrates to the cell surface and makes contact with plasma membrane This is the nucleation site for the outer doublets of axoneme after assembly begins it is a basal body Axonemes of cilia have 9 2 Primary cilia in embryos have 9 0 Axonemes side arms that reach out to doublet have dynein Sliding microtubule model sliding movement is converted to localized bending because the doublets are connected radialy to central pair and circumfrecially to one another cannot slide freely takes the form of a wave that begins at base of organell and proceeds toward tip Stalk of dyenein arm attach and deattach from B