MCB 252 Topic 23 MicrotubulesProf David Rivier MCB 252 Spring 2015 MCB 252 Cell Biology Topic 23 Microtubules Reading Lodish 18 0 18 3 MCB 252 Topic 23 Microtubules Microtubules Structure of monomer polymer Dynamics of assembly Dynamic instability Microtubule associated proteins MAPs Microtubule based structures Paramecium ciliated epithelium oviduct mitotic spindle flagellum Centrosomes Lodish 18 5 c Spindle Poles Lodish 18 5c Basal bodies Lodish 18 5f In vivo imaging of mitotic spindles in Drosophila embryos Express GFP Tau fusion protein in embryos visualized by confocal microscopy Dr Andrea Brand Cambridge University UK Movie T23M1 06 cell divisions avi Microtubules First defined by EM 1963 Assembled from heterodimers of alpha and beta tubulin Dimers are stable rarely dissociate alpha and beta tubulin 40 identical residues Tubulins are highly conserved 75 identical between plants and animals Structure of tubulin dimer exchangeable non exchangeable Lodish Fig 18 3 Structure of tubulin polymer Lodish Fig 18 3 Polarity of tubulin polymerization Nucleate assembly in presence of fragments of flagella Observe longer MTs at one end Subunits add preferentially to plus end of MTs Lodish Fig 18 8 MT assembly Lodish 18 3 EM views of MT assembly and disassembly Dynamics of pure tubulin Tim Mitchison and Marc Kirschner www ergito com Dynamic instability observed by visualizing individual MTs in vitro Lodish Fig 18 9 Time lapse fluorescence microscopy of MTs in 3T3 cells Dr Gary Borisy Northwestern Univ Med School Movie T23M2 06 microtubules avi Dynamic Instability Movie T23M3 tubulin02 mov Dynamic Instability MT dynamics GTP hydrolysis results in Structural Strain GTP protofilaments straight GDP protofilaments curved Structural Strain is Constrained by the GTP cap at the plus end See also Lodish 18 11 Role of GTP cap in MT dynamics See also Lodish 18 11 MCB 252 Topic 8 Microtubules Microtubules Structure of monomer polymer Dynamics of assembly Dynamic instability Microtubule associated proteins MAPs Microtuble associated Proteins MAPs MAPs play roles in Assembly Disassembly Stability Spacing and arrangement of MTs Search and capture EB 1 Binds Sides of GTP Cap EB 1 Stabilizes the GTP cap and thereby promotes elongation Search and Capture Movie T23M4 17 4 microtubule dynamics mov Kinesin 13 and stathmin promote disassembly Energy of binding of Kinesin 13 and Stathmin tips the balance toward curvature Katanin regulates disassembly by severing MTs 1 Adhere rhodaminelabeled MTs to surface of slide 2 Add Katanin 3 30 seconds later A 4 3 minutes later B Alberts Figs 16 45 MT associated proteins MAPs regulate MT dynamics and interactions Co purify with MTs isolated from cells MAPs that stabilize MTs MAP2 localized to dendrites assembles cross links MTs to MTs and IFs Tau localized to axons assembles stabilizes and cross links MTs Alberts Figs 16 32 and 100 Tau MAP2 Tau MAP2 MAP4 1 structures Flexible MT binding sites of Tau Pollard and Earnshaw Fig 37 11 MAPs can regulate MT spacing Express MAP2 or Tau in cultured insect cells extend axon like process MT spacing depends upon MAP that is expressed Lodish Fig 18 13 MAPs can regulate MT spacing Lodish 18 13 MAPs often connect to Intermediate Filaments Drugs that disrupt MT dynamics Colchicine from autumn crocus colcemid bind alpha beta dimers prevent polymerization at low concentrations block cells in metaphase commonly used for arresting cells to count chromosomes and to treat gout Drugs that disrupt MT dynamics Taxol generic Paclitaxel from Pacific Yew binds and stabilizes MTs chemotherapeutic agent used to treat certain types of cancers taxol taxol Alberts Figs 16 21 Regulation of MAPs Many MAPs regulated by phosphorylation Cell cycle regulation of the kinases responsible for phosphorylating MAPs allows for rapid disassembly and rebuilding of the microtubule structures associated with the different phases of the cell cycle