Microtubules or actions of microtubule motor protein → polymerization, Harvey Lodish • Arnold Berk • Paul Matsudaira • Chris A. Kaiser • Monty Krieger • Matthew P. Scott • depolymerization → movement Lawrence Zipursky • James Darnell MTOC (microtubule-organizing center): contributing to cell motility; Located near the nucleus, assembly an orientation of microtubules ,the direction of vesicle trafficking, and orientation of organelles. Organelles and vesicles are transported along microtubules, the MTOC becomes Molecular Cell Biology responsible for establishing the polarity of cell an direction of cytoplasmic processes in both interphase and mitotic cells. Fifth Edition Chapter 20: Cytoskeleton II: Microtubules Kinesin powered movement of a vesicle along a microtubule Copyright © 2004 by W. H. Freeman & Company Microtuble organized around the MTOC and spindle Cells contain stable and unstable microtubules (MTs). --Orientation of cellular SEM of the surface of ciliated microtubule epithelium of rabbit oviduct most microtubules have a constant orientation relative to MTOC (-) end: close to the MTOC (+) end: distal to the MTOC Long-distance movement microtubule-organizing center is “-” Assembly and disassembly cause microtubules → probe →…. 1 Heterodimeric tubulin subunits compose the wall of a MT. 1 2 3 Tubulin subunit are formed by α and β One subunit bind to two GTP, has GTPase Fig 5-29 activity. 55kDa monomers in all eukaryotes γ-tubulin are formed by α and β. In mammals at least 6 alpha and 6 beta isoforms have been GTP identified Irreversibl reversibly Polymer of globular Tubulin → arranged → microtubule The proteins are highly conserved y, does not Two populations of MT: (75% homology between yeast hydrolyze 1. stable, long-lived: nonreplicating cell; cilia, flagella, RBC and and human) add plaelets pass through small vessel, axon Most variability is found in the C- terminal region of the molecules 2. unstable, short-lived; assemble and disassemble quickly, replicating and is likely to affect interactions _ + cell with accessory proteins A tubulin homologue, FtsZ, is expressed in prokaryotes Arrangement of protofilaments in singlet, doublet, and triplet MTs. + α β β-GTP GTP GTP α-GTP tubulin heterodimer β-GDP − α-GTP α β α β seam GTP GDP GTP GTP The tubule is a microtubule 3-start helix complete (−) protofilament (+) microtubule cylinder, made of Thus the minus end of an a subunit may serve as GAP (GTPase activating protein) 13 protofilaments for b-tubulin of the adjacent dimer in a protofilament 2 Addition of MT fragments demonstrates polarity of tubulin Temperature affects whether MTs assemble or disassemble. polymerization. (MT assembly and disassembly take place preferentially at the + end) High Temp: assembly (need GTP) Low Temp: disassembly Slowed 2 fold than + Cc: critical concentration Stages in assembly of MTs. Up: dimers polymerize into microtubules; below: depolymerize Free αβ tubulin dimers →short protofilaments → unstable and quickly associate more stable curved sheets → wraps around microtubule with 13 protofilaments →composing the microtuble wall → incorporate αβ (β hydrolysis GTP) → bind + end Fluroresence microscopy reveals in vivo growth and shrinkage of Cryoelectron microscopy allows individual MTs. observation of disassembled MTs. Rate of MT growth in vitro is much slower than shrinkage (收 縮). 急轉直下 縮短 Fluorescently-labeled tubulin microinjection into fibroblasts. Microtubule is dynamic instability. Two factor influence the stability of MT: 1. Cc (critical concentration): > or = Cc → growth; < Cc → shrink (收縮). 2. β subunit bind to GTP or GDP. Disassembly quick (7μm/min) Assembly slowly (1μm/min) 3 Dynamic instability model of MT growth and shrinkage (β subunit --properties of polymerization/depolymerization of microtubule (fig 19-10) bind to GTP/GDP). --with a nuclei: accelerates the initial polymerization rate -- [tubulin] > Cc polymerization + end is most important for MT [tubulin] < Cc depolymerization (β subunit) growth and shrinkage [tubulin] ~ Cc dynamic -- microtubule assembly/disassembly occurs preferentially at the (+) end Dissociation of GDP-tubulin dimer >>>> GTP-tubulin dimer When GDP-tubulin → depolymerizes and unstabilized When GTP-tubulin add to + end → rescued shortening MT. Two factor influence stability of MT: 1. Cc 2. GTP or GDP –β subunit Numerous proteins regulate MT dynamics and cross-linkage to other Spacing of MTs depends on length of projection domain in structures. bound MAPs. MAPs: microtubule-associated protein, influence the assembly and stability of MAP2 and Tau can regulate microtubule spacing microtubule and their related structure. MAP2 Tau Basis of their function has two groups: When MT bundles are induced in cells overexpressing MAP2 (left) and Basic microtubule binding and acidic projection domain tau (right), the bundles formed by MAP2 have wider spacing between MT than those formed by tau. MAP2 COOH end binds along Cross-link MT the MT lattice while NH2 terminal end projects out from the microtubule Tau binds similarly but its projection arm is much shorter than arm of MAP2 Related with Alzheimer’s disease 4 Colchicine and other drugs disrupt MT dynamics. MAP kinase (MAPK): A key enzyme for phosphorylating MAPs → phosphorylated MAPs → unable to bind to microtubules Cyclin-dependent kinase → phosphorylation of MAP4 → controlling the activity of various proteins in the course of the cell cylce. MTs can assemble in vitro from purified tubulin, but MAPs are found with MTs isolated from cells; Blocked at metaphase most found only in brain tissue; MAP4 has wider distribution Have globular head domain that attaches to MT side & filamentous tail protruding from MT surface May interconnect MTs to help form bundles (cross- bridges), increase MT stability, alter MT rigidity, influence MT assembly rate Drugs involved in microtubule dynamics MTOCs orient most MTs and determine cell polarity. (1) colchicines/colcemid: mitotic inhibitors --its effect is reversible --binds to tubulin dimer blocks the addition or removal of other tubulin subunits to the ends of microtubule disruption of microtubule dynamics --cells are blocked at “metaphase” after colchicines treatment cytogenetic studies cell synchronization (時間一致) (2) taxol, vinblastin: --bind to microtubules and stablize microtubule by inhibiting the lengthening and shortening of microtubules --used for cancer treatment 5 MTOCs orient most MTs and determine cell polarity. MT (doublets) are found in cilia or flagella that are used for cell movement MT in axon are plus end out, MT in dendrites are plus and minus ends out. These MT are not attached to any nucleating structure. Cells have stable (half life >1 hr) and unstable/dynamic (half- life = 5-10 min) microtubules. These subsets differ in post- translational modification of tubulin. The centrosome (MTOC) is usually located near the nucleus during interphase. Microtubules grow out from the MTOC Centrosome contains a pair of orthogonal (直角) centrioles in The γ-tubulin ring complex (γ-TuRC) nucleates polymerization most animal cells. of tubulin subunits. MTOC = microtubule organizing center MTOC organization: pericentriolar Microtubules assemble from the MTOC. material (including protein and γ In mammals, the centrosome (中心體) is tubulin) →nucleating (以核為 the MTOC, with the MT minus (-) ends 中心) microtubule assembly → inserted into the centrosome and the plus anchoring Stain with an anti-gamma tubulin antibody. (+) ends directed towards the cell Gamma-tubulin at initiates synthesis at one γ-TuRC the has 8 polypeptides and periphery. Centrosome is a collection of end (-) (green). 25 nm diameter microtubule orienting proteins within a Under Cc, γ-TuRC directly cloud of material termed the nucleate microtubule assembly pericentriolar or centrosomal matrix. Sometimes, inside will be a pair of γ-TuRC Formed only one end (-). centrioles (中心粒) that serve to organize the matrix. The microtubules emanate from γ-tubulin ring complexes inside the centrosome. Centrioles (C) a pair, C and C’ Pericentriolar (PC) matrix: γ-tubulin & pericentrin; surrounding the centrioles 6 The center of the center of the cell… Microtubules nucleated by the γ-tubulin ring complex Centrioles in a centrosome connect to centromeres appear capped at one end, assumed from other data to MTOC=microtubule organizing center be the minus end. γ-Tubulin, which is homologous to α & β tubulins, nucleates microtubule assembly within the centrosome. Several (12-14) copies of γ-tubulin associate in a complex with other proteins called “grips” (gamma ring proteins). This γ-tubulin ring complex is seen by EM to have an open ring-like structure resembling a lock washer, capped on one side. Grip proteins of the cap may be involved in mediating binding to the lockwasher centrosome. shape Phosphorylation of a conserved tyrosine residue of γ-tubulin has cap been shown to regulate microtubule nucleation in yeast cells. γ-tubulin ring complex Cytoplasmic organelles and vesicles are organized by MTs. Kinesin and dynenin powered movements Colocalization of endoplasmic DiOC6, ER reticulum membranes and cytosolic binding MTs. fluorescent Others organelles also colocalized. dye Anti-tubulin Progression of organelles along axons requires microtubules and the motor The alignment of the ER network proteins: kinesin and dynein. and microtubules in many but not all regions of the cytoplasm is Also dependent on motor proteins: evident because the cell has Transport of vesicles for exocytosis/endocytosis or between the endoplasmic sparse