MOLECULAR CELL BIOLOGY SIXTH EDITION. CHAPTER 18 Cell Organization and Movement II: Microtubules and Intermediate Filaments

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1 Lodish Berk Kaiser Krieger Scott Bretscher Ploegh Matsudaira MOLECULAR CELL BIOLOGY SIXTH EDITION CHAPTER 18 Cell Organization and Movement II: Microtubules and Intermediate Filaments 2008 W. H. Freeman and Company Copyright 2008 W. H. Freeman and Company

2 Chromosomes (blue) Kertain intermediated filament (red) Centrosomes (magenta) Microtubule (green) Lung cell in mitosis

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4 Microtubule are found in many different locations and all have similar structure SEM of the surface of ciliated epithelium of rabbit oviduct Microtubule base motor protein Cells contain stable and unstable microtubules (MTs).

5 Heterodimeric tubulin subunits compose the wall of a MT. Tubulin subunit are formed by α and β One subunit bind to two GTP, has GTPase 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 identified The proteins are highly conserved (75% homology between yeast and human) Most variability is found in the C- terminal region of the molecules and is likely to affect interactions with accessory proteins A tubulin homologue, FtsZ, is expressed in prokaryotes Irreversibl y, does not hydrolyze _ Silimar to actin GTP reversibly + add

6 Arrangement of protofilaments in singlet, doublet, and triplet MTs. The tubule is a complete microtubule cylinder, made of 13 protofilaments

7 Microtubule are assembled from MTOCs to generate diverse organizations Microtubules or actions of microtubule motor protein polymerization, depolymerization movement 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 responsible for establishing the polarity of cell an direction of cytoplasmic processes in both interphase and mitotic cells.

8 Microtubules are assembled from microtubule organization centers (MTOC) 中心體 During mitosis, MTOC=spindle pole In non-mitotic cell, the MTOC=centrosome near nucleus (+) end of microtubule toward to periphery

9 Centrosome contains a pair of orthogonal ( 直角 ) centrioles in most animal cells MTOC = microtubule organizing center Microtubules assemble from the MTOC. + In mammals, the centrosome ( 中心體 ) is the MTOC, with the MT minus (-) ends inserted into the centrosome and the plus - (+) ends directed towards the cell periphery. Centrosome is a collection of - microtubule orienting proteins within a cloud of material termed the pericentriolar matrix or centrosomal + matrix. Sometimes, inside will be a pair of centrioles ( 中心粒 ) that serve to organize the matrix. The microtubules emanate from γ-tubulin ring complexes Two centrioles (γ-turc) inside the centrosome. Nucleating structures 9 Triplet microtubule Centrioles (C) a pair, C and C Pericentriolar (PC) matrix: γ-tubulin & pericentrin; surrounding the centrioles

10 Centrosomes and the γ tubulin ring complex pericentriolar matrix MTOC

11 γ-turc The center of the center of the cell Centrioles in a centrosome connect to centromeres MTOC=microtubule organizing center

12 Microtubules nucleated by the γ-tubulin ring complex appear capped at one end, assumed from other data to 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 咬緊 (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. Formins assemble unbranched filament + - lockwasher shape cap γ-tubulin ring complex

13 γ-tubulin ring complexes (γ-turc) assembly The γ-turc nucleates microtubule assembly

14 The γ-tubulin ring complex (γ-turc) nucleates polymerization of tubulin subunits. MTOC organization: pericentriolar material (including protein and γ tubulin) nucleating ( 以核為中心 ) microtubule assembly anchoring γ-turc the has 8 polypeptides and 25 nm diameter Under Cc, γ-turc directly nucleate microtubule assembly γ-turc Formed only one end (-). Stain with an anti-gamma tubulin antibody. Gamma-tubulin at initiates synthesis at one end (-) (green).

15 Microtubule are dynamic structures due to kinetic differences at their ends Similar to actin Treadmilling More easy polymerization Intracellular tubulin concentration: μm Cc: 0.03 μm Addition of MT fragments demonstrates polarity of tubulin polymerization. (MT assembly and disassembly take place preferentially at the + end)

16 Similar to actin, > critical concentration (Cc) formed microtubules --properties of polymerization/depolymerization of microtubule (fig 19-10) --with a nuclei: accelerates the initial polymerization rate -- [tubulin] > Cc polymerization [tubulin] < Cc depolymerization [tubulin] ~ Cc dynamic -- microtubule assembly/disassembly occurs preferentially at the (+) end

17 Temperature affects whether MTs assemble or disassemble. Cc: critical concentration Up: dimers polymerize into microtubules; below: depolymerize MAP : microtubule associated protein regulated polymerzation of microtubule High Temp: assembly (need GTP) Low Temp: disassembly

18 Addition of MT fragments demonstrates polarity of tubulin polymerization. (MT assembly and disassembly take place preferentially at the + end) Slowed 2 fold than + Stages in assembly of MTs. 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

19 Rate of MT growth in vitro is much slower than shrinkage ( 收縮 ). 急轉直下 Disassembly quick (7μm/min) Assembly slowly (1μm/min)

20 Fluroresence microscopy reveals in vivo growth and shrinkage of individual MTs. 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.

21 Dynamic instability depends on the presence or absence of a GTP-β-tubulin cap 大災難

22 Microtubule growth from MTOC Colchicine disassembled wash out colchicine Immunofluorescent microscopy

23 Drugs involved in microtubule dynamics (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

24 Colchicine and other drugs disrupt MT dynamics. Blocked at metaphase

25 Regulation of microtubule structure and dynamic Player : MAPs (microtubule associate proteins) Microtubule are stabilized by side and end binding protein Similar to actin Tau family (MAP2, Tau) spacing MAP regulating kinase (MARK): A key enzyme for phosphorylating MAPs phosphorylated MAPs unable to bind to microtubules Cyclin-dependent kinase (CDK) phosphorylation of MAP4 controlling the activity of various proteins in the course of the cell cycle.

26 Spacing of MTs depends on length of projection domain in bound MAPs. MAP2 and Tau can regulate microtubule spacing When MT bundles are induced in cells overexpressing MAP2 (left) and tau (right), the bundles formed by MAP2 have wider spacing between MT than those formed by tau. MAP2 COOH end binds along 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 MAP2 Tau Cross-link MT Related with Alzheimer s disease

27 Some MAP bind to (+) end of microtubule, such as TIPs Tubulin: green + TIP protein EB1 EB1 specific bind to (+)

28 Microtubules are disassembled by end binding and severing protein Kinesin-13 enhanced the disassembly ATPase Stathmin bind to protofilaments enhances dissociation

29 microfilaments regulatory proteins G-actin binding proteins Thymosin β 4 (inhibits polymerization) Profilin (accelerates polymerization) Capping proteins Cap Z (+) Tropomodulin (-) Severing proteins Gelsolin (severs and caps (+) ends)) ADF (actin depolymerizing factor); cofilin microtubules regulatory proteins Stabilizing proteins MAPs (phosphorykated MAPs do not bind to microbutules) Tip proteins Destabilizing proteins kinesin-13 (+) stathmin (+) katanin (-)

30 Motor proteins are enzymes that couple the hydrolysis of ATP to a conformational change Myosins: Motor proteins that walk along actin filaments Kinesin and dynein: Motor proteins that walk along microtubules MT associated motor proteins: kinesins: towards + end (anterograde transport) Golgi to ER traffic dyneins: towards - end (retrograde transport) ER to Golgi traffic wave-like motion of flagella and cillia

31 The rate of axonal transport in vivo can be determined by radiolabeling and gel electrophoresis. Anterograde transport goes towards the axon terminal (cell body synaptic terminals), such as vesicles. Retrograde transport goes towards the axon hillock (synaptic terminal cell body), such as old membrane Fast: membrane-limited vesicles, ~250 mm/day. Slow: tubulin subunits, neurofilaments. Intermediate: mitochondria.

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33 Organelles in axons are transported along microtubule in both direction Progression of organelles along axons requires microtubules and the motor proteins: kinesin (toward +) and dynein (toward -). Also dependent on motor proteins: Transport of vesicles for exocytosis/endocytosis or between the endoplasmic reticulum and Golgi Extension of the endoplasmic reticulum Integrity and reassembly of the Golgi apparatus

34 DIC microscopy demonstrates MT-based vesicle transport in vitro. Squid axon Has anterograde and retrograde

35 Kinesin-1 powers anterograde transport of vesicles down axons toward (+) end of microtubule The structure of kinesin-1 microtubule motor protein cargo - +

36 Kinesin I powers anterograde transport of vesicles in axons. Structure of kinesin: two heavy chain and two light chain most common structure comprised of two heavy chains and two light chains; and processive + end directed motor protein (most) MT bind to the helix region in the head; binding is regulated by ATP hydrolysis Plastic bead coated with kinesin will slide along a microtubule towards an end 10 families identified - mainly 2 types, cytosolic and mitotic kinesins; Some structural similarity to myosin

37 Dimer of a heavy chain complexed to a light chain Mr= 380kD Three domains: Large globular head Binds microtubules and ATP 2) Stalk 3) Small globular head Binds to vesicles Step size 8 nm, Force 6 piconewtons Speed 3 μm/s Model of kinesin-1 catalyzed vesicle transport cargo binds only one monomer at a time in a processive manner ATP hydrolysis coupled to movement EM data suggests binding primarily to β- tubulin Model of kinesin-catalyzed vesicle transport.

38 Kinesins form a large protein family with diverse functions 14 classes Not all function has known Cargo: organelle, RNA, chromosomes Kinesin-13 do not motor, for depolymerization ATP Kinesin-14 move to (-) Not all kinesins have the same subunit structure but all have the globular head domain 4 chain

39 Kinesin-1 uses ATP to walk down a microtubule

40 Motor protein Convergent structural evolution of the ATP-binding core of myosin head an kinesin

41 How does kinesin move? ATP binding to the leading head initiates neck linker docking catalytic core tightly docked neck linker detached neck linker tubulin heterodimer α-tubulin β-tubulin Neck linker docking is completed by the leading head, which throws the partner head forward by 160 Å toward the next tubulin binding site The trailing head hydrolyzes ATP to ADP-Pi The new leading head docks tightly onto the binding site The trailing head, which has released its Pi and detached its neck linker (red) from the core, is in the process of being thrown forward. Adapted from: Figure 1 in Vale & Milligan (2000) Science, Vol 288, Issue 5463, 88-95

42 Converter Domain switch loop Head-motor The Way Things Move: Looking Under the Hood of Molecular Motor Proteins Ronald D. Vale and Ronald A. Milligan Science, :80-95 Lever arm Linker region Power stroke

43 Dynein motors transport organelles toward the (-) end of microtubules (retrograde) Cytoplasmic dynein retrograde toward (-) Dynein did not directly power cargo move Need dynactin Dynein Not directly attached to cargo

44 Dynactin complex Dynactin is complex: Besides dynamitin, dynactin contains a filament made of Arp1 that is actin like and binds with spectrin Spectrin binds ankyrin which associates with the vesicle/ organelle p150 Glued binds microtubules and vesicles ankyrin, spectrin, and Arp1 are thought to form a planar cytoskeletal array. Dynein needs dynactin to link vesicles and chromosomes to the dynein light chain

45 Cytosolic dyneins are (-) end-directed motor proteins that bind cargo through dynactin. Dynein also has heavy chains like kinesin but it mediates transport towards the (-) end of the microtubule; Its light chain associates dynamtin, that is part of a large protein complex called dynactin, which is responsible for interacting with the organelles, vesicles, or chromosomes that are being transported. Transport require dynactin, to links vesicles and chromosomes to dynein light chain Arp1 actin related protein, interact with spectrin Dynactin interact with light chains of dynein Very large multimeric complex

46 The power stroke of dynein

47 Kinesin and dyneins cooperate in the transport of organelles throughout the cell ERGIC: ER to Golgi intermediate compartment

48 Cooperation of myosin and kinesin at the cell cortex. actin

49 Motor proteins are enzymes that couple the hydrolysis of ATP to a conformational change Kinesin and dynein: Motor proteins that walk along microfilaments Myosins: Motor proteins that walk along actin filaments Organelle transport uses motor proteins RER to Golgi vesicle Golgi to RER vesicle Cytoplasm + - Dynein Kinesin

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51 Movement of pigment granules in frog melanophores Microtubule: green Nucleus: blue Pigment: red

52 Cilia an flagella: microtubule based surface structure

53 Eukaryotic cilia and flagella contain a core of doublet MTs studded with axonemal dyneins. Structure of an axoneme ( 軸絲 )

54 Structural organization of cilia and flagella

55 Video microscopy shows flagellar movements that propel sperm and chlamydomonas forward

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59 Ciliary and flagellar beating are produced by controlled sliding of outer double MTs. In vitro dynein-mediated sliding of doublet MTs requires ATP.

60 Intrafllagellar transport (IFT) moves material up and down cilia and flagella Retrograde anterograde For growth Intraflagellar transport

61 Defects in IFT cause disease by affecting sensory primary cilia Autosomal recessive polycystic kidney disease (ADPKD) 自體顯性多囊性腎臟 Primary cilia (epithelial cell, kidney) loss mechanochemial sensors Bardet-biedl sndrome Retial degeneration, can not smell

62 SEM of epithelial cell Primary cilia Short stubs( 未端 ) Defective primary cilium in mouse mutants lacking components of the IFT particle