Chapter 20:

Molecular Cell Biology

Fifth Edition

Chapter 20:

Cytoskeleton II: Microtubules

Copyright © 2004 by W. H. Freeman & Company

Harvey Lodish • Arnold Berk • Paul Matsudaira • Chris A. Kaiser • Monty Krieger • Matthew P. Scott •

Lawrence Zipursky • James Darnell

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.

Kinesin powered movement of a vesicle along a microtubule

Assembly and disassembly cause microtubules → probe →….

Long-distance movement

Microtuble organized around the MTOC and spindle

microtubule-organizing center is “-”

--Orientation of cellular microtubule

most microtubules have a constant orientation relative to MTOC

(-) end: close to the MTOC (+) end: distal to the MTOC

Cells contain stable and unstable microtubules (MTs).

SEM of the surface of ciliated epithelium of rabbit oviduct

Fig 5-29

1 2 3

Polymer of globular Tubulin → arranged → microtubule Two populations of MT:

1. stable, long-lived: nonreplicating cell; cilia, flagella, RBC and plaelets pass through small vessel, axon

2. unstable, short-lived; assemble and disassemble quickly, replicating cell

Heterodimeric

tubulin subunits compose the wall of a MT.

Tubulin subunit are formed by

α and β

activity.

55kDa monomers in all eukaryotes γ-tubulin are formed by α and β.

Irreversibl y, does not hydrolyze

GTP reversibly

+

add

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

_

seam microtubule 3-start helix

β-GDP α-GTP β-GTP α-GTP

+

− α β

GTP GTP

tubulin heterodimer

α β α β

protofilament

(−) (+)

Thus the minus end of an a subunit may serve as GAP(GTPase activating protein) for b-tubulin of the adjacent dimer in a protofilament

Arrangement of protofilaments in singlet, doublet, and triplet MTs.

The tubule is a complete microtubule cylinder, made of 13 protofilaments

Temperature affects whether MTs assemble or disassemble.

High Temp: assembly (need GTP) Low Temp: disassembly

Cc: critical concentration

Up: dimers polymerize into microtubules; below: depolymerize

Addition of MT fragments demonstrates polarity of tubulin polymerization. (MT assembly and disassembly take place preferentially at the + end)

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 Slowed 2 fold than +

Cryoelectron microscopy allows observation of disassembled MTs.

Rate of MT growth in vitro is much slower than shrinkage (收 縮).

縮短

急轉直下

Disassembly quick (7μm/min) Assembly slowly (1μm/min)

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.

Dynamic instability model of MT growth and shrinkage (β subunit bind to GTP/GDP).

+ end is most important for MT growth and shrinkage Dissociation of GDP-tubulin

dimer >>>> GTP-tubulin dimer

When GDP-tubulin →

depolymerizes and unstabilized When GTP-tubulin add to + end

→ rescued shortening MT.

(β subunit)

Two factor influence stability of MT:

1. Cc

2. GTP or GDP –β subunit

--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

Numerous proteins regulate MT dynamics and cross-linkage to other structures.

MAPs: microtubule-associated protein, influence the assembly and stability of microtubule and their related structure.

Basis of their function has two groups:

Basic microtubule binding and acidic projection domain

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

Related with

disease

Cross-link MT

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;

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

Colchicine and other drugs disrupt MT dynamics.

Blocked at metaphase

Drugs involved in microtubule dynamics

--its effect is reversible --binds to tubulin dimer

blocks the addition or removalof other tubulin subunits to the ends of microtubule

disruptionof 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 microtubuleby inhibiting the lengthening and shortening of microtubules

--used for cancer treatment

MTOCs orient most MTs and determine cell

polarity.

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 most animal cells.

Centrioles (C) a pair, C and C’

Pericentriolar (PC)matrix: γ-tubulin & pericentrin; surrounding the centrioles 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 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-tubulinring complexes inside the centrosome.

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

Stain with an anti-gamma tubulinantibody.

Gamma-tubulin at initiates synthesis at one end (-) (green).

The center of the center of the cell…

Centrioles in a centrosome connect to centromeres MTOC=microtubule organizing center

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” (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.

γ-tubulin ring complex lockwasher shape cap

Gripproteins of the cap may be involved in mediating binding to the centrosome.

Phosphorylationof a conserved tyrosine residue of γ-tubulin has been shown to regulate microtubule nucleation in yeast cells.

Cytoplasmic organelles and vesicles are organized by MTs.

Colocalization of endoplasmic reticulum membranes and cytosolic MTs.

Others organelles also colocalized.

DiOC6, ER binding fluorescent dye

Anti-tubulin The alignment of the ER network

and microtubules in many but not all regions of the cytoplasm is evident because the cell has sparse microtubules.

Kinesin and dynenin powered movements

Progression of organelles along axons requires microtubules and the motor proteins: kinesin and dynein.

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

The rate of axonal transport in vivo can be determined by radiolabeling and gel electrophoresis.

Anterograde transportgoes towards the axon terminal (cell body → synaptic terminals), such as vesicles.

Retrograde transportgoes 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.

Motors carrying different cargoes in different directions

fibroblast neuron

+

-

DIC microscopy demonstrates MT-based vesicle transport in vitro.

Has anterograde and retrograde

Squid axon

Kinesin and Dynein-powered movement

Melanosomes in fish pigment cells aggregate or disperse by moving along a network of MTs.

cargo

microtubule Two major mediator for

transport along microtubules:

1. Kinesins (驅動蛋白) and dyneins (動力蛋白) 2. Cilia and flagella

Mucus secretion sperm

Transport of GFP-tagged neurofilaments down axons exhibits periodic pauses.

Every 5 sec

15μm

????

Speed down

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

Microtubule based motor protein

Kinesin I powers anterograde transport of vesicles in axons.

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

Not all kinesins have the same subunit structure but all have the

globular head domain Structure of kinesin: two heavy chain and two light chain

α-helix region

cargo

Model of kinesin-catalyzed vesicle transport.

cargo

Most kinesins are processive + end-directed motor proteins

binds only one monomer at a time in a processive manner

ATP hydrolysis coupled to movement EM data suggests binding primarily to β-

tubulin 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

How does kinesin move?

tubulin heterodimer α-tubulin β-tubulin ATP binding to the leading head initiates neck linker docking

Neck linker docking is completed by the leading head, which throws the partner head forward by 160 Å toward the next tubulin binding site catalytic core

tightly docked neck linker

detached neck linker

The new leading head docks tightly onto the binding site The trailing

head hydrolyzes ATP to ADP-Pi

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

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

Bind to MT Very large multimeric 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.

Dynactin complex

Dynein needs dynactin to link vesicles and chromosomes to the dynein light chain

Multiple motor proteins sometimes move the same cargo

General model of kinesin and dynein mediated transport in a typical cell

注意有些是錯的,原則是dyneins direct to -, and kinesins direct to +

Cooperation of myosin and kinesin at the cell cortex.

actin

Kinesinand dynein: Motor proteins that ‘walk’ along microfilaments

Myosins: Motor proteins that ‘walk’ along actin filaments

Motor proteins are enzymes that couple the hydrolysis of ATP to a conformational change

Cytoplasm

+ -

Organelle transport uses motor proteins

Dynein Kinesin

RER to Golgi vesicle Golgi to RER vesicle

Eukaryotic cilia and flagella contain a core of doublet MTs studded with axonemal dyneins.

Structure of an axoneme (軸絲)

Freeze-etching reveals structure of axonemal

dynein. _motor

domain

Video microscopy shows flagellar movements that propel sperm and

chlamydomonas

forward

Ciliary and flagellar beating are produced by controlled sliding of outer double MTs.

In vitro dynein- mediated sliding of doublet MTs requires ATP.

Comparison of the mechanochemical cycles of kinesin and myosin II.

Short time

Microtuble dynamics and motor protein in mitosis

The stages of mitosis and cytokinesis in an animal cell.

Fluorescence microscopy reveals changes in the organization of chromosomes and MTs at four mitotic stages.

Mitotic apparatus is a microtubule machine for separating chromosomes

Electron microscopy visualizes components of the mitotic apparatus in a metaphase mammalian cell.

交叉

Connected chromosome

Prophase signals must convert interphase array to mitotic apparatus - increase in short dynamic microtubules -mitotic MT turnover 5-10 fold fasterthan interphase MT - less polymer and more monomer tubulin during M phase than at other times 動粒

星狀體 紡錘體 Chromosomes align at the

equatorial plane

Kinetochore is a centromere(著絲點)-based protein complex that mediates attachment of chromosomes to MTs.

All eukaryotes, three components participate in attaching chromosomes to microtubules:

1. Centromere

2. Kinetochore and spindle proteins

3. Cell cycle mechinery Inner

kinetocho re layer

Outer kinetocho re layer

kinesins CENP-E: keeps the kinetochore

tethered to the kinetochore microtubule

MACK: plusends of spindle microtubules attach to chromosomes

A common sort of model for the kinetochore and its MT attachment: the action is in a sleeve

Relation of centrosome duplication to the cell cycle.

Parent centrioles

Daughter centrioles

Grow complete

Centrosome cycle or centriole cycle

Duplicated centrosomes align and begin separating in prophase

G1 Phase Æ 1st growth phase S Phase Æ DNA duplicated G2 Phase Æ Final growth phase Mitosis

Cytokinesis

Model for participation of MT motor proteins in centrosome movements at

prophase.

Centrosomes must be positioned properly with microtubules that are shorter and more dynamic

Kinesins implicated in organizing polar MT into a bipolar array

Mitotic kinesin, Kin-C, is (-) end directedand aids in alignment of polar MT

Bipolar kinesin, BimC, cross-links antiparallel MT and pushes them apart

Dynein also helps to tether astral MT and orient the poles

BimC motor domains walk toward the plus ends of overlapping polar microtubules, pushing the poles apart, as tubulin heterodimers add to the plus ends.

Model for participation of microtubule motor proteins in centrosome movements at prophase

Cytosolic dynein participates in the formation and stabilization of mitotic spindle poles.

Tubuline dynein

Capture of chromosomes by MTs in prometaphase.

The kinetochore contacts lateral side of MT and then slides along to the (+) end, powered by dynein and mitotic kinesins on the kinetochore. Alternatively, kinetochore might contact (+) end directly

Kinetochore “caps” the (+) end but there is polymerization and depolymerization of tubulin such that chromosomes exhibit oscillating behavior

Model of the forces stabilizing metaphase chromosomes at the equatorial plate.

Cytoplamic dynein – end directed motor

+ end directed motor

+ end directed motor

CENP-E, a kinesin, do not mediated movement, keeps the kinetochore tethered to the kinetochore microtubule

Chromosome alignment

(1) rapid polymerization/depolymerization at the (+) end of kinetochore microtubule (2) motor proteins pull chromosomes towards pole

(i) (+) end-directed motor protein at spindle pole (ii) (-) end-directed motor protein at kinetochore (dynein) (iii) CENP-E: tethers the kinetochore the shrinking microtubule (3) polar microtubule polymerization push chromosomes away from pole

chromokinesin, a non-kinetchore (+) end-directed motor on the chromatid arm:

pushing chromosome away

Poleward flux of tubulin subunits during metaphase is visualized by fluorescence speckle microscopy.

Fluorescent tubulin subunit

Metaphase live cell

Do not has fluorescence

Anaphase chromoseomes separate and spindle elongates

Anaphase A (early): shortening of kinetochore at +, pulls chromosomes toward poles

Anaphase B (late): two poles move farther apart, brining attached chromoseomes with them into two daughter

Shortening at the (+) end of kinetochore MTs moves chromosomes

pole ward in anaphase A

Shortening at + end (attach kinetochores) of microtubule by disassembly.

In vivo fluorescent-tagging experiment Kinetochore associated kinesin, MCAK →

promotes disassembly at the + end, CENP- E (also at kinetochore) binds to

progressively shortening end.

Chromosome move toward to “-” Loss fluorescent

Shortening at the + end of kinetochore microtubules moves chromosomes poleward in anaphase A

Model of spindle elongation and movement of poles during anaphase B.

Three processes of anaphase B for separation of chromosomes:

1. Pushing force by kinesin-mediated (BimC, attach microtubule) sliding of polar microtubules (move toward +)

2. Pulling force by cortex- associated cytosolic dynein (move toward -) 3. Lengthening of polar

microtubules at + end

Aster

The latter stages of anaphase usually include Significant spindle elongation: anaphase B

Micromanipulation experiments can determine whether the spindle or the asters control location of the cleavage plane during cytokinesis.

Small glass ball Asters determine two cell separate

Two asters determines where cleavage occurs in fertilized sand dollar eggs, whereas the spindle determines the cleavage planein animal cells

CDK1 (cyclin-dependent kinase) → entry into mitosis, by phosphorylation of the regulatory light chanis in myosin II.

Block microtubule elongation

Regulation of myosin light chain by mitosis-promoting factor (MPF).

MPF: CDK1 and mitotic cyclin protein

Plant cells reorganize their MTs & build a new cell wall in mitosis.

Interphase plant cells: lack a single perinuclear microtubule-organizing center Similar to animal:

Prophase: Bundle their cortical microtubules and reorganize, without centrosomes Metaphase: golgi-derived vesiclesare transported into the mitotic apparatus along

microtubules

Telophase: vesicles line up near the center of the dividing cell and to form the phragmoplast, a membrane structure similar to animal cell contractile ring → vecome the plasma membrane of daughter cells; vesicles contains cellulose pectin for cell wall

I. Microtubule structure

1. tubulins and microtubule structure 2. Microtubule-organizing center (MTOC) II. Microtubule dynamics & associated proteins

1. Assembly/disassembly of microtubule 2. Dynamic instability

3. Temperature influences microtubule stability 4. Drugs involved in microtubule dynamics 5. Microtubule associated protein (MAP) III. Motor proteins and intracellular transport

1. Motor proteins

--microtubule motor proteins: Kinesin family, Dynein family

2.Multiple motor proteins are associated with membrane vesicles Chapter 20 summary

IV. Microtubules & motor proteins during mitosis 1. Mitotic apparatus

2. Centromere and kinetochore 3. Centrosome duplication

4. Microtubule dynamics during mitosis 5. Centrosome movement during mitosis

6. Formation of spindle poles and capture of chromosomes 7. Chromosome separation & spindle elongation

8. Cytokinesis

各位同學實在是辛苦了 再撐完下週的考試，就功德圓滿。

一分努力一分收獲，相信自己也相信老師 一切都會是值得的

想想，人生就是一直創造不可能，

老師很幸運，有這麼多的修課同學陪我又創造一次不可能

各位同學，細胞生物學這門課，

可能是您這輩子從頭考到尾的一門課

這個記錄就在人生求學生涯中----最美麗的中山大學裡 創造出來

敬祝大四同學鵬程萬里 大三同學立足中山放眼未來

大二同學福樂智慧