Microtubules and Intermediate Filaments

MOLECULAR CELL BIOLOGY SIXTH EDITION

MOLECULAR CELL BIOLOGY SIXTH EDITION

Copyright 2008 ©W. H. Freeman and Company

CHAPTER 18

Cell Organization and Movement II:

Microtubules and Intermediate Filaments

CHAPTER 18

Cell Organization and Movement II:

Microtubules and Intermediate Filaments

Lodish • Berk • Kaiser • Krieger • Scott • Bretscher •Ploegh • Matsudaira

© 2008 W. H. Freeman and Company

Lung cell in mitosis

Chromosomes (blue) Kertain intermediated filament (red)

Centrosomes (magenta)

Microtubule (green)

Microtubule are found in many different locations and all have similar structure

Cells contain stable and

unstable microtubules (MTs).

SEM of the surface of ciliated epithelium of

rabbit oviduct Microtubule base

motor protein

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.

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

_

Silimar to actin

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

The tubule is a complete

microtubule cylinder, made of 13

protofilaments

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.

Microtubule are assembled from MTOCs to generate

diverse organizations

Microtubules are assembled from microtubule organization centers (MTOC)

In non-mitotic cell, the MTOC=centrosome near nucleus (+) end of microtubule toward to periphery

During mitosis, MTOC=spindle pole

中心體

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 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 (γ-TURC) inside the centrosome.

Nucleating structures

- -

+

+

Two centrioles 9 Triplet microtubule

Centrosomes and the γ−tubulin ring complex

pericentriolar matrix

MTOC

The center of the center of the cell…

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

γ-TURC

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.

γ-tubulin ring complex lockwasher shape

Formins assemble unbranched filament

cap

-

+

γ-tubulin ring complexes (γ-TURC)

The γ-TURC nucleates microtubule assembly

assembly

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).

Intracellular tubulin

concentration: 10-20 μM

Cc: 0.03

“Treadmilling”

More easy polymerization

Microtubule are dynamic structures due to kinetic differences at their ends

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

Similar to actin

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

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

MAP : microtubule associated protein regulated polymerzation of

microtubule

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 +

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)

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 depends on the presence or absence of a GTP-β-tubulin cap

大災難

Colchicine → disassembled wash out colchicine Microtubule growth from MTOC

Immunofluorescent microscopy

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

Colchicine and other drugs disrupt MT dynamics.

Blocked at metaphase

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.

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

Some MAP bind to (+) end of microtubule, such as TIPs

Tubulin: green

+ TIP protein EB1

EB1 specific bind to (+)

Microtubules are disassembled by end binding and severing protein

Kinesin-13 enhanced the disassembly

Stathmin bind to protofilaments → enhances dissociation

ATPase

microfilaments

regulatory proteins

• G-actin binding proteins Thymosin β₄ (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 (-)

Myosins: Motor proteins that ‘walk’ along actin filaments

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

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

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.

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

Organelles in axons are transported along microtubule in

both direction

DIC microscopy demonstrates MT-based vesicle transport in vitro.

Has anterograde and retrograde

Squid axon

Kinesin-1 powers anterograde transport of vesicles down axons toward (+) end of microtubule

cargo

The structure of kinesin-1 microtubule 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

Structure of kinesin: two heavy chain and two light chain

Model of kinesin-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 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

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

Kinesin-14 move to (-)

4 chain ATP

Not all kinesins have the

same subunit structure

but all have the globular

head domain

Kinesin-1 uses ATP to “walk” down a microtubule

Motor protein

Convergent structural evolution of the ATP-binding core of

myosin head an kinesin

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

The Way Things Move: Looking Under the Hood of Molecular Motor Proteins

Ronald D. Vale and Ronald A. Milligan Science, 2000 288:80-95

Head-motor

Lever arm Converter Domain

“switch loop”

“Power stroke”

Linker region

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

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

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

The power stroke of dynein

Kinesin and dyneins cooperate in the transport of organelles throughout the cell

ERGIC: ER to Golgi

intermediate compartment

Cooperation of myosin and kinesin at the cell cortex.

actin

Kinesin and 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

Movement of pigment granules in frog melanophores

Microtubule: green Nucleus: blue

Pigment: red

Cilia an flagella: microtubule based surface structure

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

Structure of an axoneme (軸絲)

Structural organization of cilia and flagella

Video microscopy shows flagellar

movements that propel sperm and

chlamydomonas

forward

nexin

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

In vitro dynein-mediated sliding of doublet MTs

requires ATP.

Intrafllagellar transport (IFT) moves material up and down cilia and flagella

Intraflagellar transport Retrograde

anterograde

For growth

Defects in IFT cause disease by affecting sensory primary cilia

Autosomal recessive polycystic kidney disease (ADPKD)

Bardet-biedl sndrome 自體顯性多囊性腎臟

Primary cilia (epithelial cell, kidney) loss mechanochemial sensors

Retial degeneration, can not smell

Defective primary cilium in mouse mutants lacking components of the IFT particle SEM of epithelial cell

Short stubs(未端)

Primary cilia