Cytoskeleton System

CytoskeletonCytoskeleton SystemSystem

CytoskeletonCytoskeleton SystemSystem

XiamixinuerXiamixinuer··Yilike Yilike

Chapter 8

Teaching Requirements:

1. Mastering: concepts of the cytoskeleton; structure, chemical composition, and assembly of microtubules and microfilaments.

2. Comprehending: functions of microtubules and microfilaments.

3. Understanding: functions of the cytoskeleton; types and functions of intermediate filaments.

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The cytoskeleton

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1. IntroductionA. Conception of Cytoskeleton (Narrow sense)A complex network of interconnected microfilaments, microtubules and intermediate filaments that extends throughout the cytosol.

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The comparison among three types of the cytoskeleton

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B. Techniques for studying the cytoskeleton

Fluorescent microscopy and Electron

microscopy :

Immunofluorescence: fluorescently-labeled antibody

Fluorescence: microinject into living cells

Video microscopy: in vitro motility assays

Electron: Triton X-100, Metal replica

Drugs and mutations (about functions)

Biochemical analysis(in vitro)

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Fluorescence microscopy

actin

microtubules

filamin

microfilaments

cytoskeleton

microtubules

microtubules

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C. The self-assembly and dynamic structure of cytoskeletal filaments

Each type of cytoskeletal filament is constructed

from smaller protein subunits.

The cytoskeleton is a network of three filamentous

structures.

The cytoskeleton is a dynamic structure with many

roles.

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D. The function of the cytoskeleton

Structural support

Internal framework maintaining position of the organelles

Machinery required for movement of materials and organelles within cells

Force generating elements responsible for movement of cells from one place to another

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2.Microtubule, MTA. Structures:

Hollow

Tubular structures 25nm in diameter

Assembled from protein tubulin

The tubulin consists of alpha-beta tubulin

heterodimers arranged in rows (protofilaments)

Form cytoskeleton, mitotic spindle, centrioles, core

of cilia and flagella

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a and ßTubulin heterodimers are the protein building blocks of MTs

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Arrangement of protofilaments in singlet, doublet, and triplet MTs

Singlet Doublet Triplet

A

B

A

B

CIn cilia and flagella

In centrioles and basal bodies

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Assembling process of MT

+

-

PE

DA

L

OUTSIDE OF THE BODY

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tubulin tubulin

heterodimerassemble Head tail connection

profilament

MT（ 13）

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4

5

6

7

8910

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CROSSSECTION

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B. MTs assemble from microtubule-organizing centers

(MTOCs)

Microtubule-organizing centers (MTOCs):is the region to assemble MT,Where

includes-tubulin.MTOCs:include Centrosome, Mitotic spindle

and Basal body.

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Microtubule-organizing centers (MTOCs)

(1) Interphase: Centrosome

Dynamic instability

(2) Dividing cell:

Mitotic spindle

Dynamic instability

(3) Ciliated cell: Basal body

Stability

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Centrosome is a microtubule organizing center, MTOCs

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Centrosome containing a pair of centrioles

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Centrioles

Centrioles are short cylinders with a 9 + 0 pattern of microtubule triplets.

Centrioles may be involved in microtubule formation and disassembly during cell division and in the organization of cilia and flagella.

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MT are nucleated by a protein complex containing -tubulin

The centrosome is the major MTOC of animal cells

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Experiments supporting that centrosome is the MTOC

Treat cell with colcemid

Cytosolic MTs depoly, except those in centrosome

Remove colcemid

Tublin repoly

Expla I: MTOC nucleate poly of tubulins

Expla II: MTOC gather MTs in cytosol

centrosome + Tubulins MT

+ Tubulins No

A

B

Why the centrosome can act as MTOC?

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Cilia and flagella

Cilia (small and numerous) and flagella (large and single) have a 9 + 2 pattern of microtubules and are involved in cell movement.

Cilia and flagella move when the microtubule doublets slide past one another.

Each cilium and flagellum has a basal body at its base.

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Basal body structure

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C. Characteristics of MT assembly

Dynamic Dynamic instability due to instability due to the structural the structural differences differences between a growing between a growing and a shrinking and a shrinking microtubule end.microtubule end.

GTP cap;GTP cap;

Catastrophe: Catastrophe: accidental loss of accidental loss of GTP cap;GTP cap;

Rescue: regain Rescue: regain of GTP capof GTP cap

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Microtubules have a Microtubules have a

plus and minus ends.plus and minus ends.

Typically the minus is Typically the minus is

for anchoring and the for anchoring and the

plus is for growing.plus is for growing.

The transition The transition

between MT growth between MT growth

and MT shrinking is and MT shrinking is

controlled in cells by controlled in cells by

special proteins..special proteins..

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Drugs affect the assembly of MTs

(1) Colchicine

Binding to tubulin dimers, prevent MTs polymerization

(2) Taxol

Binding to MTs, stabilize MTs

These compounds are called antimitotic drugs, and have application in medical practice as anticancer drugs

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D. Microtuble-associated proteins (MAPs)

MAPs modulate MT structure, assembly, and function

Katanin like proteins MAPs

Tau: In axon, cause MTs to form tight bundles

MAP2: In dendrites, cause MTs to form looser bundles

MAP1B: In both axons and dendrites to form crossbridge

between microtubules

Control organization

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MAP2 associated with brain MTs

5. Functions of MTs• A. Maintenance of cell shape(constitute the

centriols and cilia or flagella).• B. Cell motility (see in cilia or flagella).• C. Chromosome movements in cell division • D. Organelle movement (MT associated motor

proteins: kinesins: towards + end (anterograde transport) Golgi to ER or PM traffic;dyneins: towards - end (retrograde transport) ER to Golgi traffic.)

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5. Functions of MTs

A. Maintenance of cell shape(constitute the centriols and cilia or flagella).

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No centrioles in Plant and fungi

A pair of centrioles are surrounded by electron dense pericentriolar material.

Centrioles contain nine evenly spaced fibrils, each containing three microtubules, A, B and C tubules.

A tubule is connected to the center of the centriole by a radial spoke.

Centrioles are in pairs and at right angles to each other.

Structure

Constitute the centriols and cilia or flagella

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Constitute the centriols and cilia or flagella

5. Functions of MTs• B. Cell motility (see in cilia or

flagella).

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A comparison of the beating of flagella and cilia

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Ultrastructure of a eukaryotic flagellum or cilium

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      SPERM MOVEMENT

   CILIA MOVEMENT

Motility of M

T（

CIL

IA,F

ILA

GE

LA

MO

VE

ME

N

T

）

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Motor molecules and the cytoskeleton

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Motor molecules and the organelle

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Dyenin arms responsible for sliding

Crosslinks and spokes responsible for bending

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B. Transport in the cytoplasm

MT associated motor proteins:

kinesins: towards + end (anterograde transport) Golgi to ER or PM traffic

dyneins: towards - end (retrograde transport) ER to Golgi traffic

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C. Movement of chromosomes

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3. Microfilament, MFA. MFs are made of actin and involved in cell motility.

Using ATP, G-actin polymerizes to form MF(F-actin)

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G-actin

F-actin

PLUS END

MINUS END

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G-actin Dimer Trimer

F-actin+end

-endAssembly of MF

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B. MF assembly and disassembly

Characteristics:

(1) Within a MF, all the actin monomers are oriented in the same direction, so MF has a polarity

Myosin is molecular motor for actins.

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(2) In vitro, (Polymerization) both ends of the MF grow, but the plus end faster than the minus.

Because actin monomers tend to add to a filament’s plus end and leave from its minus end---- “Tread-milling”

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(3) Dynamic equilibrium between the G-actin and polymeric forms, which is regulated by ATP hydrolysis and G-actin concentration.

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(4) Dynamic equilibrium is required for the cell functions. Some MFs are temporary and others permanent.

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C. Specific drugs affect polymer dynamics

Cytochalasins:

Prevent the addition of new monomers to existing MFs, which eventually depolymerize.

Phalloidin:

A cyclic peptide from the death cap fungus, blocks the depolymerization of MF

Those drugs disrupt the monomer-polymer equilibrium, so are poisonous to cells

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D. Actin-binding proteins

The structures and functions of cytoskeleton are mainly controlled by its binding proteins

(1) Monomer-sequestering proteins

Bind with actin monomers and prevent them from polymerizing.

thymosin and ( profilin) Promoting the assembly of MF

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(2) MF-binding proteins

Functions of MF• (1) Maintenance of cell shape and enforce

PM to change cell shape( i.e.Microvillus: Support the projecting membrane of intestinal epithelial cells)

• (2) Cell migration or motility (as in pseudopodia)

• (3) To form contractile ring in cell division: At cytokinesis

• (4) Muscle contraction ： Sarcomere is the unit of the muscle cells.

• (5)Cytoplasm streaming54

Functions of MF• (1) Maintenance of cell shape

and enforce PM to change cell shape( i.e.Microvillus: Support the projecting membrane of intestinal epithelial cells)

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Microvillus: Support the projecting membrane of intestinal epithelial cells

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A structure role of microfilaments

Microfilaments (Actin filaments)

Microvilli

Intermediate filaments

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E. Functions of MFs

(2) Cell migration (Fibroblast et al)

Platelet activation is a controlled sequence of actin filament severing,uncapping, elongation,recapping, and cross-linking.

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(3) To form contractile ring in cell division :

At cytokinesis

E. Functions of MFs

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E. Functions of MFs

(4) Muscle contraction

Organization of skeletal muscle tissue

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Sarcomere

5)Cytoplasm streaming:The streaming of cytoplasm in a circular motion around the cell observed in some plants, particularly young sieve tube elements.

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Proteins play important roles in muscle contraction

Myosin: The actin motor protein

ATPase

Binding sites

Myosin II--Dimer

Mainly in muscle cells

Thick filamemts

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Tropomyosin, Tm and Tropnin, Tn

Ropelike molecule

Regulate MF to bind to the head of myosin

Complex, Ca2+-subunit

Control the position of Tm on the surface of MF

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Excitation-contraction

coupling process

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Intermediate filaments, IFs

IFs are the most abundant and stable components of the cytoskeleton

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Class I. + II. (MW 40 - 70 000) Cytokeratinsepithelial cells ( > 20 isoforms, skin, hair, nails)

Class III. (MW ~53 000) Vimentin cells of mesenchymal originDesmin muscle GFAPs astroglial cells (= Glial fibrillary acidic proteins)

Class IV. (MW 130 , 100 and 60 000)Neurofilament proteins in neural cells

Class V. (MW = 65-75 000)Nuclear lamins inside surface of the inner nuclear membranemost dynamic

INTERMEDIATE FILAMENT PROTEIN MONOMERS: (cell-type-specific)

(~ 310 amino acids)

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monomer

coiled-coil dimer

staggeredanti-parallel tetramer

twotetramers

helical arrayoftetramersmade of8 protofilaments

Functions of IF1.Maintenance of cell shape,2.Anchore of nucleus and certain

other organelles,3.Formation of nuclear lamina

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The comparison among three types of the cytoskeleton

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Summary of cytoskeletonSummary of cytoskeleton

• Three types of cytoskeletal filaments are common to many Three types of cytoskeletal filaments are common to many eucaryotic cells and are fundamental to the spatial eucaryotic cells and are fundamental to the spatial organization of these cells.organization of these cells.

• The set of accessory proteins is essential for the controlled The set of accessory proteins is essential for the controlled assembly of the cytoskeletal filaments(includes the motor assembly of the cytoskeletal filaments(includes the motor proteins: myosins, dynein and kinesin)proteins: myosins, dynein and kinesin)

• Cytoskeletal systems are dynamic and adaptable.Cytoskeletal systems are dynamic and adaptable.

• Nucleation is rate-limiting step in the formation of a Nucleation is rate-limiting step in the formation of a cytoskeletal polymer.cytoskeletal polymer.

• Regulation of the dynamic behavior and assembly of the Regulation of the dynamic behavior and assembly of the cytoskeletal filaments allows eucaryotic cells to build an cytoskeletal filaments allows eucaryotic cells to build an enormous range of structures from the three basic filaments enormous range of structures from the three basic filaments systems. systems.

Homework for cytoskeleton system

1. Conception types and the functions of the cytoskeleton

2. Structures of MT

3. building blocks of MTs and MFs

4. Arrangement of protofilaments

5. MTOC and its elements

6. Specific drugs stabilize MTs or MF

7. Functions of MTs and MFs

8. Cytoskeletal systems are dynamic and adaptable.

Nucleation is rate-limiting step in the formation of a

cytoskeletal polymer.Regulation of the dynamic behavior

and assembly of the cytoskeletal filaments allows eucaryotic

cells to build an enormous range of structures from the

three basic filaments systems. 72