Cytoskeleton System A. Conception of Cytoskeleton (Narrow sense) A complex network of interconnected microfilaments, microtubules and intermediate filaments.

Cytoskeleton System

A. Conception of Cytoskeleton (Narrow sense) A complex network of interconnected microfilaments,

microtubules and intermediate filaments that extends throughout the cytosol.

Chapter 9

Microbubules Microfilamemts Intermediate filaments

1. Introduction

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

Quick freezing-deep etching EM

Biochemical analysis (in vitro)

Difference centrifugation; SDS-PAGE

Drugs and mutations (about functions)

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 strucrure with many roles.

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

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

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.

(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----

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

2.2 Assembly

◆Mechanism of actin polymerization: 3 phases of G-actin polymerization.

◆ Critical concentration (Cc). In steady state, G-actin monomers only exchange with subunits at the filament ends but there is no net change in the total mass of filaments.

◆ During the elongation state, one end of the filament, the (+) end, elongates five to ten times faster than does the opposite (-) end. This is because Cc value is much lower for G-actin addition at the (+) end than for addition at the (-) end.

Figure 6-17 The three phases of G-actin polymerization in vitro.

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

(5)The nucleation of actin filaments at the PM is frequently regulated by external signals, allowing the cell to change its shape and stiffness rapidly in response to changes in its external environment.

This nucleation is catalyzed by a complex of proteins that includes two actin-related proteins, or ARPs(Arp2 and Arp3).

Actin arrays in a cell.

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

D. Actin-binding proteins

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

2.4 microfilament-binding proteins

Actin binding proteins control the structure and behavior of actin filament.

◆ actin binding proteins

e.g. proflin (promote acting assembly), thymosin beta4 (inhibits actin assembly).

Some cytosolic proteins control actin polymerization.

◆ microfilament-binding proteins

◆ 3 different types of stalbe actin filament structures ： ◆ Parallel bundle: MFs isotactic parallel arrange ， mainly found in microvillus and filopodium ( 丝

状伪足 ).

◆ Contractile bundle: MFs anti-parallel arrange, mainly found in stress fibers ( 应力纤维 ) and cont

ractive ring of mitosis( 有丝分裂收缩环 ) 。

◆ Gel-like network: MFs cross-linked arrange, most be found in cell cortex (cytosol, 细胞皮层 ).

(2) MF-binding proteins

成核蛋白（ nucleating proteins ） : actin-related proteins, ARPs

单体–隔离蛋白（ monomer-sequestering protein ） : thymosin

封端（加帽）蛋白（ End-blocking(capping) proteins ） :capZ

单体–聚合蛋白（ monomer-polymerizing proteins ） : 抑制蛋白（ profilin ）是一种与 ATP- 肌动蛋白单体结合的蛋白质

肌动蛋白纤维解聚蛋白（ actin filament-depolymerizing proteins ） : cofilin 、 ADF 以及蚕食蛋白与肌动蛋白纤维的减端结合，大大促进肌动蛋白纤维解聚成单体。

交联蛋白（ cross-linking proteins ） : ABP280 和细丝蛋白 , 促进形成近于正交相互联系的纤维松散网络

纤维–切割蛋白（ filament-severing proteins ） : gelsolin

膜结合蛋白（ membrane-binding proteins ） : 连接膜与肌动蛋白的蛋白质包括联结蛋白（ vinculin ）， ERM 家族的成员包括埃兹蛋白（ ezrin ）、根蛋白（ radixin ）和膜突蛋白（ moesin ）

Model of the complementary roles of profilin and thymosin β4 in regulating polymerization of G-actin.

Actin filaments are likewise strongly affected by the binding of accessory proteins along their sides.

Actin filaments in most cells are stabilized by the binding of tropomyosin, an elongated protein. Which can prevent the filament from interacting with other proteins.

Another important actin filament binding protein, cofilin, present in all eucaryotic cells, which destabilized actin filaments(also called actin depolymerizing factor).

Cofilin binds along the length of the actin filament, forcing the filament to twist a little more tightly.

In addition, cofilin binding cause a large increase in the rate of actin filament treadmilling.

The modular structures of four actin-cross-linking proteins

The formation of two types of actin filament bundles:

Contractile bundle mediated by α-actinin

parallel bundle mediated by fimbrin.

Gel-like network Actin filaments are often nucleated at the plasma membrane. The highest density of actin filaments is at the cell periphery forming cell cortex.

Filamin cross-links actin filaments into a three-dimensional network with the physical properties of a gel.

Loss of filamin causes abnormal cell motility

E. Functions of MFs

(1) Maintain cell ’ s shape and enforce PM

(2) Cell migration (Fibroblast et al)

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

(3) Microvillus: Support the projecting membrane of intestinal epithelial cells

(4) Stress fibers

Composed of actin filaments and myosin-II

Stress FibersFocal contacts Focal contacts MFs

Response to tensionResponse to tension

(5) Contractile ring: For cytokinesis

(6) Muscle contraction

Organization of skeletal muscle tissue

Sarcomere

皮肤 ? 蛋白

同伴皮肤 ? 蛋白

平原皮肤球 ??蛋白

Proteins play important roles in muscle contraction

Myosin: The actin motor portein

ATPase

Myosin II--Dimer

Mainly in muscle cells

Thick filamemts

Light-chain phosphorylation and the regulation of the assembly of myosin II into thick filaments

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

Thick and thin filaments sliding model

Excitation-contraction coupling process

Action potential

Ca2+ rise in cytosol

Tn

Tm

Sliding

Schematic diagram showing how a Ca2+-release channel in the sarcoplasmic reticulum membrane is thought to be opened by a voltage-sensitive transmembrane protein in the adjacent T-tubule membrane

F. Smooth muscle cell( 平滑肌细胞 ) contraction

Smooth and nonmuscle cell contraction are regulated in a manner distinct from that of skeletal muscle cells

Ca2+ rise Ca2+ -calmodulin Bind to MLCK

Regulate light chain Phosphorylate

Myosin interact with actin Contraction

SLOW

3.Microtubule, MT Tubulin heterodimers

are the protein building

blocks of MTsA. Structures:

Arrangement of protofilaments in singlet, double, and triplet MTs

Singlet Double Triplet

A

B

A

B

CIn cilia and flagella

In centrioles and basal bodies

B. MTs assemble from microtubule-organizing centers (MTOCs)

(1) Interphase: Centrosome

Dynamic instability

(2) Dividing cell:

Mitotic spindle

Dynamic instability

(3) Ciliated cell: Basal body

Stability

Basal body structure

C. Characteristics of MT assembly

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

GTP cap;

Catastrophe: accidental loss of GTP cap;

Rescue: regain of GTP cap

Why the centrosome can act as MTOC

Structure

No centrioles in Plant and fungi

MT are nucleated by a protein complex containing ?-tubulin

The centrosome is the major MTOC of animal cells

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

Microtuble-associated proteins (MAPs)

MAPs modulate MT structure, assembly, and function

Motor MAPs Nonmotor MAPs

Tau: In axon, cause MTs to form tight bundles

MAP2: In dendrites, cause MTs to form looser bundles

Control organization

The importance of MAPs for neurite formation

Like axon Like dendrite

Organization of MT bundles by MAPs . Spacing of MTs depends on MAPs

Insect cell expressing MAP2

Insect cell expressing tau

From J. Chen et al. 1992. Nature 360: 674

The effects of proteins that bind to MT ends

(A)The transition between Mt growth and Mt shrinking is controlled in cells by special proteins.

(B)Capping proteins help to localize Mt in budding yeast cell.

?? 蛋白

5. Functions of MTs

1. Maintain cell shape

2. Motor proteins and intracellular transport

（ 1 ） Motor proteins: 3 superfamily

Kinesin dependent-MT (2KHC+2KLC),

N-/M-Kinesin 向正极运动； C-kinesin 向负极运动

头部具 MT 和 ATP 结合位点；迄今已报道 600 余种

Cytoplasmic Dynein dependent-MT (2/3HC+ more LC)

14um/s ; C 端为马达结构域（结合 ATP ） ;N 端为易变尾部

Axonemal arm dyneins: 与纤毛鞭毛运动有关

Myosin dependent-MF

Intracellular transport of membrane-bounded vesicles, proteins: Directionality

(2) Kinesin is a plus-end directed MT motor protein

Hand-over-hand model

1985 年发现的驱动蛋白分子 --“ 常规驱动蛋白”（ conventional kinesi

n ），只是相关蛋白超家族中的一个成员，驱动蛋白 - 相关蛋白（ kinesin-rel

ated proteins ）超家族简称为 KRPs

或 KLPs （ kinesin-like proteins ）。根据基因组序列分析，估计哺乳动物能产生 50 种以上不同的 KLPs 。 KLPs

的头部都含有相关的氨基酸序列，反映出它们具有共同的进化祖先，并且在沿微管运动方面具有相似的作用。比较而言， KLPs 的尾部有较大变异，具有各种不同的序列，反映出不同的驱动蛋白

成员运送不同的货物。

Comparison of the mechanochemical cycles of kinesin and myosin II.

Motor proteins generate force by coupling ATP hydrolysis to conformational changes.

?? 星期三解释的 1 分子 ATP, 先前 ?8 nm, 相当于 ? 秒 1 um.

(3) Dynein is a minus-end directed motor protein

Axonemal and cytoplasimic dyneins

MW=1500KDa 胞质动力蛋白至少有两种作用：

1. 在有丝分裂期间作为纺锤体定位和染色体移动的产力装置 。

2. 作为在胞质中定位高尔基复合体和将小泡和细胞器移向减端的微管马达蛋白。

Mediate: 动力蛋白激活蛋白复合体 （ dynactin complex ）

Intracellular transport in nerve cells

Mt organization in fibroblasts and neurons.

Movement of pigment granules: color adjustment

The placement of organelles

3. Movement of mitotic spindle and chromosomes

4. Cilia and flagella: Structure and movement

Size and length: The same diameter, flagella

are often much longer

Movement: Cilia: Beating;

Flagella: Bending motion

Ciliary dynein

Structure:

Microtubule sliding causes cilia/flagella to bend

Dyneins Crosslinks and spokes

Intermediate filaments, IFs

IFs are the most abundant and stable components of the cytoskeleton

1. IFs assemble from fibrous subunits

Assembly Characteristics of IFs

Monomers: Fibrous proteins

Antiparallel tetramer: No polarity

Almost no IF monomers within cell

But IFs are still dynamic polymers in the cell

IF typing serves as a diagnostic tool in medicine

2. IF proteins are tissue-specific

3. Function of IFs: Confer mechanical strength on tissues

Disruption of keratin networks causes blistering

IFs are cross-linked and bundled into strong arrays;

IFs are ropelike fibers with a diameter of around 10nm;

IFs are made of IF proteins, which constitute a large and heterogeneous family.

Less is understood about the mechanism of assembly and disassembly of IFs than of actin filaments and microtubules, but they are clearly highly dynamic structures in most cell types.

Summary: Cytoskeletal functions

Summary of cytoskeleton

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

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

3. Cytoskeletal systems are dynamic and adaptable.

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

5. 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.