Cytoskeleton SystemA. Conception of Cytoskeleton (Narrow sense) A complex network of interconnected microfilaments, microtubules and intermediate filaments that extends throughout the cytosol.Chapter 9MicrobubulesMicrofilamemtsIntermediate filaments1. Introduction

B. Techniques for studying the cytoskeletonFluorescent 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 EMBiochemical analysis (in vitro) Difference centrifugation; SDS-PAGEDrugs and mutations (about functions)

C. The self-assembly and dynamic structure of cytoskeletal filamentsEach 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 disassemblyCharacteristics:(1) Within a MF, all the actin monomers are oriented in the same direction, so MF has a polarityMyosin 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 dynamicsCytochalasins: 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 proteinsThe 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 arrangemainly found in microvillus and filopodium (). Contractile bundle: MFs anti-parallel arrange, mainly found in stress fibers () and contractive ring of mitosis( ) Gel-like network: MFs cross-linked arrange, most be found in cell cortex (cytosol, ).

(2) MF-binding proteins

nucleating proteins: actin-related proteins, ARPs monomer-sequestering protein: thymosinEnd-blocking(capping) proteins:capZ monomer-polymerizing proteins:profilinATP- actin filament-depolymerizing proteins: cofilinADF cross-linking proteins: ABP280, filament-severing proteins: gelsolin membrane-binding proteins:vinculinERMezrinradixinmoesin

Model of the complementary roles of profilin and thymosin 4 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 proteinsThe formation of two types of actin filament bundles:Contractile bundle mediated by -actinin parallel bundle mediated by fimbrin.

Gel-like networkActin 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 fibersComposed of actin filaments and myosin-II Stress FibersFocal contactsFocal contacts MFsResponse to tensionResponse to tension

(5) Contractile ring: For cytokinesis

(6) Muscle contractionOrganization of skeletal muscle tissue

Sarcomere

? ? ??

Proteins play important roles in muscle contraction Myosin: The actin motor porteinATPaseMyosin II--DimerMainly in muscle cellsThick filamemts

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

Tropomyosin, Tm and Tropnin, TnRopelike moleculeRegulate MF to bind to the head of myosinComplex, Ca2+-subunitControl the position of Tm on the surface of MF

Thick and thin filaments sliding model

Excitation-contraction coupling processAction potentialCa2+ rise in cytosolTnTmSliding

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( ) contractionSmooth and nonmuscle cell contraction are regulated in a manner distinct from that of skeletal muscle cellsCa2+ riseCa2+ -calmodulinBind to MLCKRegulate light chain PhosphorylateMyosin interact with actinContractionSLOW

3.Microtubule, MTTubulin heterodimersare the protein building blocks of MTsA. Structures:

Arrangement of protofilaments in singlet, double, and triplet MTsSingletDoubleTripletABABCIn cilia and flagellaIn centrioles and basal bodies

B. MTs assemble from microtubule-organizing centers (MTOCs)(1) Interphase: CentrosomeDynamic instability(2) Dividing cell: Mitotic spindleDynamic instability(3) Ciliated cell: Basal bodyStability

Basal body structure

C. Characteristics of MT assemblyDynamic 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 MTOCStructureNo centrioles in Plant and fungi

MT are nucleated by a protein complex containing ?-tubulinThe centrosome is the major MTOC of animal cells

Drugs affect the assembly of MTs(1) ColchicineBinding to tubulin dimers, prevent MTs polymerization(2) TaxolBinding to MTs, stabilize MTsThese compounds are called antimitotic drugs, and have application in medical practice as anticancer drugs

Microtuble-associated proteins (MAPs)MAPs modulate MT structure, assembly, and functionMotor MAPsNonmotor MAPsTau: In axon, cause MTs to form tight bundlesMAP2: In dendrites, cause MTs to form looser bundlesControl organization

The importance of MAPs for neurite formationLike axonLike dendrite

Organization of MT bundles by MAPs . Spacing of MTs depends on MAPsInsect cell expressing MAP2Insect cell expressing tauFrom 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.??

5. Functions of MTs1. Maintain cell shape

2. Motor proteins and intracellular transport1Motor proteins: 3 superfamily Kinesin dependent-MT (2KHC+2KLC), N-/M-KinesinC-kinesin MT ATP600 Cytoplasmic Dynein dependent-MT (2/3HC+ more LC) 14um/s ; CATP;N Axonemal arm dyneins: Myosin dependent-MF

Intracellular transport of membrane-bounded vesicles, proteins: Directionality(2) Kinesin is a plus-end directed MT motor protein Hand-over-hand model1985--conventional kinesin-kinesin-related proteinsKRPsKLPskinesin-like proteins50KLPsKLPsKLPs

Comparison of the mechanochemical cycles of kinesin and myosin II.Motor proteins generate force by coupling ATP hydrolysis to conformational changes.?? 1 ATP, ?8 nm, ? 1 um.

(3) Dynein is a minus-end directed motor proteinAxonemal and cytoplasimic dyneinsMW=1500KDa 1. 2. Mediate: dynactin complex

Intracellular transport in nerve cellsMt 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 movementSize and length: The same diameter, flagella are often much longerMovement: Cilia: Beating; Flagella: Bending motionCiliary dynein

Structure:

Microtubule sliding causes cilia/flagella to bendDyneinsCrosslinks 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 IFsMonomers: Fibrous proteinsAntiparallel tetramer: No polarityAlmost 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 tissuesDisruption 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 cytoskeleton1. Three types of cytoskeletal filaments are common to many eucaryotic cells and are fundamental to the spatial organization of these cells.The set of accessory proteins is essential for the controlled assembly of the cytoskeletal filaments(includes the motor proteins: myosins, dynein and kinesin)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.

SSDS