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1、Cytoskeleton System,A. Conception of Cytoskeleton (Narrow sense) A complex network of interconnected microfilaments, microtubules and intermediate filaments that extends throughout the cytosol.,Chapter 10,Microbubules,Microfilamemts,Intermediate filaments,1. Introduction,Figure 10-2. The three types
2、 of protein filaments that form the cytoskeleton.,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-
3、100, Metal replica Drugs and mutations (about functions) Biochemical analysis(in vitro),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.
4、 The cytoskeleton is a dynamic strucrure with many roles.,2. Microfilament, MF,A. MFs are made of actin and involved in cell motility.,Using ATP, G-actin polymerizes to form MF(F-actin),Figure 16-51 The trapping of ADP in an actin filament.,B. MF assembly and disassembly,Characteristics:,(1) Within
5、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 filaments plus end and leave from its m
6、inus end- “Tread-milling”,(3) Dynamic equilibrium between the G-actin and polymeric forms, which is regulated by ATP hydrolysis and G-actin concentration.,(4) Dynamic equilibrium is required for the cell functions. Some MFs are temporary and others permanent.,(5)The nucleation of actin filaments at
7、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
8、a cell.,Figure 16-55 Lamellipodia and microspikes at the leading edge of a human fibroblast migrating in culture. The arrow in this scanning electron micrograph shows the direction of cell movement. As the cell moves forward, lamellipodia and microspikes that fail to attach to the tissue culture dis
9、h sweep backward over its dorsal surface - a movement known as ruffling. (Courtesy of Julian Heath.),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, b
10、locks the depolymerization of MF,Those drugs disrupt the monomer-polymer equilibrium, so are poisonous to cells,Figure 16-52 The effect of cytochalasin on the leading edge of the growth cone of a nerve cell in culture. A living growth cone is viewed by Nomarski differential-interference-contrast mic
11、roscopy both before (A) and after (B) treatment with cytochalasin. The cell in (B) has then been stained with rhodamine phalloidin to reveal the actin filaments (C). Note how the region behind the leading edge of the cytochalasin-treated growth cone is devoid of actin filaments. Cytochalasin B (D).
12、(A, B, and C, courtesy of Paul Forscher.),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 assembl
13、y of MF,Figure 16-53 Two possible mechanisms by which an actin-monomer-binding protein could inhibit actin polymerization. It is thought that thymosin inhibits actin polymerization in one of these ways.,(2) MF-binding proteins,Actin filaments are likewise strongly affected by the binding of accessor
14、y 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 desta
15、bilized 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
16、actin-cross-linking proteins,The formation of two types of actin filament bundles: Contractile bundle mediated by -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
17、 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 cells shape and enforce PM,Figure 10-75 A model for how integrins in the plasma membrane co
18、nnect intracellular actin filaments to the extracellular matrix at a focal contact. The formation of a focal contact occurs when the binding of matrix glycoproteins (such as fibronectin) on the outside of the cell causes the integrin molecules to cluster at the contact site, as illustrated schematic
19、ally in (A). A possible arrangement of some of the intracellular attachment proteins that mediate the linkage between an integrin and actin filaments is shown in (B).,(2) Cell migration (Fibroblast et al),Platelet activation is a controlled sequence of actin filament severing,uncapping, elongation,r
20、ecapping, and cross-linking.,(3) Microvillus: Support the projecting membrane of intestinal epithelial cells,Figure 16-77 Freeze-etch electron micrograph of an intestinal epithelial cell, showing the terminal web beneath the apical plasma membrane. Bundles of actin filaments forming the core of micr
21、ovilli extend into the terminal web, where they are linked together by a complex set of cytoskeletal proteins that includes spectrin and myosin-II. Beneath the terminal web is a layer of intermediate filaments. (From N. Hirokawa et al., J. Cell Biol. 91:399-409),(4) Stress fibers,Composed of actin f
22、ilaments and myosin-II,Stress Fibers,Focal contacts,Focal contacts IFs,Response to tension,Response to tension,(5) Contractile ring: For cytokinesis,(6) Muscle contraction,Organization of skeletal muscle tissue,Sarcomere,Figure 16-84 Electron micrographs of an insect flight muscle viewed in cross-se
23、ction. The myosin and actin filaments are packed together with almost crystalline regularity. (From J. Auber, J. de Microsc. 8:197-232),Proteins play important roles in muscle contraction,Myosin: The actin motor portein,ATPase,Binding sites,Myosin II-Dimer,Mainly in muscle cells,Thick filamemts,Ligh
24、t-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,Excita
25、tion-contraction coupling process,Action potential,Ca2+ rise in cytosol,Tn,Tm,Sliding,3.Microtubule, MT,Tubulin heterodimers are the protein building blocks of MTs,A. Structures:,Arrangement of protofilaments in singlet, double, and triplet MTs,Singlet,Double,Triplet,A,B,A,B,C,In 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,