B. Techniques for studying the cytoskeleton 2. Microfilament, MF A. MFs are made of actin and involved in cell motility. B. MF assembly and disassembly C. Specific drugs affect polymer dynamics D. Actin-binding proteins E. Functions of MFs (1) Maintain cell’s shape and enforce PM (2) Cell migration (Fibroblast et al) (3) Microvillus: Support the projecting membrane of intestinal epithelial cells (4) Stress fibers (5) Contractile ring: For cytokinesis (6) Muscle contraction Sarcomere Proteins play important roles in muscle contraction Thick and thin filaments sliding model Excitation-contraction coupling process 3.Microtubule, MT Tubulin heterodimers are the protein building blocks of MTs Arrangement of protofilaments in singlet, double, and triplet MTs B. MTs assemble from microtubule-organizing centers (MTOCs) Basal body structure C. Characteristics of MT assembly Why the centrosome can act as MTOC Experiments supporting that centrosome is the MTOC MT are nucleated by a protein complex containing -tubulin Drugs affect the assembly of MTs Microtuble-associated proteins (MAPs) Organization of MT bundles by MAPs . Spacing of MTs depends on MAPs 5. Functions of MTs 1. Maintain cell shape Intermediate filaments, IFs IFs are the most abundant and stable components of the cytoskeleton 3. Function of IFs: Confer mechanical strength on tissues Summary: Cytoskeletal functions Fig. 10-31 Microtubule dynamics in a living cell. A fibroblast was injected with tubulin that had been covalently linked to rhodamine, so that approximately 1 tubulin subunit in 10 in the cell was labeled with a fluorescent dye. Note, for example, that microtubule #1 first grows and then shrinks rapidly, whereas microtubule #4 grows continuously. (P.J. Sammak et al., Nature 332: 724-736) 细胞内物质运输Motor Protein 1. Kinesin Family 2. Dynein Family 染色体运动Figure 16-84 Electron micrographs of an insect flight muscle viewed in cross-section. The myosin and actin filaments are packed together with almost crystalline regularity. (From J. Auber, J. de Microsc. 8:197-232) Myosin: The actin motor portein ATPase Binding sites 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 Action potential Ca2+ rise in cytosol Tn Tm Sliding A. Structures: Singlet Double Triplet A B A B C In cilia and flagella In centrioles and basal bodies (1) Interphase: Centrosome Dynamic instability (2) Dividing cell: Mitotic spindle Dynamic instability (3) Ciliated cell: Basal body Stability 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 Structure No centrioles in Plant and fungi 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 The centrosome is the major MTOC of animal cells (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 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 MAPs MAP1A, MAP1B, MAP1C MAP2, MAP2c MAP3 ，MAP4 Tau The importance of MAPs for neurite formation 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. * * 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 of protein filaments that form 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) 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. Using ATP, G-actin polymerizes to form MF(F-actin)  Figure 16-51 The trapping of ADP in an actin filament. 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---- “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 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. 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 dish sweep backward over its dorsal surface - a movement known as ruffling. (Courtesy of Julian Heath.) 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 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 microscopy 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). (A, B, and C, courtesy of Paul Forscher.) 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 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 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