复旦大学：《细胞生物学 Cell Biology》课程教学资源（PPT课件）第九章 Cytoskeleton System 细胞骨骼系统

Chapter 9 Cytoskeleton System 1. Introduction A Conception of Cytoskeleton(Narrow sense) A complex network of interconnected microfilaments, microtubules and intermediate filaments that extends throughout the cytosol Microtubules Microfilaments 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 o Fluorescent microscopy and Electron microscopy: Immunofluorescence: fluorescently-labeled antibod 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 4 Drugs and mutations(about functions

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 o The cytoskeleton is a dynamic strucrure with many roles

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 MF A MFS are made of actin and involved in cell motility. Using ATP, g-actin polymerizes to form MF(F-actin) plus end actin molecule plus end COOH (ADP when in filament) minus end

2. Microfilament, MF A. 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: (I)Within a me, all the actin monomers are oriented in the same direction, so MF has a polarity Light chains Minus end Myosin ..ooooo.o Myosin is (a) Bret treatment with trypsin Hinge egion Heads Actn molecular motor for 。4+ Myosin segmen actins Tht meromyosin Heavy meromyosin (b)Further treatment with trypsin Subfragment 1 Subfragment 2 (s2) Plus end 025m Plus(barbed)end Subfragment 1 (c) EM and diagram of S1 fragments'decoratingactin microfilaments

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 mfgrow, 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---r 0×080

(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 ADPATP actin with actin with bound ADP bound ATP

(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 Q 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

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

Nucleus Nucleus Nucleus Factin G-actin @(lend (+1 endo Nucleation Elongation Steady state Elongation-外k- Steady state-→ Filament + nucle Monomer Actin concentration Figure 6-17 The three phases of G-actin polymerization in vitro

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. (B) (C)

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