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Slide 2 Cytoskeleton System Xiamixinuer Yilike Chapter 8 Slide 3 Teaching Requirements: 1. Mastering: concepts of the cytoskeleton; structure, chemical composition, and assembly of microtubules and microfilaments. 2. Comprehending: functions of microtubules and microfilaments. 3. Understanding: functions of the cytoskeleton; types and functions of intermediate filaments. 2 Slide 4 3 The cytoskeleton Slide 5 4 1. Introduction A. Conception of Cytoskeleton (Narrow sense) A complex network of interconnected microfilaments, microtubules and intermediate filaments that extends throughout the cytosol. Slide 6 5 The comparison among three types of the cytoskeleton Slide 7 6 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 Drugs and mutations (about functions) Biochemical analysis(in vitro) Slide 8 7 Fluorescence microscopy actin microtubules filamin microfilaments cytoskeleton microtubule s Slide 9 8 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 structure with many roles. Slide 10 9 D. The function of the cytoskeleton Structural support Internal framework maintaining position of the organelles Machinery required for movement of materials and organelles within cells Force generating elements responsible for movement of cells from one place to another Slide 11 10 2.Microtubule, MT A. Structures: Hollow Tubular structures 25nm in diameter Assembled from protein tubulin The tubulin consists of alpha-beta tubulin heterodimers arranged in rows (protofilaments) Form cytoskeleton, mitotic spindle, centrioles, core of cilia and flagella Slide 12 11 a and Tubulin heterodimers are the protein building blocks of MTs Slide 13 12 Arrangement of protofilaments in singlet, doublet, and triplet MTs SingletDoubletTriplet A B A B C In cilia and flagella In centrioles and basal bodies Slide 14 13 Assembling process of MT + - PEDAL OUTSIDE OF THE BODY Slide 15 14 tubulin tubulin heterodimer assemble Head tail connection profila ment MT 13 1 23 4 5 6 7 89 10 11 12 13 CROSSSECTION Slide 16 15 B. MTs assemble from microtubule- organizing centers (MTOCs) Microtubule-organizing centers (MTOCs) :is the region to assemble MT,Where includes - tubulin. MTOCs:include Centrosome, Mitotic spindle and Basal body. Slide 17 16 Microtubule-organizing centers (MTOCs) (1) Interphase: Centrosome Dynamic instability (2) Dividing cell: Mitotic spindle Dynamic instability (3) Ciliated cell: Basal body Stability Slide 18 17 Centrosome is a microtubule organizing center, MTOCs Slide 19 18 Centrosome containing a pair of centrioles Slide 20 19 Centrioles Centrioles are short cylinders with a 9 + 0 pattern of microtubule triplets. Centrioles may be involved in microtubule formation and disassembly during cell division and in the organization of cilia and flagella. Slide 21 20 Slide 22 21 MT are nucleated by a protein complex containing -tubulin The centrosome is the major MTOC of animal cells Slide 23 22 Experiments supporting that centrosome is the MTOC 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 + TubulinsMT + TubulinsNo A B Why the centrosome can act as MTOC? Slide 24 23 Cilia and flagella Cilia (small and numerous) and flagella (large and single) have a 9 + 2 pattern of microtubules and are involved in cell movement. Cilia and flagella move when the microtubule doublets slide past one another. Each cilium and flagellum has a basal body at its base. Slide 25 24 Slide 26 25 Basal body structure Slide 27 26 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 Slide 28 27 Microtubules have a plus and minus ends. Typically the minus is for anchoring and the plus is for growing. The transition between MT growth and MT shrinking is controlled in cells by special proteins.. Slide 29 28 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 Slide 30 29 D. Microtuble-associated proteins (MAPs) MAPs modulate MT structure, assembly, and function Katanin like proteinsMAPs 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 Slide 31 30 MAP2 associated with brain MTs Slide 32 5. Functions of MTs A. Maintenance of cell shape(constitute the centriols and cilia or flagella). B. Cell motility (see in cilia or flagella). C. Chromosome movements in cell division D. Organelle movement (MT associated motor proteins: kinesins: towards + end (anterograde transport) Golgi to ER or PM traffic;dyneins: towards - end (retrograde transport) ER to Golgi traffic.) 31 Slide 33 32 5. Functions of MTs A. Maintenance of cell shape(constitute the centriols and cilia or flagella). Slide 34 33 No centrioles in Plant and fungi A pair of centrioles are surrounded by electron dense pericentriolar material. Centrioles contain nine evenly spaced fibrils, each containing three microtubules, A, B and C tubules. A tubule is connected to the center of the centriole by a radial spoke. Centrioles are in pairs and at right angles to each other. Structure Constitute the centriols and cilia or flagella Slide 35 34 Constitute the centriols and cilia or flagella Slide 36 5. Functions of MTs B. Cell motility (see in cilia or flagella). 35 Slide 37 36 A comparison of the beating of flagella and cilia Slide 38 37 Ultrastructure of a eukaryotic flagellum or cilium Slide 39 38 SPERM MOVEMENT CILIA MOVEMENT Slide 40 39 Motor molecules and the cytoskeleton Slide 41 40 Motor molecules and the organelle Slide 42 41 Dyenin arms responsible for sliding Crosslinks and spokes responsible for bending Slide 43 42 B. Transport in the cytoplasm MT associated motor proteins: kinesins: towards + end (anterograde transport) Golgi to ER or PM traffic dyneins: towards - end (retrograde transport) ER to Golgi traffic Slide 44 43 C. Movement of chromosomes Slide 45 44 3. Microfilament, MF A. MFs are made of actin and involved in cell motility. Using ATP, G-actin polymerizes to form MF(F-actin) Slide 46 45 G-actin F-actin PLUS END MINUS END Slide 47 46 G-actinDimerTrimer F-actin +end -end Assembly of MF Slide 48 47 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. Slide 49 48 (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 minus end---- Tread-milling Slide 50 49 (3) Dynamic equilibrium between the G-actin and polymeric forms, which is regulated by ATP hydrolysis and G-actin concentration. Slide 51 50 (4) Dynamic equilibrium is required for the cell functions. Some MFs are temporary and others permanent. Slide 52 51 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 Slide 53 52 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 assembly of MF Slide 54 53 (2) MF-binding proteins Slide 55 Functions of MF (1) Maintenance of cell shape and enforce PM to change cell shape( i.e.Microvillus: Support the projecting membrane of intestinal epithelial cells) (2) Cell migration or motility (as in pseudopodia) (3) To form contractile ring in cell division: At cytokinesis (4) Muscle contraction Sarcomere is the unit of the muscle cells. (5)Cytoplasm streaming 54 Slide 56 Functions of MF (1) Maintenance of cell shape and enforce PM to change cell shape( i.e.Microvillus: Support the projecting membrane of intestinal epithelial cells) 55 Slide 57 56 Microvillus: Support the projecting membrane of intestinal epithelial cells Slide 58 57 A structure role of microfilaments Microfilaments (Actin filaments) Microvilli Intermediate filaments Slide 59 58 E. Functions of MFs (2) Cell migration (Fibroblast et al) Platelet activation is a controlled sequence of actin filament severing,uncapping, elongation,recapping, and cross-linking. Slide 60 59 (3) To form contractile ring in cell division : At cytokinesis E. Functions of MFs Slide 61 60 E.Functions of MFs (4) Muscle contraction Organization of skeletal muscle tissue Slide 62 61 Sarcomere Slide 63 5)Cytoplasm streaming:The streaming of cytoplasm in a circular motion around the cell observed in some plants, particularly young sieve tube elements. 62 Slide 64 63 Proteins play important roles in muscle contraction Myosin: The actin motor protein ATPase Binding sites Myosin II--Dimer Mainly in muscle cells Thick filamemts Slide 65 64 Tropomyosin, Tm and Tropnin, Tn Ropelike molecule Regulate MF to bind to the head of myosin Complex, Ca 2+ -subunit Control the position of Tm on the surface of MF Slide 66 65 Excitation- contraction coupling process Slide 67 66 Intermediate filaments, IFs IFs are the most abundant and stable components of the cytoskeleton Slide 68 67 Class I. + II. (MW 40 - 70 000) Cytokeratins epithelial cells ( > 20 isoforms, skin, hair, nails) Class III. (MW ~53 000) Vimentin cells of mesenchymal origin Desmin muscle GFAPs astroglial cells (= Glial fibrillary acidic proteins) Class IV. (MW 130, 100 and 60 000) Neurofilament proteins in neural cells Class V. (MW = 65-75 000) Nuclear lamins inside surface of the inner nuclear membrane most dynamic INTERMEDIATE FILAMENT PROTEIN MONOMERS: (cell-type-specific) (~ 310 amino acids) Slide 69 68 monomer coiled-coil dimer staggered anti-parallel tetramer two tetramers helical array of tetramers made of 8 protofilaments Slide 70 Functions of IF 1.Maintenance of cell shape, 2.Anchore of nucleus and certain other organelles, 3.Formation of nuclear lamina 69 Slide 71 70 The comparison among three types of the cytoskeleton Slide 72 71 Summary of cytoskeleton Three types of cytoskeletal filaments are common to many eucaryotic cells and are fundamental to the spatial organization of these cells. 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) 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. Cytoskeletal systems are dynamic and adaptable. Nucleation is rate-limiting step in the formation of a cytoskeletal polymer. 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. 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. Slide 73 Homework for cytoskeleton system 1.Conception types and the functions of the cytoskeleton 2.Structures of MT 3.building blocks of MTs and MFs 4.Arrangement of protofilaments 5.MTOC and its elements 6.Specific drugs stabilize MTs or MF 7.Functions of MTs and MFs 8.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. 72