Chapter 9.2 PDF - Cytoskeleton and Cell Motility

CHAPTER 9-part 2 The Cytoskeleton and Cell Motility: Actin and Intermediate Filaments Required reading: relevant sections of Chapter 9 9.2-1 9.1 | Overview of the Major Functions of the Cytoskeleton...

CHAPTER 9-part 2 The Cytoskeleton and Cell Motility: Actin and Intermediate Filaments Required reading: relevant sections of Chapter 9 9.2-1 9.1 | Overview of the Major Functions of the Cytoskeleton Properties of cytoskeletal components 9.2-2  9.7 | Intermediate Filaments Intermediate filaments have only been identified in animal cells Intermediate filaments are strong, flexible, ropelike fibers that provide mechanical strength to cells that are subjected to physical stress Unlike actin filaments and microtubules, IFs are a chemically heterogeneous group of structures that are encoded by approximately 70 different genes IFs can be divided into five major classes based on the type of cell in which they are found (as well as biochemical, genetic, and immunologic criteria) 9.2-3 Intermediate filament assembly does not involve ATP or GTP hydrolysis 9.7 | Intermediate Filaments IFs radiate through the cytoplasm of a wide variety of animal cells and are often interconnected to other cytoskeletal filaments by thin, wispy cross-bridges In many cells, these cross- bridges consist of an elongated dimeric protein called plectin Each plectin molecule has a binding site for an intermediate filament at one end and, depending on the isoform, a binding site for another intermediate filament, microfilament, or microtubule at the other end 9.2-4  9.7 | Intermediate Filaments Intermediate Assembly and Disassembly The basic building block of IF assembly is thought to be a rodlike tetramer Eight tetramers associate with one another in a lateral arrangement to form a filament that is one unit in length (about 60 nm) Unit lengths of filaments associate with one another in an end-to-end fashion to form the highly elongated intermediate filament None of these assembly steps require the direct involvement of either ATP or GTP The tetrameric building blocks lack polarity as does the assembled filament, which distinguishes IFs from other cytoskeletal elements 9.2-5  9.7 | Intermediate Filaments Intermediate Assembly and Disassembly Intermediate filaments are less sensitive to chemical agents than other types of cytoskeletal elements and more difficult to solubilize Localization of injected When labeled keratin subunits biotinylated are injected cells, they are keratin during a rapidly incorporated into 20-minute label existing IFs period The subunits are not incorporated at the ends of the filament but into the filament’s Distribution of interior intermediate Unlike the other two major filaments in the cytoskeletal elements, cell as revealed by anti-keratin assembly and disassembly of antibodies IFs are controlled primarily by subunit phosphorylation and dephosphorylation 9.2-6 Example of intermediate filaments: keratins Keratins: Provide strength to epithelial cells The most diverse IF family ~ 20 found in different types of human epithelial cells ~ 10 more specific to hair and nails Disulfide bonded keratin structures survive even in cell death (hair, nails, claws,scales) Keratins tend to be rich in cyteines 9.2-7  14.2 | Prophase The Dissolution of the Nuclear Envelope and Partitioning of Cytoplasmic Organelles at the end of prophase The three major components of the nuclear envelope are disassembled nuclear pores Nuclear membranes nuclear lamina composed of intermediate filaments (lamin) and membrane associated proteins The integrity of the nuclear membranes is first disrupted mechanically as holes are torn into the nuclear envelope by cytoplasmic dynein molecules associated with the outer nuclear membrane Phosphorylation of human lamin causes de-polymerization and subsequent disassembly of the lamina 9.2-8 Pathologies of intermediate filaments epidermolysis bullosa simplex (EBS), is from mutations in the gene that encodes a keratin polypeptide 9.2-9 Pathologies of intermediate filaments Example: desmin-related myopathy -mutations in the gene that encodes desmin Desmin integrates the various components of a muscle cell Leads to skeletal muscle weakness, cardiac arrhythmias, and eventual congestive heart failure Other diseases related to pathology of intermediate filaments 9.2-10 Ovrview of Cytoskeletalal organization 9.2-11  9.10 | Actin Microfilaments: (actin) Motility Shape Structural support Muscle contraction 9.2-12  9.8 | Actin and Myosin Actin Structure Minus (- ) end of G-actin The most abundant protein in cells G actin = globular F actin = filamentous In the presence of ATP actin monomers Plus (+) end of G-actin polymerize into a flexible helical filament The ATP binding cleft is oriented in the same direction in all actin subunits (monomers)in the filament Minus (-) end: the end of the filament with an exposed binding cleft (also called pointed end) binds to the plus end in a filament Plus (+) end: the other end (also called barbed end) is where the minus 9.2-13 end of an G actin binds  9.8 | Actin Actin Filament Assembly and Disassembly In-vitro: plus and minus ends have different polymerization rates In vivo: polymerization only occurs at the plus end by ATP-actin Minus end of G-actin The minus end may be anchored Only ADP-actin dissociates 9.2-14 Microfilaments form various structures (in accordance with their function) Actin polymerization and organization is regulated by Actin Binding Proteins 9.2-15 Actin binds to many, many accessory proteins in eukaryotic cells Myosins are actin-based motor proteins All share a characteristic “head” or “motor” domain (ATP-ase activity) Grouped into conventional and unconventional myosins Move towards + end (except myosin VI) Type II (conventional) myosins are best studied, responsible for muscle contraction, cytokinesis, cell migration Note the possibly confusing terminology: The type II myosins have subclasses, one of which is Myosin II One of the Unconventional myosins are associated with transport vesicles 9.2-16 and organelles  9.8 | Myosin: The Molecular Motor of Actin Conventional (Type II) Myosins Each myosin II molecule is composed of six polypeptide chains: one pair of heavy chains two pairs of light chains organized in such a way as to produce a highly asymmetric protein. Myosin II consists of (1) a pair of globular heads that contain the catalytic site of the molecule (2) a pair of necks, each consisting of a single, uninterrupted α helix and two associated light chains (3) a single, long, rod-shaped tail formed by the intertwining of long a-helical sections of the two heavy chains Assembles into fibers with the ends of the tails pointing toward the 9.2-17 center, and the globular heads pointing away 9.9 | Muscle Organization and Contraction Skeletal muscle: voluntary movement Muscle: bundles of parallel muscle fibers (cells) joined by tendons to the bones that the muscle must move Fibers: each fiber is a multinucleate cell formed during embryogenesis and specialized for contraction Myofibril: thinner cylindrical strands that make up a muscle fiber and consist of repeating units of sarcomeres 9.2-18 Sarcomere: the contractile unit of myofibrils each of which has a very specific organization 9.9 | Muscle Organization and Contraction Organization of Sarcomeres Each sarcomere extends from one Z line to the next Z line Thin filaments (actin) Thick filaments (myosin) 9.2-19 Sarcomere organization Actin has an orientation: Plus ends are anchored at Z-lines CapZ caps actin at plus end Tropomodulin caps the (-) end and regulates the length of actin filaments Nebulin: repeating actin binding motifs that binds actin filament to Z line Myomesin: bundles the myosin filaments Titin: extends through the myosin (thick) filament and attaches to the Z line – helps to prevent tearing of muscle 9.2-20 9.9 | Muscle Organization and Contraction The Sliding Filament Model of Muscle Contraction During contraction, the myosin molecules pull the surrounding thin filaments (actin), forcing them to slide toward the center of the sarcomere Individual myosins work asynchronously, so that only a fraction are active at any given instant The “neck” acts as a lever, amplifying the conformational change caused by ATP hydrolysis 9.2-21 Contractile cycle Conformational changes (mechanical) in the myosin head couple ATP hydrolysis (chemical) to movement 1) ATP binds to the cleft in the myosin head, releasing myosin from actin 2 and 3) ATP hydrolysis to ADP+Pi causes weak binding to actin 4) Pi release causes tighter binding and the power stroke that moves the thin filament toward the center of the sarcomere 5) ADP is released, freeing the ATP binding cleft 1) ATP binds to the cleft in the myosin head, releasing myosin 9.2-22 No ATP = rigor mortis Calcium ions trigger contraction via troponin and tropomyosin Tropomyosin masks the myosin Troponin complex (3 subunits): binds to binding sites on the actin filament tropomyosin Both have regulatory roles in contraction Calcium binding to troponin relieves the tropomyosin blockages of the interaction between actin and myosin head 1) Motor neuron excitation signal 2) Signal transduction pathway leads to Ca2+ release from the SR 3) Ca2+ binds to troponin (TnC subunit), causing conformation shift 4) Troponin conformation shift moves tropomyosin out of place 5) Myosin binding site on actin is exposed 6) When excitation signaling ceases, Ca2+ 9.2-23 are pumped back into SR, muscle relaxes Pathologies associated with myosins Familial hypertrophic cardiomyopathy: Genetically dominant inherited mutation in myosin (~2 per 1000 people) Over 40 different point mutations can lead to: Heart enlargement Abnormally small coronary vessels Cardiac arrhythmias 9.2-24 9.11 Actin-Binding Proteins Myosins are actin-based Actin polymerization and organization motor proteins is regulated by Actin Binding Proteins 9.2-25  9.11: Actin polymerization is regulated by G-actin binding proteins There is a (large) pool of soluble actin monomers generated in a cell whose polymerization is controlled by actin binding proteins Cofilin: binds ADP actin and severs filaments promoting depolymerization (at minus end) Profilin functions as an adenine nucleotide exchange factor Binds to ADP actin (at the plus end), changing the conformation and allowing binding of ATP Binding results in dissociation of profilin Minus end of an ATP actin then either joins a growing actin monomer filament at the plus end OR is bound by: Thymosin: sequesters G- actin preventing polymerization 9.2-26 Displacement of thymosin allows binding of G-actin to the plus end 9.11: Capping proteins stabilize F-actinCapping the plus end of the filament prevents further growth Capping the minus end prevents loss of subunits In muscle both ends of an actin filament are capped stabilizing the sarcomere and preventing either addition or loss of actin subunits plus end 9.2-27 9.11 Proteins that crosslink actin filaments Actin can be linked to other actin filaments Actin can be linked to the cell membrane (indirectly) Cell cortex: network of actin filaments and accessory proteins that underlies the plasma membrane in most eukaryotic cells 9.-26 9.2-28 9.11 Proteins that promote actin branching and growth Minus end of Plus end G-actin Arp2/3 complex: (actin related proteins) nucleates new branches off the sides of existing filaments Arps are activated by WASPs (Wiskott-Aldrich syndrome protein) Note orientation of minus and 9.2-29 plus ends and that G-actin is added to the plus end 9.12: Cell Motiity -growing filaments can push the cell membrane forward 1-2)Extracellular signal recognized and signal transduction cascade initiated 3-4) WASP activates Arp complex and Arp nucleate new actin filaments 5) result is branch formation at plus end 6) Membrane is pushed forward 7) Caps terminate elongation 8) Oldest part of filament at the minus end 9) F-actin servered and depolymerized at minus end 10) Profilin exchanges GDP to GTP 9.2-30 Regulation of the actin skeleton by Rho family of small GTPases Rho family GTPases often are bound to a guanine nucleotide dissociation inhibitor (GDI) in the cytosol 9.2-31 The GDI prevents Rho from interacting with it’s GEF at the plasma membrane The role of actin in endocytosis 9.2-32  Learning objectives Know the general characteristics of intermediate filaments Understand the role of plectins and lamins Know what regulates the assembly and disassembly of intermediate filaments Understand the structure of G-actin and F-actin and always be clear on polarity Know what myosins are, that there are two classes and the role of myosin II in muscle contraction Be clear on the role of the following structures and proteins in muscle and be clear on the polarity of skeletal muscle contraction Cap Z tropomodulin myomesin nebulin titan troponin tropomyosin Understand the structure of G-actin and assembly of F-actin in vivo (this includes the profilin, cofilin and thymosin cycle) Understand the role of the Arp complex and the fundamentals of branching and elongation and how this relates to cell motility Know what Rho is Know what a GDI is and it’s role Be able to compare and contrast microtubules, intermediate filaments and actin 9.2-33