3. Cell Biology-Cytoskeleton_Bradshaw_NOTES.pdf

Cytoskeleton Name: Amy Bradshaw Office: STB 325 Email: [email protected] Phone: 792-4959 OUTLINE: Cytoskeleton aka How Do Cells Organize and Move? A. Overview of Cytoskeleton B. Main Types of Cytoskeletal Elements 1. Microfilaments 2. Intermediate Filaments 3. Microtubules C. Microfilaments 1. Mole...

Cytoskeleton Name: Amy Bradshaw Office: STB 325 Email: [email protected] Phone: 792-4959 OUTLINE: Cytoskeleton aka How Do Cells Organize and Move? A. Overview of Cytoskeleton B. Main Types of Cytoskeletal Elements 1. Microfilaments 2. Intermediate Filaments 3. Microtubules C. Microfilaments 1. Molecular Structure 2. Assembly and Disassembly 3. Actin Binding Proteins 4. Cellular Organization 5. Examples of Microfilament Organization D. Myosin 1. Classes of Myosin 2. Molecular Structure of Myosin II 3. Regulation of Myosin II Contraction: NM II and SM II 4. Contractile Assemblies of Microfilaments and Non-muscle Myosin II (NM II) 5. Non-Conventional Myosins E. Microfilaments and Cell Motility 1. Steps in Cell Motility 2. Examples of Cell Motility 3. Role of Microfilaments in Cell Motility F. Intermediate Filaments 1. Types of Intermediate Filament Proteins 2. Assembly of Intermediate Filaments 3. Function of Intermediate Filaments 4. Intermediate Filaments and Disease G. Microtubules 1. Molecular Structure 2. Assembly and Disassembly of Microtubules 3. Dynamic Instability of Microtubules 4. Microtubule Associated Proteins (MAPs) 5. Microtubule Transport 6. Cilia and Flagella 7. Microtubule-targeted Drugs in Chemotherapy OBJECTIVES: After studying these lectures you should be able to: 1). Describe the protein subunit composition of the 3 main cytoskeletal elements. 2). Explain how microfilaments are assembled and disassembled continuously. 3). Describe how actin binding proteins alter structure and function of microfilaments. 4). Describe the molecular structure and corresponding cellular functions of myosin I versus Conventional myosin aka Non-muscle myosin II. 5). Explain how contraction of conventional myosin is regulated and specify 3 contractile assemblies that can form via interactions with microfilaments. 6). Specify the 4 basic steps involved in cell motility and describe the role of microfilaments and myosin. 7). Specify the main types of intermediate filament (IF) proteins and explain how subunits are assembled into intermediate filaments. 8). Describe how IF are linked to desmosomes and hemidesmosomes. 9). Why are identification of specific IFs in tumor pathology useful? 10). Describe the molecular structure of microtubules including subunit composition, arrangement of protofilaments and diameter. 11). Explain how microtubules are assembled and disassembled. 12). Explain the concept of dynamic instability of microtubules. 13). Describe alterations in structure and function of microtubules produced by microtubule associated proteins (MAPs). 14). Describe the molecular structure of kinesins and dyneins and their corresponding roles in intracellular transport. 15). Describe the structure and function of cilia and flagella and explain the molecular basis of movement. READING REFERENCE: Medical Cell Biology, 3rd Edition, Edited by Steven R. Goodman, Chapter 3, p. 75-100 Junqueira’s Basic Histology, 12th Edition: Text and Atlas 2010, Chapter 2. 7/25/2022 Cytoskeleton (and Molecular Motors) Amy D. Bradshaw, PhD Illustrations adapted from: The Cell: A Molecular Approach © 2000 ASM Press and Sinauer Associates, Inc.; Medical Cell Biology, 3rd Ed. © 2008, Elsevier Inc.; Molecular Biology of the Cell © 2002, Garland Science; Molecular Cell Biology © 2008, W. H. Freeman and Company; Junqueira’s Basic Histology, 12th Edition: Text and Atlas © 2010, McGraw‐Hill Main Types of Cytoskeletal Components 1. Microfilaments (F-actin) - 7-9 nm in diameter - Polymers of G-actin: DYNAMIC 2. Intermediate Filaments - 10 nm in diameter - Polymers of heterogeneous proteins: STABLE 3. Microtubules - 25 nm in diameter - Polymers of  and  tubulin subunits: DYNAMIC Microfilaments Microtubules Intermediate Filaments Fig Immunofluorescence of the same cell using antibodies that detect the 3 primary types of cytoskeletal components and the 3 cytoskeletal networks. 1 7/25/2022 Microfilaments: Actin cytoskeleton Molecular Structure: G‐actin monomers polymerize to form F‐actin nucleation F‐actin (Filamentous actin) G‐actin monomers (Globular actin) polymerization 14 G‐actin monomers per turn of helix G‐actin monomers must be bound to ATP for polymerization; ATP is hydrolyzed to ADP•Pi after polymerization into F‐actin Microfilaments Assembly and Disassembly: Microfilaments are dynamic meaning that assembly and disassembly can occur continuously in the cell a) Polarity of F-actin: G-actin•ATP is added to plus ends of F-actin at a much faster rate than the minus ends because ADP•actin dissociates more readily than ATP•actin • Plus (+) end = faster growing end • Minus (-) end = slower growing end b) Treadmilling: Results from the difference in critical concentrations of G-actin required for polymerization at the plus end versus the minus end - At intermediate G-actin concentrations, loss from the minus end is balanced by addition at the plus end c) Reversibility - F-actin can depolymerize when necessary by dissociation into G-actin Pathway 2 7/25/2022 Actin Binding Proteins: To harness and direct the energy of actin polymerization, the cell has a large, heterogeneous group of proteins that regulate structure and function of F‐actin in all cell types ‐ Examples of proteins and their function are listed in the Table below: Actin Binding Protein Function Formin F-actin nucleation & polymerization Arp 2/3 complex F-actin branching Fimbrin; -Actinin; Villin F-actin cross-linking (bundles) Filamin F-actin cross-linking (networks) Gelsolin; ADP/cofilin F-actin severing & depolymerization Cap Z Capping of F-actin plus ends -Catenin; Spectrin; Vinculin; Talin Linkages to other cytoskeletal proteins Microfilament Organization The dynamics of actin polymerization and structural support provided by actin binding proteins allows for the generation of cellular structures such as lamellipodia and filopodia. 3 7/25/2022 Cellular Organization Microfilaments are organized into a variety of structural patterns and are dependent upon the cell types as well as the intracellular location within a particular cell 1) Microfilament bundles - Formed by cross-linking of F-actin • Surface projections such as microvilli - cross-linked by Fimbrin & Villin Small intestines • Stress fibers - cross-linked by ‐Actinin Cellular Organization Microfilaments are organized into a variety of structural patterns and are dependent upon the cell types as well as the intracellular location within a particular cell 2). Microfilament networks - Crosslinking of F-actin by Filamin • Pseudopodia for phagocytosis • Lamellipodia in leading edge during cell movement 4 7/25/2022 Actin and Myosin (A Great Cellular Team) • The actin cytoskeleton (microfilaments) can generate forces through polymerization/depolymerization as well as through actin:myosin interaction. • Force generation is critical for cell movement and extracellular matrix (ECM) assembly. Myosins  Superfamily of motor proteins present in all cell types.  Interact with F-actin to function in cellular processes that require generation of force or translocation of molecules and organelles. Myosin “walking” along a microfilament Mehta A J Cell Sci 2001;114:1981-1998 5 7/25/2022 Myosins Myosin molecules can: 1) carry cargo by “walking” along microfilaments 2) generate tension on microfilaments 3) propel the sliding of microfilaments for cellular contraction and movement. Myosin “walking” along a microfilament (IRL) Mehta A J Cell Sci 2001;114:1981-1998 Myosins Classes of Myosin:  At least 20 classes of myosin have been identified. Myosin proteins are ubiquitously expressed in almost all cell types. They are divided into different classes.  Myosin II is the class containing traditional or conventional myosins such as sarcomeric myosin (skeletal and heart muscle), smooth muscle myosin (SMII) and non-muscle myosin (NM II). 6 7/25/2022 Molecular Structure of Conventional Myosin a) Myosin Heavy Chains (MHC) – MHCs contain the following 3 domains: • Motor (head) - globular domain, conserved sequence containing ATPase activity • Neck (lever) - domain contains sequences that bind to MLCs or calmodulin • Tail (rod) - a-helical domain, most variable in sequence and length - diverse functions b) Myosin Light Chains (MLC)– Bind to MHCs and regulate myosin function: Ca2+ binding proteins regulated by intracellular Ca2+ levels • Essential Light Chains (ELC) • Regulatory Light Chains (RLC) Motor Domain Tail Domain Fig RLC ELC Coiled-coil of MHC tails forms a dimer Schematic of Myosin II (conventional myosins) Regulation of Myosin II (aka Conventional Myosin) Contraction: a) Phosphorylation of RLC by Myosin Light Chain Kinase (MLCK) in response to Ca2+ Ca2+ + Calmodulin Schematic Ca2+•Calmodulin Complex MLCK•Ca2+•Calmodulin (active) MLCK (inactive) ATP ADP Pi Pi 7 7/25/2022 Regulation of Myosin II (aka Conventional Myosin) Contraction: b) Effects of RLC Phosphorylation on Myosin Activity • Increases ATPase activity in MHC motor domain • Enables assembly of bipolar filaments via interactions in the MHC tail domains filament sliding ends ends Myosin II microfilaments filament sliding Nature Reviews Molecular Cell Biology 10: 778‐790, 2009 Myosins Examples of Contractile Assemblies of Microfilaments and Conventional Myosin II (Non Muscle (NM) II) a) Stress Fibers in Focal Adhesions are Used for Cell Migration and ECM Assembly: NM II interacts with microfilaments of stress fibers to generate tension that enables the cell to “pull” and to counteract outward forces on the cell receptors generated by interaction with the extracellular matrix (ECM). ECM 8 7/25/2022 Myosin Examples of Contractile Assemblies of Microfilaments and Conventional Non‐muscle Myosin II (NM II) b) Adherens Junctions allows for contraction of sheets of cells: Adherens junctions form a continuous “belt” of microfilaments around a layer of epithelial cells - NM II interacts with these microfilaments to generate a contractile belt that is mechanically linked to the cell surface receptors the Cadherins via Vinculin,  and  Catenin. F-actin Vinculin Cadherin Myosin Examples of Contractile Assemblies of Microfilaments and Conventional Non‐muscle Myosin II (NM II) Contractile ring c) Contractile Ring: A contractile ring containing microfilaments and NM II forms during telophase of mitosis- the microfilaments are connected to the cell membrane which causes constriction and ultimately cleavage into 2 cells 9 7/25/2022 Microfilaments and Cell Motility Steps in Cell Motility 1. Extension of leading edge toward direction of movement: lamellipodia and filopodia actin based. 2. Attachment of lamellipodia and filopodia to the substratum: integrin based. 3. Development of tension and cell movement: actin/myosin based. 4. Retraction of trailing edge Examples of Cell Motility Cell migration during development, wound healing, inflammatory cell recruitment and metastatic movement of cancer cells. Microfilaments in Cell Motility Actin polymerization works with actin binding proteins and the actin:myosin motor to drive cell movement. • Rapid polymerization of F-actin network at the plus ends generates force that pushes on cell membrane to produce lamellipodia and filopodia. Formins are actin-binding proteins that link the plus ends of F-actin to the cytoplasmic surface of the cell membrane. • NM II generates sliding of F-actin in opposite direction toward the minus ends. • Cell moves forward when F-actin polymerization at the plus ends is faster than sliding of F-actin toward the minus ends. 10 7/25/2022 Non‐Conventional Myosins Non-conventional myosins are implicated in movement of proteins and organelles along microfilaments as opposed to force generation. An example is Myosin I: a) Myosin I: Smallest class- consists of single Myosin Heavy Chain, Myosin Light Chain = calmodulin. • Motor domain - binds to microfilaments • Tail domain - binds to proteins and organelles b) Functions of Myosin I: • Myosin I moves along microfilament toward plus end carrying cargo linked to the tail domain • Myosin I functions in microvilli where it forms lateral links or “struts” between bundles of microfilaments and the cell membrane + end - end Motor Domain Tail Domain Calmodulin Intermediate Filaments Main fxn(2) Intermediate filaments are composed of a heterogeneous group of proteins that are expressed in different cell types. The main function of intermediate filaments is to provide mechanical support and structural integrity to the cell. These filaments are not dynamic. Type I Table Protein Subunits Cell Types Acidic Keratins Epithelial Cells II Neutral/Basic Keratins Epithelial Cells III Vimentin Many Cell Types Desmin Muscle Cells and others Glial Fibrillary Acidic Protein (GFAP) Glial Cells Peripherin Peripheral Neurons IV Neurofilament Proteins (NFL, NFM & NFH) Neurons V Nuclear Lamins All Nucleated Cells VI Nestin Stem Cells 11 7/25/2022 Assembly of Intermediate Filaments Protein subunits of each specific type of IF proteins assemble into higher order structure • All IF proteins have a central ‐helical (rod) domain that is critical for assembly of coiled‐ coils • Anti‐parallel alignment of IF protein dimers produces tetramers • Tetramers align end to end to form protofilaments • Intermediate filaments consists of 8 protofilaments wound into a ropelike structure Function of Intermediate Filaments Immunofluorescence generated by desmin antibody staining reveals the intermediate filament network within the cell. a) Cytoskeletal network: Integrates cytoskeletal components such as microfilaments and microtubules - physically links organelles and organizes intracellular structure. 12 7/25/2022 Function of Intermediate Filaments b) Specialized Cell Contacts: Desmosomes Intermediate Filaments Plakophilin Desmoplakin Fig Plakoglobin Function of Intermediate Filaments c) Specialized Cell Contacts: Hemidesmosomes Plectin Intermediate Filaments ECM 13 7/25/2022 dermis a) Epidermolysis Bullosa Simplex: The basal layer of cells (keratinocytes) in the epidermis of the skin are fragile because of mutations in keratin genes that code for these intermediate filament proteins. The basal kerotinocytes of the epidermis peel away from the underlying connective tissue in the dermis, which causes blistering of the skin. epidermis Intermediate Filaments and Disease Mutation in keratin 14 (K14) gene Epidermolysis Bullosa: Blistering Disorders can arise from Keratinocyte Layers Detaching from Underlying Dermal Connective Tissue Keratinocyte Layer Basement Membrane Dermal Connective Tissue 14 7/25/2022 Intermediate Filaments and Disease b) Desminopathies: Cardiac and skeletal myopathies caused by mutations in the desmin gene. • Desmin is a key intermediate filament protein of the cytoskeleton in cardiac and skeletal muscle that maintains the structural integrity of the contractile apparatus. • Approximately 45 mutations in the desmin gene have been identified in humans • Mutations cause accumulation of defective desmin protein and breakdown of contractile apparatus Journal of Clinical Investigation 119:1806-1813, 2009 Intermediate Filaments and Disease c) Tumor Cell Markers: Specific antibodies are used for immunohistochemistry • Keratins – Carcinomas (epithelial cell derived) • Vimentin – Sarcomas (connective tissue and bone derived) • GFAP – Glial cell tumors http://path.upmc.edu/cases/case74/micro.html Front Lobal Mass. 15 7/25/2022 Microtubules Molecular Structure: -Tubulin and -Tubulin subunits form dimers that polymerize to produce hollow cylinders approximately 25 nm diameter a) Basic structural unit: ,-Tubulin heterodimer GTP GTP   • -Tubulin has bound GTP that does not hydrolyze • -Tubulin has bound GTP that is hydrolyzed to GDP•Pi following assembly into microtubules b) Protofilaments: Heterodimers join end to end to form protofilaments consisting of alternating & -Tubulin subunits c) Assembled Microtubule: 13 protofilaments form a cylinder with a hollow core Assembly and Disassembly of Microtubules (MT) MTs are dynamic structures that can undergo rapid assembly and disassembly end Pi + end :GTP :GDP :GTP :GTP Note that most of the MT is composed of Tubulin•GDP dimers 16 7/25/2022 Assembly and Disassembly of Microtubules (MT) a) Tubulin heterodimers bound to GTP are added onto the plus (growing) end of the MT b) A short time after polymerization, GTP bound to -Tubulin is hydrolyzed to GDP•Pi c) -Tubulin•GDP is less stable and dissociates from either end of the MT d) Growth of microtubules occurs when GTP•Tubulin dimers are added to the plus end of the MT at faster rate than hydrolysis of GTP to GDP•Pi e) Growing MTs maintain a GTP•Tubulin “cap” on the plus end; Shrinking MTs have lost GTP•Tubulin cap- GDP•Tubulin is exposed on the plus end Assembly and Disassembly of Microtubules (MT) Dynamic Instability of Microtubules: Cycles of rapid growth and shrinkage of MTs that is dependent on the concentration of GTP•Tubulin dimers GTP a) High concentration of GTP•Tubulin dimers: Rapid growth of MT occurs as GTP cap is maintained on the plus end. b) Low concentration of GTP•Tubulin dimers: Rapid shrinkage of MT occurs because the GTP cap is lost and Tubulin•GDP dimers dissociate from the plus end. + + GDP 17 7/25/2022 Microtubules Microtubule Associated Proteins (MAPs): Diverse family of proteins that regulate behavior of MTs‐ Bind to MTs or Tubulin dimers a) Assembly - Binding to Tubulin dimers to increase polymerization rate b) Stabilization - Capping of plus ends to prevent disassembly of MTs c) Cross-linking - Spacing of MTs or linking to membranes and cytoskeletal components d) Tracking - Binding to plus end and directing MTs toward specific cellular locations e) Destabilization - Binding to tubulin dimers and preventing MT assembly f) Severing - Destabilizing MTs by generating plus ends without GTP cap and minus ends without -Tubulin cap EM showing arrangement of MTs and microfilaments (MF) Microtubules Microtubule Transport • Intracellular transport of macromolecules, organelles and vesicles • Two main types of microtubule-dependent ATPases carry cargo along MTs a) Kinesins: Carry cargo outward towards the plus end of MTs (kick out) b) Dyneins: Carry cargo inward towards the minus end of MTs (drag in) • Globular head domains contain ATPase and “walks along MT • Tail domains carry cargo using sequence-specific binding to proteins in membranes and organelles 18 7/25/2022 Microtubules Cilia and Flagella Specialized motile projections of cells composed mainly of MTs • Cilia are prominent in epithelial cells lining parts of the respiratory tract and the oviduct • Flagella are vital for motility of spermatozoa (basically longer cilia). a) Axoneme: Core structure composed of microtubules in 9+2 array and associated proteins required for structural integrity (nexin) and movement (dynein). EM of an axoneme in a flagellum Fig Microtubules Cilia and Flagella b) Basal bodies: The axoneme grows out of the basal body which derives from the centrioles- The minus ends of the MTs are anchored in axoneme doublets which extending from the basal body. c) Molecular Basis of Movement: • Dynein is affixed to A tubules. • Motor heads of dynein move along adjacent B tubules towards the minus end. • Because adjacent doublets are connected by nexins, the forces generated by movement of the dynein heads cause MTs to bend. 19 7/25/2022 Microtubules Microtubule‐targeted Drugs in Chemotherapy MOA Table Used as chemotherapeutic agents because they disrupt assembly of the mitotic spindle during mitosis and block M phase of the cell cycle Drug Mechanism of Action Vinblastine Binds to Tubulin and inhibits MT assembly Vincristine Binds to Tubulin and inhibits MT assembly Colchicine Binds to Tubulin and inhibits MT assembly Taxol Binds to Tubulin and inhibits disassembly Nocodazole Binds to Tubulin and inhibits MT assembly 20