Cytoskeleton Fundamentals B Lecture Outline 2 PDF

Dr. Muriel W. Lambert February 28, 2024 CYTOSKELETON I. MICROTUBULES (MT) A. MTs are involved in cell shape and motility B. Two populations of MTs 1. Unstable, short-lived – mitotic spindle 2. Stable, long-lived – cilia, flagella, nerve cells (axons) C. Size – 24 nm in diameter D. Composition E. 1....

Dr. Muriel W. Lambert February 28, 2024 CYTOSKELETON I. MICROTUBULES (MT) A. MTs are involved in cell shape and motility B. Two populations of MTs 1. Unstable, short-lived – mitotic spindle 2. Stable, long-lived – cilia, flagella, nerve cells (axons) C. Size – 24 nm in diameter D. Composition E. 1. Polymers of globular tubulin subunits - heterodimers of α and β tubulin monomers 2. Each tubulin subunit (α and β) binds GTP Microtubule dynamics – assembly/disassembly 1. Tubulin subunits (dimers) link to form linear protofilament 2. Protofilaments associate side by side to form a sheet then a cylinder 3. 13 protofilaments form a MT 4. GTP bound to α and β tubulin in dimer; GTP bound to β tubulin hydrolyzes to GDP after dimer incorporated into MT 5. Polarity a. MTs have a (+) and a (-) end 1 b. a. 6. MT assembly and disassembly occurs twice as fast at (+) end then it does at (-) end 7. MTs can exhibit treadmilling 8. Agents that disrupt MT dynamics a. b. c. F. G. Colchicine, colcemid – bind tubulin, poison end of a MT, prevents addition of tubulin subunits Taxol – binds to and stabilizes MTs These agents have antimitotic action MT associated proteins 1. MAP1, MAP2, Tau 2. Aid in assembly and stabilization of MTs 3. Crosslink MTs to one another and to intermediate filaments 4. Have two domains Centers for MT assembly 1. Microtubule-organizing center (MTOC) 2. MTOC nucleates and organizes MTs a. b. H. One end of MT ringed by α tubulin, (-) end The opposite end ringed by β tubulin, (+) end Centrosome – primary MTOC, contains a pair of centrioles Basal body – another MTOC Roles for MTs 1. Maintenance of cell shape 2. Intracellular transport a. MTs function as tracks in intracellular transport – vesicles, proteins, organelles, the nucleus b. MT motor proteins – mediate transport along MT 2 (1) (2) Kinesin (a) A dimer of 2 heavy chains, each complexed to a light chain (b) Divided into 3 domains (c) Responsible for (+) end-directed movement Dynein (a) Multimeric – composed of 2 heavy chains and light chains (b) Responsible for (-) end-directed movement (c) Dynein cannot mediate transport by itself - requires a large protein complex (dynactin complex) that links dynein to vesicles and MTs 3. Ciliary and flagellar movement a. Structure cilia and flagella (1) (2) (3) b. Movement (1) (2) (3) (4) 4. I. 9 + 2 pattern of MTs 9 outer doublet tubules Central pair of MTs Cilia/flagella bending produced by sliding between pairs MTs Bends are propagated Dynein arms generate sliding force – ATP required Radial spokes and central pair MTs control bending of flagella Microtubule involvement in mitosis Consequences of defects in MTs 1. Primary ciliary dyskinesia (Immotile cilia syndrome) a. Absence of dynein arms on microtubules b. Loss of ciliary and flagellar movement 2. Alzheimer’s disease a. Hyperphosphorylation of tau (P-tau) b. P-tau forms paired helical filaments c. Breakdown of microtubules and loss of function 3 II. MICROFILAMENTS (MF) A. Actin 1. A major component of MF – very abundant 2. Important in cell membrane movements and changes in cellular morphology – must rapidly assemble and disassemble to carry out these functions 3. 4. Size – 7-9 nm in diameter Composition and structure a. b. c. 5. Polarity and polymerization a. b. c. d. e. 6. Cellular motility Phagocytosis Stress fibers - support Actin binding proteins involved in regulation of actin polymerization, length and stability a. b. c. 8. Actin filaments have polarity Filaments have a (+) end and a (-) end (+) end of actin filament elongates faster than (-) end Treadmilling – subunits released at (-) end of filament, reassemble at the leading edge A steady state is reached Function a. b. c. 7. Made up of globular actin monomers (G-actin) Momomers assemble to generate an actin filament (F-actin) – form a helix Each actin monomer has a binding site for ATP Inhibition of actin assembly – thymosin β4 Promotion of actin assembly – profilin Severing of actin filaments – gelsolin, cofilin Agents which affect actin filament polymerization/depolymerization a. b. c. Cytochalasin D – depolymerizes actin filaments Latrunculin – leads to actin filament dissociation Phalloidin – prevents filament depolymerization 4 9. Organization a. b. Actin filaments organized into bundles and networks Actin cross-linking proteins (1) Connect two filaments (2) Determine whether bundles or networks form (3) Different cross-linking proteins (4) Length and flexibility of cross-linking protein determines whether bundles or networks form (5) Fibrin, fascin crosslink actin filaments to form bundles (6) Filamin crosslinks actin filaments to form networks c. Proteins cross-linking actin to cell membrane (1) (2) B. Proteins connect the cell membrane to the underlying cortical actin network Help determine shape of cell Myosin 1. An actin motor protein – it interacts with actin 2. Member of a gene family a. b. c. d. 3. 13 members Best characterized – myosin I, II, V Myosin I and V involved in cytoskeleton-membrane interactions Myosin II involved in muscle contraction and cytokinesis Structure and function a. b. Myosins are composed of heavy and light chains Heavy chains - organized into 3 domains (head, α-helical neck, tail) (1) (2) c. d. Myosin I – 1 heavy chain Myosin II and V - 2 heavy chains Light chains – regulate the activity of the head domain Functions of the 3 domains of myosin (1) Head domain – contains actin and ATP binding sites 5 (2) α-helical neck region – associated with light chains (3) Tail domain - dictates specific role of each myosin - Tail domains of myosin I and V bind plasma membrane or membrane bound intracellular organelles and vesicles - Tail domains of myosin II associates with each other – involved in muscle contraction 4. Movement of myosin along actin filaments a. b. c. C. D. Myosin head slides/walks along actin filament towards (+) end Myosin head moves in discrete steps – hydrolysis of ATP required Conformational changes in myosin head occur during movement along actin filament Role of actin and myosin in muscle contraction 1. Actin and myosin associate into a complex (actomyosin) 2. Myosin II involved 3. Sliding – filament model of muscle contraction Role of actin and myosin in nonmuscle cells 1. Noncontractile actin bundles - present in microvilli and filopodia 2. Actin aids in cell membrane movements 2. Myosin is a component of cortical actin networks - stiffens plasma membranes 3. Actin and myosin involved in cytokinesis - form a contractile ring at equator of a dividing cell 4. Myosin involved in moving vesicles along actin filaments 5. Actin and myosin involved in cell locomotion a. b. c. d. Polymerization and rearrangements of actin filaments in leading edge pushes membrane (lamellipodium) forward Lamellipodium adheres to substratum Bulk contents of cell body translocated forward Myosin plays a role in forward extension – myosin II contraction in the rear of the cell draws the body of the cell forward 6 E. Consequences of defects in MFs 1. Mutation in gene coding for profilin 2. Defect in profilin and decrease in actin filament assembly 3. Amyotrophic lateral sclerosis III. INTERMEDIATE FILAMENTS (IF) A. IFs are more abundant in epidermal cells and axons of neurons than MFs and MTs B. IF are extremely stable and typically form a network throughout the cytoplasm, also present in nucleus C. Structure and composition 1. Size – 10 nm in diameter 2. α-helical rods – assemble into rope-like filaments 3. 6 types a. Type I – acidic keratins b. Type II – basic keratins c. Type III – vimentin, desmin, glial fibrillary acidic protein, peripherin d. Type IV – neurofilament proteins, internexin e. Nonstandard type IV f. Type V – lamins 4. Expression of each IF protein is characteristic of a certain tissue or cell type 5. All IFs have a common domain structure a. b. D. Central α-helical core Globular N- and C-terminal domains differ Assembly 1. IF protein monomer forms a dimer 2. Pair dimers associate laterally to form tetramer 7 E. F. G. 3. Two tetramers bind end to end 4. Eight tetramers bind to form an IF – very stable structure 5. α-helical core is common to all Ifs; N- and C-terminal domains differ IF associated proteins (IFAPs) 1. Cross-link IFs with one another to form a bundle (tonofilament) or network or with the plasma membrane 2. Cross-link IFs to MTs and actin filaments - plectin Function 1. Support cellular membranes – lamins 2. Form internal framework to help support cell shape and resilience 3. Function in cell junctions 4. Stabilize sarcomeres in muscle 5. Maintain structural integrity of tissues Consequences of defects in IFs 1. Mutations in genes coding for keratins 2. Mutations in genes coding for nuclear lamins SUGGESTED READING (Reading chapters in one of these texts is recommended) Essential Cell Biology, Alberts et al., Second Addition, Cytoskeleton, Chapter 17. Molecular Cell Biology, Lodish et. al., Chapters: 3 (p.79-82), 5 (p.173-178), 19 and 20 Molecular Biology of the Cell (5th edition) - Alberts et al. Chapter 16 - The Cytoskeleton - p. 965-1052 8 9