Molecular Cell Biology and Genetics THE CYTOSKELETON Mitch Denning, Ph.D. Molecular Cell Biology and Genetics August 31, 2018 The cytoskeleton is a network of 3 intertwined, interconnected and dynamic filament systems involved in cell movement, cell shape, and cell integrity Actin Microtubules Nucleus

Cytoskeleton Overview Functions Structure Assembly/Disassembly Drugs Motors Actin Filaments 5-9 nm Functions Structure Assembly/Disassembly Drugs Motors Microtubules 25 nm In my lecture, I will discuss each cytoskeletal element individually. Actin Filaments: smallest diameter Microtubules: largest diameter Intermediate Filaments: Intermediate diameter Each cytoskeletal element is assembled or polymerized from soluble subunit. Polymerization and depolymerization of cytoskeletal elements is highly regulated. The polymerized filaments can be organized into different types of arrays localized in different parts of the cell. Functions Structure Diversity Intermediate Filaments 10 nm

Actin (Microfilaments) Functions Cell Shape Surface area Cell adhesion Cell-Cell (Adherens Junction) Cell-Matrix (Focal Contact) Polarization Apical vs Basolateral Phagocytosis Phagosome Muscle contraction Cortical Actin Actin Endocytosis, contractile ring in cytokinesis

Actin (Microfilaments) Functions Cell Migration Development Wound Healing Immune System Cancer Metastasis

Neutrophil Chase

Structure of Actin Filaments Actin monomers are the soluble subunits of actin filaments. Sequence is highly conserved. Actin filaments are flexible and polar. Plus end is more dynamic. ATP becomes hydrolyzed in the filament Actin monomers bind ATP/ADP Helix of 2 protofilaments

Filament Assembly and Dynamic Structure Dynamic regulated equilibrium between soluble and filamentous actin Accessory proteins regulate assembly/disassembly Accessory proteins regulate assembly/disassembly

Direct addition of actin “nuclei” shifts kinetics Actin Filament Assembly Kinetics Direct addition of actin “nuclei” shifts kinetics Nucleation is rate-limiting One of the obstacles a cell must overcome is that the initiation of polymerization (nucleation) is slow and thus rate limiting. Cells utilize pre-formed oligomers or nuclei to initiate rapid filament polymerization.

Actin Assembly and Disassembly • Actin nucleation at cell membrane: Cortical actin Cell Surface Structures: Microvilli, Filopodia, Lamellipodia • Actin Related Protein complex, ARP, nucleates at minus end Binds pre-existing filaments at 70° angle Subunit Binding Proteins: Profilin (+); Thymosin (-) Cofilin bind ADP Actin filaments to promote depolymerization End Binding Proteins (ARP/Cap Z) modulate filament growth and localization Efficient stoichiometry Subunit Binding Proteins modulate filament elongation Less efficient Filament Binding Proteins modulate filament stability and orientation Cofilin: Actin Depolymerization Factor

Consequence of Nucleotide Hydrolysis Treadmilling High Cc Low Cc In addition to filament and subunit binding proteins, actin filament are regulated by ATP hydrolysis. Treadmilling

Actin Protrusion in Lamellipodia (Cap Z) 70° Angle Binds ADP form of F-Actin to cause depolymerization

Actin Arrays in Cells Microvilli Stress Fibers Striated Muscle a-Actinin Striated Muscle Spectrin Filamin Fimbrin

Actin Filament Cross-Linking

Global Actin Rearrangements Rho family GTPases Stress Fibers Focal Contacts Lamellipodia Membrane Ruffles Microspikes Filopodia

Actin Targeted Drugs Drugs Effects on Actin Phalloidin Binds and stabilizes filaments Cytochalasin Caps filament plus ends Latrunculin Binds subunits and prevents their polymerization   Death Cap Mushroom (Phalloidin) www.heraldsun.com.au Phalloidin from Death Cap mushroom. Most mushroom poisoning deaths worldwide. Clostridium difficile: TcdA TcdB disrupt Rho and thus tight junctions Bacterial toxins also target actin polymerization (Rho Family) Induces cell death Disrupts tight junctions (diarrhea)

Phalloidin Binds Actin Filaments Actin cytoskeleton stained with fluorescent phalloidin Effects of phalloidin on actin filaments

Actin Molecular Motors: Myosin Actin-based motor proteins Myosin II Plus-end directed Bipolar Thick Filament + Myosin II found in muscle 2 heavy chains + 2 copies of 2 light chains Majority of myosins are plus end directed +

Myosin Motor Mechanisms Myosin “Rowing” on Actin High speed Heads work independently Attached Released Cocked Force Attached Starts in “rigor” state. Phosphate release triggers power stroke Familial Hypertrophic Cardiomyopathy Myosin b heavy chain mutations Enlarged heart Cardiac arrhythmia Sudden death in young athletes

Movie 16.6 Myosin moving along actin (Cartoon) Movie 16.8 Myosin motors moving actin

Microtubules Functions Positioning of organelles Golgi and Endoplasmic Reticulum Intracellular transport Mitotic Spindles Vesicle and membrane transport between ER, Golgi and plasma membrane Anterograde/retrograde axonal transport of organelles and proteins Cell motility Flagella Cilia (motile and non-motile) Golgi Microtubules Membranes are recycled after moving from ER to Golgi ER to Golgi: Anterograde Golgi to ER: Retrograde

Structure of Microtubules GTP Hydrolysis 13 More Dynamic Alpha/Beta tubulin dimers are the soluble subunits for microtubules. Centrosome (MTOC)

Microtubule Organizing Center (MTOC) Basal bodies organize cilia and flagella microtubules. Similar to centrioles

Microtubule Assembly/Disassembly Dynamic Instability GTP form favors assembly Minus end protected in MTOC (centrosome) GDP form favors disassembly

Microtubule Dynamics – EB-1 and Tubulin GFP 16.3 Movie Microtubule Dynamics – EB-1 and Tubulin GFP 16.2 Movie Microtubule Leading Edge and ER Extension

Higher Order Microtubule Organization Centrosomes: Astral arrays MAPs: Filament spacing, Stabilization Neurofibrillary Tangles Hyperphosphorylated Tau aggregates in Alzheimer’s Disease

Microtubule Targeted Drugs MICRTUBULE DRUGS Taxol Colchicine, Colcemid Vinblastine, Vincristine Nocodazole (Mitotic Spindle Poisons) Binds and stabilizes microtubules Binds subunits and prevents their polymerization Microtubule-targeting drugs useful for cancer therapy Anaphase of Mitosis

Microtubule Stabilization by Taxol

Microtubule Molecular Motors Kinesins and Kinesin Related Proteins Move toward plus end Endoplasmic reticulum Dyneins Move toward negative end Cytoplasmic: Vesicle trafficking, golgi locomotion Axonemal: Rapid movement of cilia/flagella Both kinesins and dyneins are involved in chromosome segregation. Both are also involved in melanosome movement in fish pigment cells (figure16-61) High cAMP Low cAMP

Hand-Over-Hand/Step-by-Step Microtubule Motor Mechanisms Kinesin Dimer “Walking” on Microtubules Cargo Cargo Cargo Cargo Nucleotide exchange throws other head forward. High Processivity Hand-Over-Hand/Step-by-Step

Movie 16.7 Kinesin moving along Microtubules (Cartoon) Movie 16.5 Organelles moving along microtubules

9 doublets around 2 singlets Flagella and Cilia Axonemal Dyneins Flagella: Wave-like motion 1-2 per cell Protozoa and sperm Cilia: Whip-like motion Many per cell Respiratory epithelium Flagella: 1 or 2 per cell. Longer than cilia Cilia: hundreds per cell. Shorter than flagella Axoneme 9 doublets around 2 singlets

Basal Bodies Anchor cilia and flagella at cell surface 9 Microtubule Triplets No center pair of singlets Basal Bodies similar to centrioles inside centrosomes. Kahr-tag′ĕ-nĕr syndrome Kartagener’s Syndrome: Primary Ciliary Dyskinesia (Ciliary Dynein) Infertility (Male: Sperm/ Female: Fallopian tubes) Respiratory/Sinus Infections Situs Inversus (organ positions inverted)

Intermediate Filaments Functions Mechanical Strength Cell and tissue integrity Cell Adhesion Cell-cell adhesion (Desmosome) Cell-matrix adhesion (Hemidesmosome) Axon Diameter and Strength (Neurofilaments) Amyotropic Lateral Sclerosis Nuclear Lamins Nucleus integrity Laminopathies Epidermolysis Bullosa Simplex ALS: Neurofilament accumulations found in neurons

Intermediate Filament Structure and Assembly Rope-Like High Physical Strength - Non-Polar Soluble Subunit A tetramer (two coiled-coil dimers) is the soluble subunit for Intermediate Filaments.

Intermediate Filament Cross-Linking C-terminal domains binds neighboring filaments Bundled cytokeratins are called tonofilaments Accessory proteins Filaggrin (Filament Aggregation) Eczema and/or Ichthyosis Vulgaris Plectin (Vimentin, Actin, Microtubules) Muscular Dystrophy with Epidermolysis Bullosa Simplex

TYPES OF INTERMEDIATE FILAMENTS COMPONENT POLYPEPTIDES Diversity of Intermediate Filament Proteins Large gene family Tissue specific expression Useful for tumor diagnosis TYPES OF INTERMEDIATE FILAMENTS COMPONENT POLYPEPTIDES CELLULAR LOCATION Nuclear Lamins A, B, and C Defect: Progeria Nuclear Lamina (Inner lining of nuclear envelope) Vimentin-like Vimentin Desmin Glial fibrillary acidic protein Peripherin Many cells of mesenchymal origin; Muscle Glial cells (Astrocytes, Schwann cells) Some neurons Epithelial Keratins, type I (acidic) (~10 genes) Keratins, type II (basic) (~10 genes) Hair Keratins (~10 genes) Epithelial cells Hair and Nails (Epithelial Appendages) Axonal Neurofilament proteins (NF-L, NF-M, and NF-H) Neurons