Molecular Cell Biology Actin including Principles of Assembly

Molecular Cell Biology Actin, including Principles of Assembly Cooper

Introduction n Handouts n Readings • Text • Mini. Reviews - PDF files online n Homework

Reading n Textbook Chapters • Lodish et al. , Molecular Cell Biology, 6 th ed. , 2008, Freeman. Chaps. 17, 18. • Pollard & Earnshaw, Cell Biology, updated ed. , 2004, Saunders. Chaps. 35 -42, 47. n Articles on the Course Web Site • Original Articles • Reviews

Older Advanced / Reference Materials n 1. Cell Movements, 2 nd ed. , Dennis Bray, 2001, Garland. n 2. Guidebook to the Cytoskeletal and Motor Proteins. Kreis and Vale, eds. 1999, Oxford Univ. Press. n 3. Video Tape of Motility. Sanger & Sanger, Cell Motility & the Cytoskeleton, Video Supplement 2, 1990. A one-hour tape of examples of microtubule-based motility. Short segments shown in class. Available at the Media Center in the Becker (medical) library.

Chemotaxis of neutrophil to bacteria

Phagocytosis of bacteria by Dictyostelium amoebae

Biological Scope of Cell Motility & the Cytoskeleton n Shape n Translocation n Contraction n Intracellular Movements n Mechanical & Physical Properties

Elements of the Cytoskeleton n Structural • Filaments - Actin, Microtubules, Intermediate Filaments, Septins • Crosslinkers n Motors • Actin - Myosin • Microtubules - Dynein, Kinesin n Regulators

Higher Order Structures and Functions n Actin • Muscle sarcomere • Epithelial cell brush border • Cortex of motile cells n Microtubules • Cilia & Flagella • Mitotic spindle apparatus • Radiate from MTOC - organize membranes n n Septins - cytokinesis Major Sperm Protein in nematode sperm

Self-Assembly by Proteins Entropy & the Hydrophobic Effect n n n High Order in Assembled State Implies Lower Entropy, which is Unfavorable ∆G = ∆H - T∆S must be <0 for a Reaction to Occur But ∆H>0, ∆S>>0 ! Higher Entropy => Disorder in Assembled State Ordered Water on Hydrophobic Surface of Protein Subunit is Released

Self-Assembly by Proteins - Specificity n n Hydrophobic Surfaces of Proteins Must Fit Snugly to Exclude Water Assorted Non-covalent Bonds • Van der Waals • Coulombic • H-bond

Why Use Subunits to Make Large Molecules? n Efficient Use of the Genome n Error Management n Variable Size n Disassembly / Reassembly

Equivalence and Quasi-Equivalence n n Subunits in Polymer Must be Indistinguishable from Each Other Helical Arrangement Produces Linear Filament Some Flexibility in Structure Produces Loss of Equivalence Quasi-Equivalence: Similar with Distortion

Assembly of Helical Filaments n Add & Lose Subunits Only at Ends n ON Rate = k+ c 1 N n OFF Rate = k- N c 1 = Concentration of Monomers N = Concentration of Filament Ends

Assembly of Helical Filaments n At Steady State, by Definition • ON Rate = OFF Rate n k+ c 1 N = k - N n c 1 = k- / k+ n Subunit Concentration is Constant? !

Steady-state Concentrations of Polymer & Monomer [Polymer] Critical Concentration [Monomer] [Total]

Critical Concentration and Binding Affinity A 1 + N j Ka = Nj+1 [Nj+1] _ c 1 [Nj]

Critical Concentration and Binding Affinity Ka = Kd = 1 _ c 1 [Nj] [Nj+1] = _ c 1

Treadmilling n n Polar Filaments have Two Different Ends Can Have Different Critical Concentrations at the Two Ends Steady State Critical Concentration is an Intermediate Value Net Addition at One End, Net Loss at the Other End

Microtubule Photobleaching Experiment In Vivo Fluorescent Tubulin Microinjected into Cell as Tracer Laser Bleaches a Vertical Stripe

Cells Regulate Polymers n Cells Have Unexpectedly High Concentrations of Subunits n Cells Change their Subunit / Polymer Ratio Dramatically n Filament Lengths in Cells are Short

How do Cells Regulate the Level of Polymerization? n Total Concentration of Protein n Covalent Modification of Subunits n Binding of Small Molecules n Binding of Another Protein

How do Cells Regulate the Number and Length of Filaments? n Limit Growth • • n Intrinsic to Protein Deplete Subunits Capture by Capping End Template Create New Filaments • Nucleation - End or Side • Bolus of Subunits - High Concentration

Nucleation n Creation of New Filament from Subunits is Unfavorable n Subunit Prefers End of Filament to One or Two Other Subunits n Allows Cell to Control Where & When Filaments Form

“Dynamic Instability” of Microtubules GFP-tubulin in Cells Pure proteins in vitro

Nucleotides Can Generate “Dynamic Instability” n The Basic Facts. . . • • • Tubulin Binds GTP or GDP GTP Tubulin Polymerizes Strongly GDP Tubulin Polymerizes Poorly Subunits Exchange w/ Free GTP on Tubulin Hydrolyzes to GDP over Time after Addition to Microtubule

The Implication of All those Facts, taken together is. . . n n At Steady State, at any given time. . . • Most Ends have a GTP “Cap” and Grow Slowly • A Few Ends – Lose their GTP Cap – Exposing GDP-tubulin subunits – so the Microtubule Shrinks Rapidly Occurs In Vitro and In Vivo for Tubulin - Extensive and Relevant

Steps in Cell Movement Extension Adhesion Retraction Lodish et al. Molecular Cell Biology

Types of Actin Structures in a Migrating Cell

Scanning EM of the Front of a Migrating Cell

Small G-Proteins Regulate Different Assemblies of Actin Stress Fibers Lamellipodia Filopodia

GFP-Actin in a Migrating Melanoma Cell Text

Fish Keratocyte - Gliding Across a Surface 0. 1 - 1 µm per second

Fish Keratocytes Stationary Moving

End-to-Side Branches Svitkina et al. 1997.

Free Ends toward Direction of Movement Svitkina et al. 1997.

Arp 2/3 Complex at Filament Branches in vitro in vivo

Arp 2/3 Complex Structure, at a Filament Branch Point Hanein, Robinson & Pollard. 2001.

Creation & Growth

Termination

Destruction & Recycling

Model for Listeria Actin Motility Jon Alberts. Center for Cell Dynamics, Friday Harbor, U Wash. Cell. Dynamics. Org.

Fluorescence Microscopy of Living Cells n n GFP technology - colors, aggregation, multiple labels, FRET Sensitive video cameras - increased time until bleaching • Speed and sensitivity n Confocality • Laser scanning • Two-photon • Spinning disk • TIRF

Speckles to Single Molecules

Evidence for Single Molecules Fluorescence Intensity of Single Speckles over Time

Speckle Microscopy in Living Cells

Two-Color Speckle Microscopy Microtubules Actin

TIRF (Total Internal Reflection Fluorescence) Microscopy

Watching Single Actin Filaments Polymerize

Movies of Actin Filaments Polymerizing

Actin Assembly Regulators Bind Monomers n Cap Ends of Filaments • Barbed, Pointed n Bind Sides of Filaments • Univalent, Divalent n

Monomer Binding Proteins n Thymosin • Very small protein • Binds tightly • Simple buffer n Profilin • Small protein • Stimulates exchange of ADP to ATP • Promotes / permits addition at Barbed Ends

Barbed End Binding Proteins n Capping Protein • Terminates growth of free barbed ends • Enables “funneling” to free barbed ends in Dendritic Nucleation Model • Nucleation activity in vitro - probably irrelevant in vivo

Barbed End Binding Proteins n Gelsolin • Severs filaments, as well as caps • Needs high Ca 2+ • Knockout mouse grossly normal, but cells show poor induced actin polymerization • Extracellular (plasma) version - respond to cell necrosis

Barbed End Binding Proteins n Formins • Cap, Nucleate and Bind near Barbed Ends • Variable Level of Capping – Actin can add, unlike “Capping Protein” • Variable Level of Inhibition of Binding of Capping Protein • Profilin Combination - Increases Actin Polym Rate • Properties Combine to Keep Barbed Ends Growing Longer

Formin Mechanism Capping Protein Formin

Formin: Caps and Grows

Formin Mechanism

Pointed End Binding Proteins n Tropomodulin • Caps pointed end in muscle sarcomere • Caps much better if tropomyosin present • Role in nonmuscle cells uncertain

Arp 2/3 Complex n Complex of 7 proteins, including two actin-related proteins

Arp 2/3 Complex n Caps pointed end and nucleates with barbed end free

Arp 2/3 Complex n Binds side of filaments at same time, creating branching network

Side Binding Proteins n Univalent - Tropomyosin • Inhibits depolymerization • Makes filament stronger n Divalent • Crosslinkers - Filamin/ABP, α-actinin • Bundlers - Fimbrin, Fascin

Cofilin n Complicated Mechanism • Severs filaments • Binds monomers Essential for Viability n Present in High Concentrations n Regulated by a Specific Kinase n

Model for Actin Polymerization in Cells

Wiskott-Aldrich Syndrome n Human genetic disease: X-linked recessive n Immunodeficiency, thrombocytopenia n T and B cells and platelets have abnormal shape and motility n Gene product, WASp, activates Arp 2/3

Activation of WASp

Dorsal Closure of the Drosophila Embryo

Filopodial Formation n n Thin extensions Bundle of long unbranched actin filaments Can arise from an Arp 2/3 branched network Inhibit capping in one region • Formins • Inhibitors of Capping Protein

Actin-binding Toxins Used in Experiments Phalloidin n • Binds Actin Filaments • Permeates Cells n Cytochalasin • Caps Barbed Ends n – Induces Polymerization – Fluorescent Derivatives for Microscopy Latrunculin • Binds (Sequesters) Actin Monomers • Not Permeant • Permeates Cells • Binds Actin Filaments n Jasplakinolide • Permeates Cells

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