molecular cell biology actin, including principles of assembly cooper
TRANSCRIPT
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Molecular Cell Biology
Actin, including Principles of Assembly
Cooper
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Introduction
Handouts
Readings
• Text
• MiniReviews - PDF files online
Homework
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Reading
Textbook Chapters• Lodish et al., Molecular Cell Biology, 6th ed., 2008,
Freeman. Chaps. 17, 18.• Pollard & Earnshaw, Cell Biology, updated ed., 2004,
Saunders. Chaps. 35-42, 47. Articles on the Course Web Site• Original Articles• Reviews
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Older Advanced / Reference Materials
1. Cell Movements, 2nd ed. ,Dennis Bray, 2001, Garland. 2. Guidebook to the Cytoskeletal and Motor Proteins. Kreis
and Vale, eds. 1999, Oxford Univ. Press. 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.
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Chemotaxis of neutrophil to bacteria
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Phagocytosis of bacteria by Dictyostelium amoebae
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Biological Scope of Cell Motility & the Cytoskeleton
Shape
Translocation
Contraction
Intracellular Movements
Mechanical & Physical Properties
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Elements of the Cytoskeleton
Structural• Filaments - Actin, Microtubules, Intermediate Filaments, Septins• Crosslinkers
Motors• Actin - Myosin
• Microtubules - Dynein, Kinesin
Regulators
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Higher Order Structures and Functions
Actin• Muscle sarcomere• Epithelial cell brush border• Cortex of motile cells
Microtubules• Cilia & Flagella• Mitotic spindle apparatus• Radiate from MTOC - organize membranes
Septins - cytokinesis Major Sperm Protein in nematode sperm
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Self-Assembly by Proteins -Entropy & the Hydrophobic Effect
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
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Self-Assembly by Proteins - Specificity
Hydrophobic Surfaces of Proteins Must Fit Snugly to Exclude Water
Assorted Non-covalent Bonds • Van der Waals• Coulombic• H-bond
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Why Use Subunits to Make Large Molecules?
Efficient Use of the Genome
Error Management
Variable Size
Disassembly / Reassembly
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Equivalence and Quasi-Equivalence
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
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Assembly of Helical Filaments
Add & Lose Subunits Only at Ends
ON Rate = k+ c1 N
OFF Rate = k- N
c1 = Concentration of Monomers
N = Concentration of Filament Ends
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Assembly of Helical Filaments
At Steady State, by Definition
• ON Rate = OFF Rate k+ c1 N = k- N
c1 = k- / k+
Subunit Concentration is Constant?!
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Steady-state Concentrations of Polymer & Monomer
[Monomer]
[Polymer]
[Total]
CriticalConcentration
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Critical Concentration and Binding Affinity
A1 + Nj Nj+1
Ka = [Nj+1]
[Nj]_c1
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Critical Concentration and Binding Affinity
Ka = 1_c1
Kd = [Nj+1]
[Nj]=
_c1
_c1
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Treadmilling
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
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Microtubule PhotobleachingExperiment In Vivo
Fluorescent Tubulin Microinjected into Cell as Tracer
Laser Bleaches a Vertical Stripe
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Cells Regulate Polymers
Cells Have Unexpectedly High Concentrations of Subunits
Cells Change their Subunit / Polymer Ratio Dramatically
Filament Lengths in Cells are Short
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How do Cells Regulate the Level of Polymerization?
Total Concentration of Protein
Covalent Modification of Subunits
Binding of Small Molecules
Binding of Another Protein
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How do Cells Regulate the Number and Length of Filaments?
Limit Growth• Intrinsic to Protein• Deplete Subunits• Capture by Capping End• Template
Create New Filaments• Nucleation - End or Side• Bolus of Subunits - High Concentration
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Nucleation
Creation of New Filament from Subunits is
Unfavorable
Subunit Prefers End of Filament to One or Two
Other Subunits Allows Cell to Control Where & When
Filaments Form
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“Dynamic Instability” of Microtubules
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GFP-tubulin in Cells
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Pure proteins in vitro
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Nucleotides Can Generate “Dynamic Instability”
The Basic Facts...• Tubulin Binds GTP or GDP• GTP Tubulin Polymerizes Strongly• GDP Tubulin Polymerizes Poorly• Subunits Exchange w/ Free GTP• GTP on Tubulin Hydrolyzes to GDP over Time after Addition to
Microtubule
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The Implication of All those Facts, taken together is...
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
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Steps in Cell Movement
Extension
Adhesion
Retraction
Lodish et al. Molecular Cell Biology
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Types of Actin Structures in a Migrating Cell
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Scanning EM of the Front of a Migrating Cell
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Small G-Proteins Regulate Different Assemblies of Actin
StressFibers
FilopodiaLamellipodia
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GFP-Actin in a Migrating Melanoma Cell
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Fish Keratocyte - Gliding Across a Surface
0.1 - 1 µm per second
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Fish Keratocytes
Moving
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Stationary
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End-to-Side Branches
Svitkina et al. 1997.
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Free Ends toward Direction of Movement
Svitkina et al. 1997.
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Arp2/3 Complex at Filament Branches
in vitro
in vivo
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Arp2/3 Complex Structure, at a Filament Branch Point
Hanein, Robinson & Pollard. 2001.
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Creation & Growth
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Termination
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Destruction & Recycling
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Model for Listeria Actin Motility
Jon Alberts. Center for Cell Dynamics, Friday Harbor, U Wash. CellDynamics.Org.
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Model for Listeria Actin Motility
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Jon Alberts. Center for Cell Dynamics, Friday Harbor, U Wash. CellDynamics.Org.
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Fluorescence Microscopy of Living Cells
GFP technology - colors, aggregation, multiple labels, FRET
Sensitive video cameras - increased time until bleaching• Speed and sensitivity
Confocality• Laser scanning •Spinning disk• Two-photon •TIRF
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Speckles to Single Molecules
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Evidence for Single Molecules
Fluorescence Intensity of Single Speckles over Time
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Speckle Microscopy in Living Cells
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Two-Color Speckle Microscopy
MicrotubulesMicrotubules
ActinActin
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TIRF (Total Internal Reflection Fluorescence) Microscopy
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Watching Single Actin Filaments Polymerize
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Movies of Actin Filaments Polymerizing
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Actin Assembly Regulators
Bind Monomers Cap Ends of Filaments• Barbed, Pointed
Bind Sides of Filaments• Univalent, Divalent
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Monomer Binding Proteins
Thymosin• Very small protein• Binds tightly• Simple buffer
Profilin• Small protein• Stimulates exchange of ADP to ATP• Promotes / permits addition at Barbed Ends
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Barbed End Binding Proteins
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
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Barbed End Binding Proteins
Gelsolin• Severs filaments, as well as caps
• Needs high Ca2+
• Knockout mouse grossly normal, but cells show poor
induced actin polymerization
• Extracellular (plasma) version - respond to cell
necrosis
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Barbed End Binding Proteins 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
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Formin Mechanism
Formin
Capping Protein
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Formin: Caps and Grows
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Formin Mechanism
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Pointed End Binding Proteins
Tropomodulin
• Caps pointed end in muscle sarcomere
• Caps much better if tropomyosin present
• Role in nonmuscle cells uncertain
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Arp2/3 Complex Complex of 7 proteins, including two actin-related proteins
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Arp2/3 Complex Caps pointed end and nucleates with barbed end free
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Arp2/3 Complex Binds side of filaments at same time, creating branching
network
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Side Binding Proteins
Univalent - Tropomyosin• Inhibits depolymerization• Makes filament stronger
Divalent• Crosslinkers - Filamin/ABP, α-actinin• Bundlers - Fimbrin, Fascin
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Cofilin
Complicated Mechanism• Severs filaments• Binds monomers
Essential for Viability Present in High Concentrations Regulated by a Specific Kinase
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Model for Actin Polymerization in Cells
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Wiskott-Aldrich Syndrome
Human genetic disease: X-linked recessive Immunodeficiency, thrombocytopenia T and B cells and platelets have abnormal shape and motility Gene product, WASp, activates Arp2/3
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Activation of WASp
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Dorsal Closure of the Drosophila Embryo
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Filopodial Formation
Thin extensions Bundle of long unbranched actin filaments Can arise from an Arp2/3 branched network Inhibit capping in one region • Formins• Inhibitors of Capping Protein
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Actin-binding Toxins Used in Experiments
Cytochalasin• Caps Barbed Ends
• Permeates Cells
Latrunculin• Binds (Sequesters) Actin
Monomers
• Permeates Cells
Phalloidin
• Binds Actin Filaments– Induces Polymerization– Fluorescent Derivatives for
Microscopy
• Not Permeant
Jasplakinolide
• Binds Actin Filaments
• Permeates Cells
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