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Molecular Cell Biology
Actin, including Principles of Assembly
Cooper
Introduction
Handouts
Readings
• Text
• MiniReviews - PDF files online
Homework
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
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.
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
Elements of the Cytoskeleton
Structural• Filaments - Actin, Microtubules, Intermediate Filaments, Septins• Crosslinkers
Motors• Actin - Myosin
• Microtubules - Dynein, Kinesin
Regulators
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
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
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
Why Use Subunits to Make Large Molecules?
Efficient Use of the Genome
Error Management
Variable Size
Disassembly / Reassembly
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
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
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?!
Steady-state Concentrations of Polymer & Monomer
[Monomer]
[Polymer]
[Total]
CriticalConcentration
Critical Concentration and Binding Affinity
A1 + Nj Nj+1
Ka = [Nj+1]
[Nj]_c1
Critical Concentration and Binding Affinity
Ka = 1_c1
Kd = [Nj+1]
[Nj]=
_c1
_c1
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
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
How do Cells Regulate the Level of Polymerization?
Total Concentration of Protein
Covalent Modification of Subunits
Binding of Small Molecules
Binding of Another Protein
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
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
“Dynamic Instability” of Microtubules
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GFP-tubulin in Cells
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Pure proteins in vitro
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
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
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
StressFibers
FilopodiaLamellipodia
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.
Free Ends toward Direction of Movement
Svitkina et al. 1997.
Arp2/3 Complex at Filament Branches
in vitro
in vivo
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
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.
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
Speckles to Single Molecules
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Evidence for Single Molecules
Fluorescence Intensity of Single Speckles over Time
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
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
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
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
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
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
Formin Mechanism
Formin
Capping Protein
Formin: Caps and Grows
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Formin Mechanism
Pointed End Binding Proteins
Tropomodulin
• Caps pointed end in muscle sarcomere
• Caps much better if tropomyosin present
• Role in nonmuscle cells uncertain
Arp2/3 Complex Complex of 7 proteins, including two actin-related proteins
Arp2/3 Complex Caps pointed end and nucleates with barbed end free
Arp2/3 Complex Binds side of filaments at same time, creating branching
network
Side Binding Proteins
Univalent - Tropomyosin• Inhibits depolymerization• Makes filament stronger
Divalent• Crosslinkers - Filamin/ABP, α-actinin• Bundlers - Fimbrin, Fascin
Cofilin
Complicated Mechanism• Severs filaments• Binds monomers
Essential for Viability Present in High Concentrations Regulated by a Specific Kinase
Model for Actin Polymerization in Cells
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
Activation of WASp
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
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
End
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