Molecular Cell Biology
Actin, including Principles of Assembly
Cooper
Introduction
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Handouts
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Readings
• Text
• MiniReviews - PDF files online
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Homework
Reading
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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.
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Articles on the Course Web Site
• Original Articles
• Reviews
Older Advanced / Reference Materials
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1. Cell Movements, 2nd ed. ,Dennis Bray, 2001, Garland.
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2. Guidebook to the Cytoskeletal and Motor Proteins.
Kreis and Vale, eds. 1999, Oxford Univ. Press.
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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
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Shape
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Translocation
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Contraction
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Intracellular Movements
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Mechanical & Physical Properties
Elements of the Cytoskeleton
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Structural
• Filaments - Actin, Microtubules, Intermediate Filaments, Septins
• Crosslinkers
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Motors
• Actin - Myosin
• Microtubules - Dynein, Kinesin
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Regulators
Higher Order Structures and Functions

Actin
• Muscle sarcomere
• Epithelial cell brush border
• Cortex of motile cells
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Microtubules
• Cilia & Flagella
• Mitotic spindle apparatus
• Radiate from MTOC - organize membranes
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Septins - cytokinesis
Major Sperm Protein in nematode sperm
Self-Assembly by Proteins Entropy & the Hydrophobic Effect
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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

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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?
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Efficient Use of the Genome
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Error Management
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Variable Size
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Disassembly / Reassembly
Equivalence and Quasi-Equivalence
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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
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Add & Lose Subunits Only at Ends
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ON Rate = k+ c1 N

OFF Rate = k- N
c1 = Concentration of Monomers
N = Concentration of Filament Ends
Assembly of Helical Filaments
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At Steady State, by Definition
• ON Rate = OFF Rate

k+ c1 N = k- N
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c1 = k - / k +
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Subunit Concentration is Constant?!
Steady-state Concentrations of Polymer &
Monomer
[Polymer]
Critical
Concentration
[Monomer]
[Total]
Critical Concentration and Binding Affinity
A1 + Nj
Ka =
Nj+1
[Nj+1]
_
c1 [N ]
j
Critical Concentration and Binding Affinity
Ka =
Kd =
1
_
c1
_
c1 [Nj]
[Nj+1]
=
_
c1
Treadmilling
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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
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Laser Bleaches a Vertical Stripe
Cells Regulate Polymers
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Cells Have Unexpectedly High Concentrations of
Subunits
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Cells Change their Subunit / Polymer Ratio Dramatically
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Filament Lengths in Cells are Short
How do Cells Regulate the Level of Polymerization?
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Total Concentration of Protein
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Covalent Modification of Subunits
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Binding of Small Molecules
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Binding of Another Protein
How do Cells Regulate the Number and
Length of Filaments?
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Limit Growth
•
•
•
•
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Intrinsic to Protein
Deplete Subunits
Capture by Capping End
Template
Create New Filaments
• Nucleation - End or Side
• Bolus of Subunits - High Concentration
Nucleation
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Creation of New Filament from Subunits is
Unfavorable
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Subunit Prefers End of Filament to One or Two
Other Subunits
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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”
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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...
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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
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Fish Keratocyte - Gliding Across a Surface
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0.1 - 1 µm per second
Fish Keratocytes
Stationary
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Moving
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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.
Creation & Growth
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Termination
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Destruction &
Recycling
Model for Listeria Actin Motility
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Jon Alberts. Center for Cell Dynamics, Friday Harbor, U Wash. CellDynamics.Org.
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
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GFP technology - colors, aggregation, multiple
labels, FRET
Sensitive video cameras - increased time until
bleaching
• Speed and sensitivity
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Confocality
• Laser scanning
• Two-photon
•Spinning disk
•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
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Microtubules
Actin
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
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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
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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
Capping Protein
Formin
Formin: Caps and Grows
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Formin Mechanism
Pointed End Binding Proteins
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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,  -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
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Human genetic disease: X-linked recessive

Immunodeficiency, thrombocytopenia
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T and B cells and platelets have abnormal shape and motility
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Gene product, WASp, activates Arp2/3
Activation of WASp
Dorsal Closure of the Drosophila Embryo
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Filopodial Formation
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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

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Phalloidin
Cytochalasin

• Caps Barbed Ends
• Binds Actin Filaments
• Permeates Cells
– Induces Polymerization
– Fluorescent Derivatives for
Microscopy
Latrunculin
• Binds (Sequesters) Actin
Monomers
• Permeates Cells
• Not Permeant

Jasplakinolide
• Binds Actin Filaments
• Permeates Cells
End