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

[Polymer]
Critical
Concentration
[Monomer]

[Total]
Critical Concentration and Binding Affinity

A1 + Nj Nj+1

[Nj+1]
Ka = _
c1 [N ]
j
Critical Concentration and Binding Affinity

1
Ka = _
c1

_ _
c1 [Nj] c1
Kd = =
[Nj+1]
 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 Photobleaching
Experiment In Vivo

Fluorescent Tubulin Microinjected into Cell as Tracer

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Laser Bleaches a Vertical Stripe

 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 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

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 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
 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

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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

 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

Capping Protein

Formin
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,  -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
 Phalloidin
• Binds Actin Filaments
– Induces Polymerization
– Fluorescent Derivatives for
Microscopy

• Binds (Sequesters) Actin • Not Permeant

Monomers  Jasplakinolide
• Permeates Cells
• Binds Actin Filaments
• Permeates Cells
End