Molecular Cell Biology Actin, including Principles of Assembly Cooper

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



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



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



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



Termination



Destruction & Recycling

Model for Listeria Actin Motility

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



Two-Color Speckle Microscopy

MicrotubulesMicrotubules

ActinActin

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TIRF (Total Internal Reflection Fluorescence) Microscopy

Watching Single Actin Filaments Polymerize



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



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

Documents

Molecular Cell Biology Actin, including Principles of Assembly Cooper