PowerPoint Presentation - Molecular Cell...

Molecular Cell Biology

Ac0n, including Principles of Assembly

Cooper

Wednesday, August 29, 2012

Introduc0on

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.

Ar0cles on the Course Web Site• Original Ar0cles

• 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 Mo0lity. Sanger & Sanger, Cell Mo0lity & the Cytoskeleton, Video Supplement 2, 1990. A one-‐hour tape of examples of microtubule-‐based mo0lity. Short segments shown in class. Available at the Media Center in the Becker (medical) library.

Chemotaxis of neutrophil to bacteria

Phagocytosis of bacteria by Dictyostelium amoebae

Biological Scope of Cell Mo0lity & the Cytoskeleton

Transloca0on

Contrac0on

Intracellular Movements

Mechanical & Physical Proper0es

Elements of the Cytoskeleton

Structural • Filaments -‐ Ac0n, Microtubules, Intermediate Filaments, Sep0ns

• Crosslinkers

Motors

• Ac0n -‐ Myosin

• Microtubules -‐ Dynein, Kinesin

Regulators

Higher Order Structures and Func0ons

Ac0n• Muscle sarcomere

• Epithelial cell brush border

• Cortex of mo0le cells

Microtubules• Cilia & Flagella

• Mito0c spindle apparatus

• Radiate from MTOC -‐ organize membranes

Sep0ns -‐ 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 Reac0on 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 Indis0nguishable from Each Other

Helical Arrangement Produces Linear Filament Some Flexibility in Structure Produces Loss of Equivalence

Quasi-‐Equivalence: Similar with Distor0on

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 Defini0on

• ON Rate = OFF Rate k+ c1 N = k-‐ N

c1 = k-‐ / k+

Subunit Concentra0on is Constant?!

Steady-‐state Concentra0ons of Polymer & Monomer

[Monomer]

[Total]Wednesday, August 29, 2012

[Monomer]

[Polymer]

[Monomer]

[Polymer]

[Total]

CriticalConcentration

Cri0cal Concentra0on and Binding Affinity

A1 + Nj Nj+1

Ka = [Nj+1]

[Nj]_c1

Cri0cal Concentra0on and Binding Affinity

Ka = 1_c1

Kd = [Nj+1]

[Nj] = _c1

Treadmilling

Polar Filaments have Two Different Ends Can Have Different Cri0cal Concentra0ons at the Two Ends

Steady State Cri0cal Concentra0on is an Intermediate Value

Net Addi0on 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 Concentra0ons of Subunits

Cells Change their Subunit / Polymer Ra0o Drama0cally

Filament Lengths in Cells are Short

How do Cells Regulate the Level of Polymeriza0on?

Total Concentra0on of Protein

Covalent Modifica0on 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• Nuclea0on -‐ End or Side

• Bolus of Subunits -‐ High Concentra0on

Nuclea0on

Crea0on 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

“Dynamic Instability” of Microtubules

GFP-tubulin in Cells Pure proteins in vitro

Nucleo0des 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 aqer Addi0on to Microtubule

The Implica0on of All those Facts, taken together is...

At Steady State, at any given 0me...

• 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 Ac0n Structures in a Migra0ng Cell

Scanning EM of the Front of a Migra0ng Cell

Small G-‐Proteins Regulate Different Assemblies of Ac0n

StressFibers

FilopodiaLamellipodia

GFP-‐Ac0n in a Migra0ng Melanoma Cell

Fish Keratocyte -‐ Gliding Across a Surface

0.1 - 1 µm per second

Fish Keratocytes

Stationary

Fish Keratocytes

MovingStationary

End-‐to-‐Side Branches

Svitkina et al. 1997.

Free Ends toward Direc0on 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

Termination

Destruction & Recycling

Model for Listeria Ac0n Mo0lity

Jon Alberts. Center for Cell Dynamics, Friday Harbor, U Wash. CellDynamics.Org.

Fluorescence Microscopy of Living Cells

GFP technology -‐ colors, aggrega0on, mul0ple labels, FRET

Sensi0ve video cameras -‐ increased 0me un0l bleaching• Speed and sensi0vity

Confocality• Laser scanning •Spinning disk

• Two-‐photon •TIRF

Speckles to Single Molecules

Evidence for Single Molecules

Fluorescence Intensity of Single Speckles over Time

Speckle Microscopy in Living Cells

Two-‐Color Speckle Microscopy

Microtubules

TIRF (Total Internal Reflec0on Fluorescence) Microscopy

Watching Single Ac0n Filaments Polymerize

Movies of Ac0n Filaments Polymerizing

Ac0n Assembly Regulators

Bind Monomers Cap Ends of Filaments

• Barbed, Pointed Bind Sides of Filaments

• Univalent, Divalent

Monomer Binding Proteins

Thymosin• Very small protein

• Binds 0ghtly

• Simple buffer

Profilin• Small protein

• S0mulates exchange of ADP to ATP

• Promotes / permits addi0on at Barbed Ends

Barbed End Binding Proteins

Capping Protein

• Terminates growth of free barbed ends

• Enables “funneling” to free barbed ends in Dendri0c Nuclea0on Model

• Nuclea0on ac0vity in vitro -‐ probably irrelevant in vivo

Gelsolin

• Severs filaments, as well as caps

• Needs high Ca2+

• Knockout mouse grossly normal, but cells show poor

induced ac0n polymeriza0on

• Extracellular (plasma) version -‐ respond to cell

necrosis

Formins• Cap, Nucleate and Bind near Barbed Ends

• Variable Level of Capping

– Ac0n can add, unlike “Capping Protein”

• Variable Level of Inhibi0on of Binding of Capping Protein

• Profilin Combina0on -‐ Increases Ac0n Polym Rate

• Proper0es 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 beter if tropomyosin present

• Role in nonmuscle cells uncertain

Arp2/3 Complex

Complex of 7 proteins, including two ac0n-‐related proteins

Arp2/3 Complex

Caps pointed end and nucleates with barbed end free

Arp2/3 Complex

Binds side of filaments at same 0me, crea0ng branching network

Side Binding Proteins

Univalent -‐ Tropomyosin

• Inhibits depolymeriza0on

• Makes filament stronger

Divalent

• Crosslinkers -‐ Filamin/ABP, α-‐ac0nin

• Bundlers -‐ Fimbrin, Fascin

Cofilin

Complicated Mechanism• Severs filaments

• Binds monomers

Essen0al for Viability Present in High Concentra0ons Regulated by a Specific Kinase

Model for Ac0n Polymeriza0on in Cells

Wiskot-‐Aldrich Syndrome

Human gene0c disease: X-‐linked recessive

Immunodeficiency, thrombocytopenia

T and B cells and platelets have abnormal shape and mo0lity

Gene product, WASp, ac0vates Arp2/3

Ac0va0on of WASp

Dorsal Closure of the Drosophila Embryo

Filopodial Forma0on

Thin extensions Bundle of long unbranched ac0n filaments Can arise from an Arp2/3 branched network Inhibit capping in one region

• Formins

• Inhibitors of Capping Protein

Ac0n-‐binding Toxins Used in Experiments

Cytochalasin

• Caps Barbed Ends

• Permeates Cells

Latrunculin

• Binds (Sequesters) Ac0n

Monomers

• Permeates Cells

Phalloidin

• Binds Ac0n Filaments– Induces Polymeriza0on

– Fluorescent Deriva0ves for

Microscopy

• Not Permeant

Jasplakinolide

• Binds Ac0n Filaments

• Permeates Cells

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