PowerPoint Presentation - Molecular Cell Biologymcb5068.wustl.edu/MCB/Lecturers/Cooper/s/6_Actin.pdf · Some)Flexibility)in)Structure) ... Lodish et al. Molecular Cell Biology Wednesday,

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


http://www.cooperlab.wustl.edu/MCB/Lecturers/Cooper/Cooper.html

http://www.cooperlab.wustl.edu/MCB/Lecturers/Cooper/Cooper.html

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

Shape

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]



[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

_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

Form


“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

Text


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

Actin


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


End


Documents

PowerPoint Presentation - Molecular Cell Biologymcb5068.wustl.edu/MCB/Lecturers/Cooper/s/6_Actin.pdf · Some)Flexibility)in)Structure) ... Lodish et al. Molecular Cell Biology Wednesday,