Lecture 3 Actin and myosin in non-muscle cells; Cell motility Outline: Actin polymerization in vitro...

Lecture 3

Actin and myosin in non-muscle cells; Cell motility

Outline:Actin polymerization in vitroRegulation of actin dynamics in cellsActin organizationCell motility

Paper: Self-polarization and directional motilityof the cytoplasm

Actin

roles: cell shape, polarization, locomotion, division; vesicle traffic

highly conserved 375 aa, 43 kD protein

the most abundant protein in non-muscle cells 1-5%

monomer = G-actin

polymer = F-actin, microfilaments

inhibitors:latrunculin, cytochalasin; phalloidin

platelet dynamics

resting activated retraction

Dramatic morphological changes result from reorganization of actin cross-linked to plasma membrane

Actin Structure

barbed

pointed

two-stranded helix

Actin highly conserved, binding proteins are not

Assays to measure:

1) viscometry

2) sedimentation

3) fluorescence spectroscopy-pyrene actin assembly assay

Actin polymerization dynamics in vitro

pyrene couple to C-374 of actin

Mg++, KCl

elongation

*steady state

nucleation

fluorescence

polymer

time

Pyrene actin assembly assay

*[free actin] =Cc=

0.1 M

-

Cc(- end) = 0.8 M > Cc(+ end) = 0.1 M

D D D D-Pi

D D D-Pi T

T

+

steady state - treadmilling of subunits

T D

D

T

filament turnover rate: t1/2 = 30 min

rate limiting step = dissociation of ADP actin from minus end

Actin dynamics in vivo

2. Keratocyte - epithelial cell

3. Listeria monocytogenes - intracellular bacterial pathogen

1. Spatial and temporal control of polymerization/depolymerization

2. Turnover

3. Movement of actin filaments - myosins

Parameters:

Model systems:

1. Fibroblast

Listeriamonocytogenes

Spatial Control

microinject fluorescently-labeled actin

t= 5 min

t= 1 min

polymerization occurs at leading edge of keratocyte, rear surface of Listeria

t= 0

Filament Turnover

microinject “caged” fluorescently-labeled actin

illuminate in specific location with UV light to release caging group

caginggroup

resorufin resorufin

Measure rate of fluorescence decay = actin turnover ratet1/2 = 30 sec

t = 30 sec

t = 1 min

t = 0

actin stays insame placeas cell movesforward

[Actin]= 500 M

in vivo

t1/2 = 0.5 min

in vitro

t1/2 = 30 min

Actin Cc = 0.1 M

Actin dynamics in vivo are controlled by actin binding proteins

- D D D D-Pi

D D D-Pi T

T

+

T D

D

1

1. monomer pool

2

2. nucleation

3

3. elongation

4

4. depolymerization

Regulation of the monomer pool

Thymosin 4M.W. 5000binds 1:1 - enough to buffer all the actinsequesters actin from polymerizinglocalization - diffuse

ProfilinM.W. 14,000binds 1:1 - can buffer 20% of actinpromotes nucleototide exchange and polymerizationbinds PIP2 and proline-rich sequences localization - diffuse and leading edge, Listeria surface

Nucleation

Arp2/3 complex7 subunits, include actin-related proteins 2 and 3promotes actin polymerization at listeria surfaceaccelerates actin polymerization in pyrene actin assembly assay

(with activator, eliminates lag phase)binds (-) ends and filament sides - branching functionlocalization - lamellipodia

Activators:Listeria: Act Acells: WASP family proteins

immuno-EM ofArp2/3at actinbranchpoints in leadingedge

Elongation

Capping factors:CapZ (Capping protein) - (+) endtropomodulin - (-) endgelsolin - (+) end

• can stabilize or destabilize filaments,• prevent elongation

Profilinpromotes (+) end growth

Depolymerization

gelsolinM.W. 87,000Ca++-dependent severing

ADF/cofilinM.W. 19,000binds G- and F-actinaccelerates (-) end depolymerization 25-fold

+ ADF

Question:what controls depolymerization in Listeria tails?

Listeria + cytoplasmic egg extract motility in vitro

immunodeplete gelsolin or ADF/cofilin and observe effects

control gelsolin ADF/Cofilin

Rosenblatt et al., 1997

Important Breakthrough:• Reconstitution of Listeria motility frompurified components

required:• Actin and ATP• Arp2/3 complex• ADF/cofilin• Capping protein

stimulators:• VASP -binds ActA, actin, profilin• Profilin• -actinin

- D D D D-Pi

D D D-Pi T

T

+

T D

D

end availability:CapZ, gelsolin

nucleation:Arp2/3complex

monomer regulation:thymosin 4, profilin

depolymerization:ADF/cofilin

Organization of actin filaments

Cross-linking proteins

Membrane attachments

Myosins

Assemblies: cell cortex, stress fibers, contractile ring,cell protrusions, microvilli

stationary cell - stress fibers

Dividingcell

Dictyostelium amoeba

locomoting cell - filopodia and lamellipodia

Cell Motility

SwimmingMicrotubule-based – cilia, flagella

CrawlingActin-based

purposes: 1) wound healing - epidermal cells2) immune response - leukocytes –migrate to sites of infection3) development – neural crest cells; neuronal process extension4) cancer cell metastasis –malignancy determinant

protrusion

anchorage

forward movement

tail retraction

4 processes coordinated:

protrusion

Actin polymerization at leading edge - local force

Proposed mechanisms:

1) “thermal rachet” - actin polymerization pushes

2) myosin I - movement of actin filaments

2) myosin I - dependent

myosin I could also transport assembly factors to membrane

1) thermal rachet - membrane fluctuations

anchorage

Adhesion plaques:

connect cell to substratumprevent leading lamella from retracting

forward movement

Observations:Actin networks stationary with respect to substratumCell body and nucleus rotatesmyosin II required

Proposed mechanisms:

1) sarcomere-like contractions in rear2) transport along actin arrays

crawlingDictyosteliumamoeba

tail retraction

passive - cell snaps loose from adhesion plaques

How is cell polarity established?

cell loaded with Ca++ -sensitive dye Fura-2