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ECB Chap. 17

Cytoskeleton filament systems

Actin polymerization dynamics
Actin polymerization and force generation
Myosin motors
Signals that regulate actin
how molecular properties of a single protein, along with its family of regulators,
enable the formation of elaborate and dynamic structures that underlie many of
the cells functions.

Cells have three major cytoskeletal systems:

1. actin microfilaments
2. microtubules

cell shape, movement, adhesion,

division, intracellular transport

3. intermediate filaments } shape, adhesion

Dynamic

From Lodish, Molecular Cell Biology 6e

More static

Cell shape
and
structure,
mechanical
strength

Actin: an abundant (1-5% of total protein)

and evolutionarily conserved filamentforming protein present in all eukaryotes

Cell adhesion,
cortical tension

What does
actin do?

Cell migration:
Converts ATP into motion

The actin cytoskeleton:

Assembly of filaments in different times and places enable different shapes
and functions

Epithelial
microvilli

Stress fibers in
static cells

Filopodia and
lamellopodia in
motile cells

Cytokinetic furrow in
dividing cells

Movie: actin-GFP in a moving cell

From Lodish, Molecular Cell Biology 6e

Properties of actin 1: exists in two forms

Monomer (G-actin)

From Lodish, Molecular Cell Biology 6e

Polymer (F-actin: filament)

Chain of monomers, noncovalently bound

Properties of actin 2: filaments are polar with different ends

Subunits all face the same direction: ATP-binding cleft faces (-) end
Growth can happen at both ends of a filament: are they different?

Expt: take short seed filament -> add G-actin to polymerize

Faster
growing

Slower
growing

seed

Lodish, Molecular Cell Biology 6e

Actin polymerization: nucleation, elongation and steady state

Dynamics!
In absence of seed 3 stages of polymerization
1). Nucleation: formation of stable nucleus (slow)
2). Elongation: monomer addition at ends (fast)
3). Steady state: rate of total addition=rate of total loss
this happens at Cc: critical concentration

From Lodish, Molecular Cell Biology 6e

Actin polymerization: kinetics in vitro

Fast!

Lag
phase

No change in
mass over time

From Lodish, Molecular Cell Biology 6e

Actin polymerization dynamics in vitro: treadmilling

At steady state: [g-actin]=Cc=0.2 uM
If [g-actin]>Cc..polymerize
If [g-actin]<Cc..depolymerize
At steady state: treadmilling!
(when on rate of + end = off rate at end)
(-) ends
disassemble into
ADP, actin

(+) ends assemble:

Bound to ATP

Hydrolysis
destabilizes
filaments
From Lodish, Molecular Cell Biology 6e

Actin polymerization: kinetics in vitro vs in vivo

Seeds or
nucleating
protein eliminate
log phase

From Lodish, Molecular Cell Biology 6e

Actin function in cells: controlled by actin-binding proteins

[G-actin] in non-muscle cells: ~200 uM. 50% of total actin.
This is well above critical concentration.
Why dont unorganized filaments spontaneously form?
Thymosin-B4,
Profilin
Arp2/3

Fascin

Cofilin

Myosin

ERM
CP
Figure 17-31 Essential Cell Biology ( Garland Science 2010)

Actin polymerization dynamics drive cell migration

Cells pull on substrate using actin-generated forces

Stages in the actinbased cycle

de-adhesion

extension/protrusion
adhesion
de-adhesion,
translocation

A model system for protrusion:

intracellular actin-based movement of the bacterial pathogen Listeria monocytogenes

bacteria

actin

Hijacks actin cytoskeleton!

From Lodish, Molecular Cell Biology 6e

Reconstitution of motility with pure proteins:

actin polymerization is sufficient for force generation

FORCE

Which proteins are sufficient?

(with bacteria, ATP, G-actin)
1). Arp2/3 complex: Nucleator
Organizes Y-branches (70 angle)
optimal for force generation at
bacterial surface

2). Capping protein:

Caps (+) ends of old filaments
so that newer ends (closer to
bacteria) polymerize

3). Cofilin: severs ADP filaments

->disassembly, monomer recycling
From Lodish, Molecular Cell Biology 6e

(maintain high local [ ] of monomer)

How do cells move?

A mechanism similar to Listeria movement drives lamellipodia protrusion
during cell migration.
A branched actin network with outwardpointing Plus-ends pushes PM forward.

FORCE

Cell: an Image Library

Lodish, Molecular Cell Biology 6e

Myosin motors: mechanochemical enzymes that harness ATP

hydrolysis to drive conformational changes and movement along actin

Myosin II:
Muscle contraction, cell migration

Myosin V:
Vesicle movement

http://www.youtube.com/watch?v=vJ9ffKeUCvE
From Kodera et al. Nature Nov 4;468(7320):72-6. Epub 2010 Oct 10.
http://www.nature.com/nature/journal/v468/n7320/full/nature09450.html

Most myosin motors move towards the + end of actin filaments

If you attach the myosin motor domain (head) to glass and add actin filaments
and ATP as depicted below, the actin filaments will move along the glass.
Which end of the actin filament will be leading, and which will be lagging?
A. - end leading, + end lagging
B. + end leading, - end lagging

From Lodish, Molecular Cell Biology 6e

By pulling actin filaments relative to the membrane or to each

other, myosins can generate contractile forces that are essential
for vesicle transport, locomotion, cortical tension, muscle contraction etc.

The actin cytoskeleton in migrating cells:

Localized in three distinct structures
-lamellipodia/ruffles sheet-like membrane extensions
-filopodia spike-like membrane extensions
-stress fibers long intracellular fibers

From Lodish, Molecular Cell Biology 6e

Signaling pathways that regulate actin:

the Rho GTPases Rho, Rac and Cdc42
Three related signaling proteins that regulate distinct arrangements of actin

Stress
fibers

Inject cells with

constitutively active
GTPase:
(Cant hydrolyze GTP)
Lamellipodia

Filopodia
From Lodish, Molecular Cell Biology 6e

Signaling from Rho GTPases to actin:

regulate cell migration

From Lodish, Molecular Cell Biology 6e