Cytoskeleton and cell motility

Dr. Mirvat El-Sibai

The cytoskeleton
Microtubule filaments
Microtubules
Microtubules
Microtubule dynamics
Microtubule dynamics
Microtubule-associated proteins (MAPs)
Microtubule-associated proteins (MAPs)
 Motor Proteins Produce Large Movements in Cells

Visualization of walking kinesin motor protein by high

speed atomic force microscopy

Myosin video
Kinesin video
Motor Proteins Produce Large Movements in Cells
Cytoskeleton drugs
Taxol
Microtubule organizing center
(MTOC)
Microtubule organizing center
(MTOC)
Microtubules in cytokinesis
Microtubules in cytokinesis
Microtubules in cytokinesis
Actin filaments
Actin dynamics
 Actin Binding Proteins
• Monomer binding proteins
– Profilin
– Thymosin Beta 4

• Filament binding proteins

– Capping proteins
• capZ, gelsolin, tropomodulin
– Motor proteins
• myosins
– Side binding proteins
• Cross-linking proteins filamin, alpha actinin
• Severing proteins ADF/cofilin, gelsolin
Motor Proteins Produce Large Movements in Cells
Motor Proteins Produce Large Movements in Cells
Monomer Binding proteins
Capping Proteins
Severing Proteins
Actin in cell migration
Movie Metastasis in Animal
 Carcinoma cell motility cycle
• The earliest event in defining
cell direction is a signal-
induced actin polymerization.

1. SIGNAL • Protrusion occurs off of the

substratum – initial
2. PROTRUSION protrusions
A region of highly branchedare adhesion
actin network
independent.
driven by actin nucleation
2. PROTRUSION

• Adhesion defines the

dominant lamella and fixes cell
3. ADHESION
polarity.

4. CONTRACTION • Regulation of adhesion sites

stabilizes cell polarity and
determines the consequences
of contraction.
5. DEADHESION
Actin in motility
Actin polymerization at the leading edge
Actin Polymerization
The Arp2/3 complex looks like the
barbed end of an actin dimer
The Arp2/3 complex looks like the
barbed end of an actin dimer
The Arp2/3 complex looks like the
barbed end of an actin dimer
Arp 2/3 in lamellipod of motile keratocyte
 What happens when you add together
proteins with different effects?

Green – preformed F-actin seeds

Red – G actin Ichetovkin et al., Curr Biol. 2002 12:79-84.
Mechanisms of lamellipod formation in MTLn3 cells

ABP Capping proteins Cofilin ATP Actin/profilin Arp2/3

7000 PLCCofilin
Relative number of barbed
ends at the cell edge (a.u.)

6000

5000

4000
F ACTIN AT THE
LEADING EDGE
3000
FREE BARBED ENDS
2000 AT THE LEADING
PI3K
EDGE
1000

0
0 60 120 180 240 300 360 (El-Sibai et.al, Cell motil Cytoskel. 2004)
Time after stimulation (sec)
 The GDP/GTP switch

EGF
EGF-R PI(4,5)P2 PI(3,4,5)P3

PH
GDP
Rho-GDP GEF Rho-GTP

GTP
PI3K Effectors

GDI
Rho-GDP GAP

Pi
 Signaling of Rho GTPases in actin
Rac Cdc42
IRSp53 PIR121 GTP GTP
Nap1 WAVE
HSPC Abi
300
PAK
N-WASP WASP

PI-4-P5K
MLCK
LIMK

Arp2/3 PIP2 Arp2/3

Release of capping cofilin

proteins

Actomyosin contraction Severing Actin nucleation

Actin nucleation
and barbed
and polymerization and polymerization
end formation
Activation of WASP family
 Traditional roles of Rho GTPases

Control Rac CA Cdc42 CA Rho CA

Swiss 3T3
Distance from edge Cdc42 is required for actin-mediated protrusion

Time 0 min 1 min 3 min

Fold area increase 1.6 Luciferase control

1.5
1.4
1.3 Cdc42 KnDn
1.2
1.1
1
0.9
0 100 200 300 400
(El-Sibai et. al, JCS 2008) Time (sec)
 Cdc42 KnDn cells lack the Arp2/3-rich/Tropomyosin-free
Lamellipodia:
Compartment at the leading edge
(first 1 um)
- Zone of actin nucleation
- enriched in Arp2/3, cofilin, barbed ends
- dense network of highly branched actin
- lacks tropomyosin

750 1 min
Luciferase
650 8 Luciferase

Fold increase in fluorescence

Adhesion
Pixel intensity (a.u.)

Cdc42 KnDn

Pixel intensity at the edge

550 Cdc42 KnDn
6
450

350 4
250
0 min Lamella
0 min -enriched in tropomyosin
2
150 1 min -less dense, less branched actin network
50 0
-0.2 0.3 0.8 1.3 1.8
Distance (m) 0 min 1 min et al.,3JCS
Demarais min2002
 Effect of Cdc42 knock-down on actin branching

Luciferase

v
Incidence angle
(0)
Cdc42 KnDn 1
38.2 -/+ 1.6
2 WT

Cdc42 KD 46.0 -/+ 1.8

Maly and Borisy, PNAS 2001

(El-Sibai et. al, JCS 2008)

Cdc42 is required for cell motility
Luciferase Cdc42 KnDn
Live Visualization of RhoA Activity in Carcinoma Cells
Reflects a Novel Role of the Rho/p160ROCK in Cancer
Cell Motility

Rho
GTP

ROCK mDia
G-actin

LIMK

MLC phosphatase
MLC MLC P
MLC kinase
Cofilin

Actomyosin assembly Severing Increased actin

and contraction and barbed polymerization
and stress fiber formation end formation
GTPase regulation of focal
adhesions

Kaibuchi et al., Annu. Rev. Biochem. 1999.

68:459-486
 Localization of Rho GTPases during cell motility
1. Direction of movement (Cdc42)

4. Contraction (RhoA)

3. New adhesions form

2. Lamellipodial protrusions (Rac/Cdc42)

5. Dissolution of old adhesions

and tail retraction (Rho)
RhoA/ROCK activity leads to the maturation of focal complexes into
focal adhesions

Control Y27632

Control ROCK
inhibitor

GFP-Vinculin
 Rho fixes the protrusion/sense of direction
leading to constructive motility
1) Protrusion forms 2) Rac-mediated focal 3) Rho-mediated focal
by polymerized actin complexes form at the edge adhesions form behind the edge
transmitting mechanical force
to the cell

Anchorage

Tension

GTP-Rac Focal complex

GTP-Rho Focal adhesion