Actin-based Motility - WPI

Actin-based Motility
Lamellipodium and filopodium
extension
propulsion
Listeria
actin tail
bacteria
(Vic SMALL)
(M-F Carlier et al)

actin
Actin Filament
ATP
(Ken Holmes et al, Nature 1990

Treadmilling
Rate of polymerization
0
C cB
Barbed end
Pointed end
concentration
p
T
Pi
e
D
d
C ss
C cP
ADP
ATP
At steady state the filament treadmills :
Subunit flux = d = e = p = k+B.(Css-CcB)

Lamellipodium Protrusion
Signal
TREADMILLING
ADF-F-actin
growing barbed end
shrinking pointed end
capped barbed end
cross linker
Anchorage
nucleation
elongation
capping
release

imicking lamellipodium with a glass rod ?
30 µm Glass fibre, ∅ =1 µm
Actin lamella

Treadmilling
Depolymerization
Polymerization
Vitesse = 4µm /min = 66 nm/s = 26 a/fil/s

Synergy between Profilin and ADF
ADF
D
D
T
ADF
D
K T prof
Profilin
ADP
ATP
Profilin
K D prof
Profilin
T
P
D

Treadmilling of actin filaments :
Effect of capping
Rate of polymerization
0
C ss
Barbed end
C cB C cP
Pointed end
C ss
k B + kP + (CP C -CB C )
C ss
C ss
C ss
C ss
concentration
Rate of filaments growth
+
0
-
J γ G
0 0,5 1
Extent of barbed end capping:γ
« Tous pour un et un pour tous »
Jγ ss
J γ Les trois G mousquetaires. = 1- (1- γ γ) k B A. Dumas
+ +kP +

Role of capping proteins in motility:
funneling the treadmilling
Slow pointed end disassembly
from many capped filaments
Nucleotide exchange
Fast barbed end assembly
of few uncapped filaments
and
Capping

WASP family proteins :
activators of Arp2/3 complex
« closed N-WASP »
WH1
B
A
GBD
VV C
Pro
+ X
+ Arp2/3
+G-actin
Cdc42
IcsA
TccP
Plasma membrane
« activated N-WASP »
X
WH1 B
GBD Pro VVC A
A
VV C
Pro
R. Robinson et al
(Science, 2001)
(Miki et al., 1998; Machesky and Insall, 1998; Rohatgi et
al., 1999; Egile et al., 1999;
Kim, Rosen et al., 2000; Prehoda et al;, 2000)

Filament branching array in lamellipodia
Arp 2/3
50
40
Count
30
20
Branching
(T. Svitkina and G.G. Borisy, 1999; Blanchoin et al.; Pantaloni et al., 2000)
10
0
0 1 2 3 4
Ratio D/M

Modeling barbed end branching
by Arp2/3 complex: un autocatalytic
process
2.5
Polymerised actin, µM
2
1.5
1
0.5
A+A F
F +A F
k +1
= 2.10 -9 s -1 µM -1
k -1
= 5.10 -4 s -1
k +2
= 10 s -1 µM -1
k -2
= 1 s -1
F +Y+2A ===> 2F
Arp2/3
app k +3
= 0.64 s -1 µM -3
0-71 nM
2Y Y 2
active inactive
0
0 500 1000 1500 2000
Time, s
K
4
= 10 -2 µM

Arp2/3: Branching Mechanism
(Pantaloni et al., NCB 2000)
WA or ActA

Cycles of filament attachment-detachment are coupled to branching
VCA
Pantaloni et al., NCB 2000; Science 2001

Actin polymerization and force production:
Evolution of the Brownian Ratchet .
(Oster and Mogilner, 1996-2003)

Reconstitution of actin-based
movement from pure proteins (Loisel et al.,
Nature 1999)
• Treadmilling of filaments feeds movement
• Functions required for movement:
3) Site-directed generation of barbed ends
by N-WASP (resp. ActA)-activated Arp2/3
2) Chemostat maintaining a high steady-state
concentration of ATP-G-actin : Actin,
ADF/cofilin, profilin, Capping protein
• Movement results from a balance between the
creation of new growing filaments (branching)
and death of these filaments (capping).

Movement of E. coli (IcsA) and Listeria monocytogenes with
pure components.
E. coli IcsA
Rate :
≈2 µm/min
Listeria
Rate :
≈3 µm/min
Essential Proteins :
N-WASP IcsA-bound
Arp2/3 0.1 µM
Capping Protein 0.1 µM
ADF 2 µM
ATP-actin+F-actin 8 µM
Useful Proteins :
Profilin 2 µM
α-actinin 0.5 µM
VASP 0.1 µM

Mimicking
Lamellipodium Protrusion
TREADMILLING
ADF-F-actin
cross linker
shrinking pointed end
capped barbed end

Biomimetic motility assay: actin
assembly against a functionalized solid
surface
Beads of 3 different sizes
Stuck bead

A break of symmetry in the
actin meshwork leads to a
polarized actin tail and
movement

Biomimetic motility assay: actin assembly
against a functionalized lipid membrane (GUV)

Actin tail forms and propels the liposome
following break of symmetry

03:18 pm
Encounters of the third kind
Motility medium :
N-WASP bead-bound
Arp2/3 0.1 µM
Capping Protein 0.1 µM
ADF 2 µM
ATP-actin+F-actin 8 µM
Profilin 2 µM

Four Symmetric Comets

The two helices rotate in register
and display opposite handedness
Actin insertion

The helical parameters of the actin tails
depend on the geometry of the
microsphere

The surface density of N-WASP affects bead motility
comet-forming beads [%]
[%]
100
80
60
40
20
Mean velocity [µm/min]
1.6
1.2
0.8
0.4
0
0 5 10 15 20 25
mean distance between N-WASP [nm]
100
• Movement requires a threshold
80
of N-WASP density
[%]
60
• Velocity 40 is proportional to filament
number, i.e. to N-WASP density
20
0
0 100 200 300 400 500
gelsolin concentration [nM]
mean tail density [∆ gray value]
0
60
40
20
0
0 0.04 0.08 0.12
N-WASP surface density [ molecules/nm 2 ]

Effect of external force on motility
4
3
billes 1600 N-WASP
mean velocity [ µm/min]
1.6
1.2
0.8
0.4
0
0 0.5 1 1.5 2 2.5 3 3.5
bead diameter [µm]
mean velocity [ µm/min]
2
1
billes 200 N-WASP
0
0 0.2 0.4 0.6 0.8 1
methyl cellulose concentration [% w/v]

Simulation of actin-based motility: balance between
filament branching and capping (A.E. Carlsson, 2003,
Biophys. J.)
Autocatalytic branching :
1. Growth velocity is independent of applied force
2. Branch spacing decreases with capping

Measurement of force velocity relationship for actin-based
propulsion
tail section versus force
Experimental design
force
displacement
pulling
pushing
elastic extension
Tail lengthening
Force velocity diagram
Fast pulling, detachment and regeneration of the actin tail
Marcy et al., PNAS, 2004

Arp2/3 incorporates in actin tails upon barbed
end branching at the surface of N-WASP coated
beads
Rhodamine-actin
Alexa green-Arp2/3
Phase contrast
Alexa green-Arp2/3
Addition of Alexa green-Arp2/3

Branch spacing decreases steeply
upon increasing capping (Wiesner et al.,
JCB, 2003)
Gelsolin: 25 nM 50 nM 100 nM 200 nM
Rh-actin
Alexa488
-Arp2/3
Both
. . .
. . .

Conclusions
• The velocity of beads depends on the number
of filaments pushing the bead.
• Movement is controlled by a balance between
filament branching and capping (Carlsson’s
model).
• Velocity is not sensitive to external load
(viscous drag), i.e. the force due to
polymerization at the bead surface is balanced
by the internal brake (friction) due to attached
filaments.

Importance of the detachment
of filaments following formation
of the branched junction:
role of VASP

Effect of VASP on the motility of ActA-coated
beads

Effect of VASP on the motility of ActA-coated
beads

VASP decreases the density of
filament branching
+ VASP - VASP
A
Actin
Arp2/3
Merged
(Samarin, Romero et al., J. Cell Biol. 2003)
Actin, pico­moles
Arp2/3, femto­moles
60
40
20
0
Arp2/3
(Arp2/3:actin)x1000
2
1
0
Actin
+ VASP ­ VASP
Arp2/3
+ VASP ­ VASP
Arp2/3
Actin
Actin

VASP enhances actin-based motility by accelerating
filament detachment allowing growth after branching
+ VASP
ActA on BEADS
Fluorescence, a.u.
Soluble ActA
+ VASP
ActA on BEADS
control
0 2000 4000 6000 seconds

Without VASP the rate of detachment of the branched
junction from ActA, is slow.
k = 1.510 -3 s -1
- VASP
A
YA
Y
A
Y
ActA
A
VASP
Arp2/3
Filament
V
Y
Y
A
ActA-coated-Bead
A
Y
A
Y
Y Y
A
Y
- VASP
k = 2.5 10 -4 s -1
A
Y
A
Y

Actin-based motility
• Control of filament turnover
• Site-directed generation of new filaments:
2 mechanisms:
Branching
Barbed end
nucleation (WASP/Arp2/3)
and
processive growth
(formins)

The formin family
FH3
FH1
FH2
DAD
Fmn1/2, cdc12p
Rho
cdc42
GBD
FH3
FH1
FH2
DAD
Bni1p, Bnr1p
Rac1
GBD
FH1
FH2
DAD
FHOS/FHOD1
RhoA
GBD
FH3
FH1
FH2
DAD
Dia, hDia1/mDia1
cdc42
GBD
FH3
FH1
FH2
DAD
hDia3/mDia2
RhoD
GBD
FH3
FH1
FH2
DAD
hDia2/mDia3
DAD
RhoA
GBD
FH2
FH3
FH1
Rho
GTPases
Actin
nucleation
profilin
Autoregulatory
domain
RhoA
GBD
FH3 FH1 FH2
FH1
DAD
DAD
FH2

Properties of formins
• Nucleate actin assembly (FH2 is sufficient)
• Active as FH2 or FH1-FH2 dimers
• Bind to barbed ends without greatly affecting rat
parameters for actin assembly and disassembly
• Postulated to be processive « leaky cappers »
remaining bound to growing barbed ends

Formin is a processive motor that directs barbed
end assembly of actin filaments from profilinactin
Romero et al., Cell (2004)

Formin remains bound to a growing barbed end for
1200 to 2500 seconds before detaching (Romero et al.,
Cell, 2004)
QuickTime et un décompresseur Video sont requis pour visionner cette image.
Solution of F-actin (0.5 µM) and profilin (4 µM)
Frequency of detachment of a barbed end:
kd = 7.5. 10 -4 ± 1.5.10-4 s -1

Formin increases the rate of profilin-actin
association to barbed ends
0 profilin + profilin
FH1-FH2
FH2
free
FH1-FH2
free
PA

Formin increases the rate of ATP hydrolysis
in profilin-actin assembly

Reconstitution of formin-based motility
FH1FH2
coated bead
ADF : 4µM
Prof : 3µM
F-actin : 7µM
Bead velocity, µm/min
20
15
10
5
0
0 5 10 15 20 25 30
[ADF] in µM
25
20
15
10
5
0
0 5 10 15 20
[profilin] in µM
V = 2,5 µm/min

Actin-based motility
LEBS, CNRS, Gif-sur-Yvette
▼
▼
▼
▼
▼
Dominique Pantaloni
Marie-France Carlier
Emmanuèle Helfer
Dominique Didry
Diep Lê
Stanislav Samarin
Sebastian Wiesner
Christophe Le Clainche
✆ Stéphane Romero
✆ Vincent Delatour
Collaborators
(Institut Pasteur) :
Coumaran Egile
Philippe Sansonetti
(Institut Curie):
Jacques Prost
Cécile Sykes
(Harvard medical
school) :
Christine Kocks

Capping proteins regulate the speed and
duration of formin-based motile processes

Microfilaments: Polarity,
Flexibility.
Persistance length : 12 µm
7 nm

Mimicking « hopping Listeria »:
From continuous to periodic actinbased
movement

Formin drives rapid site-directed barbed end
assembly of actin filaments from profilin-actin
Distance
Time

DPi
DPi

ATP hydrolysis occurs on Arp2 only following
branch formation and drives debranching
(Le Clainche » et al., 2003, revised, PNAS)

Model for actin-based motility
i n a c t i v e N - W A S P
1. Signal-mediated activation of N-WASp, the branching
enzyme
2. G-actin, Arp2/3 and filaments as substrates of N-WASp
3. Dissociation of the branch (product) and recycling
Question: Role of ATP exchange and hydrolysis on Arp2/3
in the branching and recycling of Arp2/3 complex ?

Dendritic nucleation on ActA
beads .
(Svitkina and Borisy)

Gallery

Localization of Arp2/3 complex at the branch
junction and on the mother filament
50
40
Count
30
20
10
0
0 1 2 3 4
Ratio D/M
Rhodamine-Actin
200 ms
Alexa488-Arp2/3
30 seconds

Arp2/3-stimulated actin polymerization is an
autocatalytic process
2.5
Polymerised actin, µM
2
1.5
1
Arp2/3
0.5
1-71 nM
Ln [(F-Fo)/(Fmax-F)]
1
0
-1
-2
-200 0 200
Time, s
0
0 500 1000 1500 2000
Time, s

Contraintes de Polymérisation
?

Comète Hélicoïdale

Forces of the order of 1 nN are developed
by actin polymerization
Effect of methylcellulose on G(f)
(Laser Tracking Microrheology)
Force-velocity relationship

Using the motility assay to understand
the mechanism of production of force
and directional movement
• Control of the concentration of soluble proteins
in the motility medium.
• Control of the surface density of filament
branching enzyme (N-WASP or ActA).
• Load/velocity relationship: control of the size of
the bead and of the viscosity of the medium.
• Frequency of filament branching during
movement: two fluorophores (actin and Arp2/3)

Actin-based motility:
How vectorial assembly of actin filaments
can generate force and movement

Microfilaments: Polarité,
Flexibilité.
7 nm

PERSPECTIVES
• Biomimetics: Reconstitution of lamellipodium protrusion
(force applied to a membrane, functionalized liposome)
• Coupling of adhesion and protrusion during cell
migration: concerted actin dynamics at focal contacts
and in lamellipodium.
• Signaling, actin-based motility and morphogenesis:
specifying different motile actin-based structures.

Arp2/3 Complex : downstream target of
multiple signaling pathways leading to actin
assembly
Arp3
21 47
20 34
16
Arp2
42
Non-zero crosslink
Two hybrid
Zero-length crosslink
40
(Higgs and Pollard, 1999)
R. Robinson et al
(Science, 2001)
• Localized in actin-based motility processes:
• Listeria and Shigella propulsion (Welch, 1997;
Egile et al., 1998)
• Lamellipodium extension (Svitkina and Borisy,
1999)
• Phagocytic cups (Machesky, 2000)
• Endocytic vesicles (Taunton et al., 1999)
• Actin patches (Li 1997; Cooper 1999)
• Cadherin-mediated adhesion (Yap, 2002)
• Morphological events in Drosophila (Cooley,
2002)
• Must interact with an activator:
• ActA on Listeria
• WASP and Scar/WAVE proteins in

03:18 pm
Encounters of the third kind
Motility medium :
N-WASP bead-bound
Arp2/3 0.1 µM
Capping Protein 0.1 µM
ADF 2 µM
ATP-actin+F-actin 8 µM
Profilin 2 µM

Nucléation Polymérisation : f(c)
Mg ++
λex
λem
polymérisation
concentration
c 3
c 2
c 0
c 1
•
•
c 4
•
•
•
c 5
•
Temps, minutes

Structure de l’Actine
Séquence des 375 acides aminés
constituant l’actine humaine
MDDDIAALVVDNGSGMCKAGFAGDDAP
RAVFPSIVGRPRHQGVMVGMGQKD
SYVGDEAQSKRGILTLKYPIEHGIVTN
WDDMEKIWHHTFYNELRVAPEEHP
VLLTEAPLNPKANREKMTQIMFETFNT
PAMYVAIQAVLSLYASGRTTGIVM
DSGDGVTHTVPIYEGYALPHAILRLDL
AGRDLTDYLMKILTERGYSFTTTA
EREIVRDIKEKLCYVALDFEQEMATAA
SSSSLEKSYELPDGQVITIGNERF
RCPEALFQPSFLGMESCGIHETTFNSI
MKCDVDIRKDLYANTVLSGGTTMY
PGIADRMQKEITALAPSTMKIKIIAPP
ERKYSVWIGGSILASLSTFQQMWI
SKQEYDESGPSIVHRKCF

Actin filaments in cell movement and
morphogenesis
• Actin filaments have a polar structure
• They are semi-flexible polymers
• Assembly dynamics is regulated in vivo
• Filament assembly is a dissipative
reaction (hydolysis of actin-bound ATP)

Cell motility and signaling
f-Met.Leu.Phe
Neutrophils
Chasing Leucocyte

How ATP hydrolysis regulates motility and the
stability/mechanical strength of branched actin
arrays
Le Clainche et al., J. Biol. Chem. Accel. Publ. 2001; PNAS 2003

TP hydrolysis on Arp2/3 drives filament debranching
WA
Pi
ADP ATP
G
ATP
2
ADP
2
ATP
3
Pi
ATP
G
ATP
2
Arp2/3
Activé
ATP
3
ATP
2
ATP
3
Arp2/3
Inactif

Control of actin dynamics
in cell motility
• Control of the [G-actin]/[F-actin] ratio
• Control of filament turnover
• Spatial control of the generation of new
filaments (link to signaling)

Structural basis for the switch from inhibition
to promotion of actin assembly in ciboulot
(Hertzog et al., Cell 2004)
Pointed End
Barbed End

Treadmilling (Wegner, 1976)
Barbed end
Pointed end
Pi
T
D
C ss
Css Filament turnover
ADP
ATP
Pure actin: 0.1 µM 3µm / 90 min
Lamellipodium: 2 µM ? 3 µm / 1 min

B
Ciboulot regulates axonal growth in Drosophila central brain
during metamorphosis by acting like profilin
(Boquet et al. Cell, 2000)
Low level of Cib expression
Overexpression of Cib
A:wild type
B: mutant

Branching Models
C
A
Autocatalytic barbed end branching
B
Arp2/3 activation and side branching
Nucleation at the membrane and side branching

Fluorescence Video Microscopy of F-actin
2000- fold dilution
in unlabeled G-actin
at Cc in F buffer
1 filament per field
= 1 filament / 50 picoliter
F-Rh-actin
+G-Rh- actin
40 µ M
F-Rh-actin
20 nM
+ G-actin
=Cc
Isambert, Venier, Maggs and Carlier (1995)

F l e xi b il i t y o f A c t i n F il ame n t s
C or r e l a t io n o f t a n ge nti al di re ct io ns
s
θ ( s )
O
< c ( s ) > = c o s [ θ ( s ) - θ ( 0 ) ]
< c ( s ) > = e - | s | / 2 L p
l n < c ( s ) > = -
s
2 L p

Reconstitution of actin-based
movement from pure proteins
Essential Proteins
N-WASP
Useful Proteins
VASP
Rate µm/min
2
1
Arp2/3
Capping
protein
ADF
2
1
α-Actinin
Profilin
0
0
0.001 0.01 0.1 1 10 100.001 0.01 0.1 1 10 100
Concentration, µM
Concentration, µM

Motile activities of living cells
Migration
Endocytosis
Division
Intracellular
transport
Directionality
Energy dissipation
Molecular motors
Directed assembly of polymer

ADF increases the treadmilling of F-actin
without ADF with ADF
Rate of polymerization
0
C cB
Barbed end
C cP
Pointed end
concentration
C cP
C ss
Pointed end
C ss

Arp2/3 interacts with barbed ends,
independently of filament length
(D.Pantaloni et al. 2000)
Polymerised actin, µM
2
1
0
20 pM
0 200 400 600
Time, s
control
Seeds length
( µm)
0
1
2 µm
Spectrin-actin
Gelsolin

VASP increases the rate of detachment of the branched
junction from ActA, allowing growth after branching
ActA in solution
V
Y
Y
V
V Y
k = 1.510 -3 s -1
± VASP
- VASP
Y
V
V
Y
Bead
ActA
VASP
V
+ VASP
Arp2/3
Y
ActA-coated beads
V
Y
Y V
k = 3.10 -2 s -1
- VASP
k = 2.5 10 -4 s -1
V
Y
+Y
Filament