Actin-based motility of
Listeria monocytogenes
Scot C. Kuo
Department of Biomedical Engineering
Johns Hopkins University
Baltimore, MD 21205
People
(Animations: //www.bme.jhu.edu/~skuo/)
Scot C. Kuo, Johns Hopkins
Dictyostelium discoideum strains
James L. McGrath (now U. Rochester) Peter Devreotes, Johns Hopkins
John Hammer, NIH
Fay Peng (actin gels; Listeria)
Charles Fisher (Listeria)
Listeria monocytogenes strains
Soichiro Yamada (COS7 cells)
Dan Portnoy, U. California, Berkeley
Rooshin Dalal (Dictyostelium phago)
Justin Skoble, U. California, Berkeley
Karthik Ganesan (LTM device)
Reconstitution & Extracts
Support: NSF (MCB), NIH (GM),
Dyche Mullins, U. California, SF
Whitaker Foundation
Tim Mitchison, Harvard
Polymer Physics
Narat John Eungdamrong, Harvard
Tom Mason, CalTech
Frank Gertler, MIT
Denis Wirtz, Johns Hopkins
Jim Harden, Johns Hopkins
Outline
Biology: actin-based cell motility
 Technology: laser-tracking and
microrheology
 Nanometer-scale stepping

– Complexity of Listeria motility

Force-velocity relationship
Cell structure determined by cytoskeleton:
Svitkina et al. 1997
plus-end
G-actin
(monomer)
~5.4 nm
F-actin (all cells; muscle)
200-4,000 G-actin subunits
not covalently associated
cytoskeleton =
network of cross-linked filaments
Dynamics of actin (GF)
=
plus-end
Life Cycle of Listeria monocytogenes
--penetration into adjacent cell (MDCK cells)
spread
Cell
boundary
listeriolysin
host
actin
Target cell
division
propulsion
MDCK columnar cells
Robbins et al. 1999 J Cell Biol 146, 1333-49
Similar Biochemistry
Listeria (ActA)
Cells (WASp)
Outline
Biology: actin-based cell motility
 Technology: laser-tracking and
microrheology
 Nanometer-scale stepping

– Complexity of Listeria motility

Force-velocity relationship
Wiggles reveal mechanical (viscoelasticity)
environment around a particle
For tracer particles
(low vol fxn, larger than pores):
big wiggles = soft/thin
small wiggles = hard/thick
Time-scale important:
liquids different from solids
*Theory: Mason & Weitz, 1995; Diffusing-wave spectroscopy (DWS)
For General Viscoelastic Materials
G* = Gd(w) exp[id(w)]
Rough Approximation:
2kBT
Gd(w) 
3pa R2(t)
d ln Gd(u)
p
d(w) 
2
Wiggles2D=R2(t)
a=particle radius
(
d ln u
t=1/w
)
u=w
Better Approximation:
2kBT
Gd(w) 
d ln R2(t)
2
3pa R (t) G( 1 + d ln t )

d ln Gd(u) u+w
1
d(w) 
ln u-w d ln u
p - d ln u

t=1/w
Laser-Tracking Instrument
Laser-Tracking Microrheology (LTM)
Mason et al. 1997 Phys Rev Lett 79, 3282-85
(a+b)-(c+d)
a+b+c+d
(b+c)-(a+d)
y =
a+b+c+d
x =
No optical forces (<0.1mW)
--not an optical tweezers
High resolution
(latex beads, lipid droplets)
~0.2 nm spatial (ms)
~20 msec temporal
Spatial Resolution
Resolution
(latex beads, lipid droplets)
~0.3 nm spatial (ms)
~20 msec temporal
Proof-of-Principle:
LTM of 3% PEO
•Very accurate over 3.5 decades
of moduli and 4.5 decades of
frequency (<15% error)
Mechanical rheometry (strain-controlled cone & plate)
Diffusing wave spectroscopy (multiply-scattered light)
•Phosphor latency of Newvicon
video introduces major phase
shift error
Mason et al. 1997, Phys Rev Lett 79, 3282-5
Non-invasive measurements:
LTM in COS7 cells (not motile)
• Natural “granules” (lipid droplets) ~300 nm
-- spherical, rigid, and very refractile
oP
ER
L
• Laser-tracking sensitivity gives non-invasive,
in situ estimate of particle size (Mie-like)
• Fast (3-30s, including calibration by PZT)
Lamellae (F-actin; 820, 28)
Endoplasmic Reticulum (vimentin; 330, 45)
Other perinuclear (~50, ~90)
F-actin, entangled (80 mM; 11, 23)
Moduli Values: Gd & d at 10 rad/s; Units: dyne/cm2
Yamada et al. 2000, Biophys J. 78, 1736-47
Kuo Lab: Current Model Systems

How do proteins generate force?
– Listeria monocytogenes – food borne pathogen that spreads
by “hijacking” host cell’s actin-based motility
– motility can be reconstituted using only purified proteins

How do cells respond to and generate forces?
– Dictyostelium discoideum



Cell division (collab: D. Robinson)
Phagocytosis
Chemotaxis (collab: P. Devreotes)
– Animal cells: adhesion, spreading, particle uptake
(collab: L. Romer, C. Chen, K. Leong)

How do cells maintain tissue integrity?
– Mouse keratinocytes (collab: P. Coulombe)

Validity of microrheology assumptions
– Continuum? Ergodic?
Listeria in COS7 cells
Yamada et al. 2000, Biophys J. 78, 1736-47
“Classic” Brownian ratchet
(Peskin et al, 1993 Biophys J 65:316-324)
Because of staggered filaments in
F-actin, the intercalation distance is
d=2.7nm (G-actin is 5.4nm).
Prediction
At t>(d/velocity),
Brownian fluctuations  d
“Elastic” Brownian
ratchet
(Mogilner and Oster, 1996 Biophys J 71:3030-45)
Prediction: If flexing filament is bound, binding
must be flexible enough to allow intercalation
(>2.7nm).
Within living host cell
Wiggles too small; Steps during motility
INSIDE
CELLS
2 mm
Kuo & McGrath (2000) Nature 407, 1026-9
2.7
Perp (nm)
RECONSTITUTED
EXTRACTS
-2.7
(Methylcellulose)
-8.1
2.2
7.6
13
18.4 23.8 29.2 34.6
40
45.4 50.8 56.2 61.6
Parallel (nm)
67
72.4 77.8
Speed controlled by duration of
pauses
• Despite presence of half-steps,
the average distance between
pauses is constant with speed
(5.21.1 nm, n>650)
• Duration of pauses increases as
bacteria slow (power law= -1)
Reconstituted extracts + methylcellulose
Steps should not be observable!
One filament is too soft, particularly if the end is fluctuating
monomer dimensions to allow monomer intercalation.
Hundreds of filaments should
not be molecularly coordinated
nor molecularly aligned!
Models that generate “steps”
#Tethering Filaments:
One (Kuo & McGrath;
Dickinson & Purich)
Few (Mogilner & Oster)
All (Mahadevan)
Models that generate “steps”
#Tethering Filaments:
One (Kuo & McGrath;
Dickinson & Purich)
Few (Mogilner & Oster)
All (Mahadevan)
Models that generate “steps”
#Tethering Filaments:
One (Kuo & McGrath;
Dickinson & Purich)
Few (Mogilner & Oster)
All (Mahadevan)
Spatial periodicity of system
Biochemical Complexity:
Two systems activated/recruited by ActA
ARP2/3
ARP2/3 (Actin-Related Protein)
 Nucleates F-actin
 Dendritic networks
 Lamellapodia-like
VASP
VASP (Vasodilator-Activated Serine Phosphoprotein)
 Delivers profilin-actin (ATP)
 Protect barbed (+) ends?
 Straighten actin filaments (debranch?)
 Filopodia-like
Removing VASP
Mutant ActA (GGG) that cannot bind VASP
Extract lacking VASP (MVD7 cell line)
Effects of removing VASP:
 Slower speeds
 Less directional persistence
 Consistent with multiple (few) tethers without VASP
Biochemical Complexity:
Two systems activated/recruited by ActA
ARP2/3
VASP
Reduced System (no recycling):
ActA (on beads)
ARP2/3
G-actin
Capping Protein
Motility with Subset of Proteins:
ARP2/3, actin, capping protein
Stepsizes not regular, but some
stretches appear very regular
~3nm steps appear often
Pure proteins very different
from extract
--Concentration of proteins?
Models that generate “steps”
#Tethering Filaments:
One (Kuo & McGrath;
Stretches of
regularity?
Dickinson & Purich)
Few (Mogilner & Oster)
All (Mahadevan)
Jerky
motion?
Spatial periodicity of system
For General Viscoelastic Materials
G*(w) = Gd(w) exp[id(w)] ; |G*| = Gd
Rough Approximation:
2kBT
Gd(w) 
3pa R2(t)
d ln Gd(u)
p
d(w) 
2
Wiggles2D=R2(t)
a=particle radius
(
d ln u
t=1/w
)
u=w
Better Approximation:
2kBT
Gd(w) 
d ln R2(t)
2
3pa R (t) G( 1 + d ln t )

d ln Gd(u) u+w
1
d(w) 
ln u-w d ln u
p - d ln u

t=1/w
Strategy to measure forces
-- use methylcellulose
Challenges:
• Interfere with biochemistry? - NOT • Quantify moduli (hence Fdrag)?
Heterogeneity
too thick to pipet: dissolve in situ 15 min
tail
Listeria
bead (0.5 mm)
==> Local Measurement (tracer particles)
(Laser-Tracking Microrheology)
Quantifying methylcellulose ‘load’
on motility
G a c3.3
Use Laser-Tracking
Microrheology
(LTM) to acquire
complete
viscoelastic spectra,
despite
heterogeneity (not at
equilibrium).
8 pN
80 pN
45 nm/s
7.3 nm/s
Fdrag=12(1.4)pa2|G*(w)|, w=v/2a
Listeria velocity with methylcellulose ‘load’
RNA polymerase (Wang et al. 1998)
Skeletal Muscle, frog (Hill 1937)
Kinesin (Visscher et al. 1999)
Skeletal: c=1.3-4
Cardiac: c=3-6.1
c=4.5
Why biphasic relationship?
1. Kinetics of “working”
vs. “attached” filaments
(Mogilner & Oster, 2003)
2. Biochemistry (VASP?)
More actin in tail with loading
Mogilner & Oster, 2003
McGrath et al, 2003
Pure Proteins Stronger than
Bovine Brain Extract
Pure Proteins (no recycling):
ARP2/3
Actin
Capping Protein
Problem: Agarose does not obey
Cox-Merz rule.
Microneedle Measurements
Marcy et al. 2004
Biochemical Differences
Using purified proteins
WASP stimulation (not ActA)
Comparison:
Force-velocity relationship very
gentle (not biphasic)
Tail wall “thickens” with load
(similar to fluor.)
Summary
Biology: actin-based cell motility
 Technology: laser-tracking and
microrheology
 Nanometer-scale stepping

– Complexity of Listeria motility

Force-velocity relationship
?
Actin-based motility of pathogens
-- Listeria monocytogenes & Shigella flexneri
spread
listeriolysin
host
actin
division
propulsion
Listeria monocytogenes:
food-borne infections
Shigella flexneri:
bacillary dysentery
Rickettsiae conorii & R. rickettsiae:
Rocky Mountain spotted fevers
Vaccinia virus:
related to smallpox virus
Listeria Outbreak, 2002



40 cases in Northeast US, including 7 deaths
10/9/02: Recall of 0.3 million pounds of
cooked poultry deli meats (Pilgrim’s
Pride/Wampler)
10/14/02: Recall of additional 27.4 million
pounds (Pilgrim’s Pride/Wampler); 6% of total
turkey production
== Largest recall in USDA history ==
Dendritic Nucleation (Arp2/3)
Biochemical Complexity:
Two systems activated/recruited by ActA
ARP2/3
VASP
Reduced System (no recycling):
VASP (Vasodilator-Activated Serine Phosphoprotein)
ARP2/3 (Actin-Related
Protein)
ActA
(on beads)
 Delivers profilin-actin (ATP)
 Nucleates F-actin
ARP2/3
 Protect barbed (+) ends?
 Dendritic networks
G-actin
 Straighten actin filaments (debranch?)
 Lamellapodia-like
Capping Protein
 Filopodia-like
“Classic” Brownian ratchet
(Peskin et al, 1993 Biophys J 65:316-324)
Paradox: How can polymerization push?
Strategy to measure forces
-- use methylcellulose
Challenges:
• Interfere with biochemistry?
• Quantify moduli (hence Fdrag)?
tail
Listeria
bead (0.5 mm)
Methylcellulose does not affect
biochemistry
VCA (C-term of WASp)
activates
ARP2/3
1.5% MCL is highest methyl
cellulose concentration
Methyl cellulose has no effect
on actin alone (not shown)
and ARP2/3-induced actin
polymerization kinetics.
More rigorous model
Oster)
Two classes of actin filaments:
• Attached (strain-dependent rate of dissociation)
• Working (elastic Brownian ratchet; not attached)
(Mogilner &
Microneedle Measurements
Marcy et al. 2004
Solvent Effects on Methylcellulose