Actin-based motility ofActin-based motility ofListeria monocytogenesListeria monocytogenes

Scot C. KuoScot C. KuoDepartment of Biomedical EngineeringDepartment of Biomedical Engineering

Johns Hopkins UniversityJohns Hopkins UniversityBaltimore, MD 21205Baltimore, MD 21205

PeoplePeople(Animations: //www.bme.jhu.edu/~skuo/)(Animations: //www.bme.jhu.edu/~skuo/)

Scot C. KuoScot C. Kuo, Johns Hopkins, Johns Hopkins

James L. McGrathJames L. McGrath (now U. Rochester) (now U. Rochester)

Fay PengFay Peng (actin gels; (actin gels; ListeriaListeria))

Charles FisherCharles Fisher ( (ListeriaListeria))

Soichiro YamadaSoichiro Yamada (COS7 cells) (COS7 cells)

Rooshin DalalRooshin Dalal ( (DictyosteliumDictyostelium phago) phago)

Karthik GanesanKarthik Ganesan (LTM device) (LTM device)

SupportSupport:: NSF (MCB), NIH (GM), NSF (MCB), NIH (GM), Whitaker Foundation Whitaker Foundation

Dan PortnoyDan Portnoy, U. California, Berkeley, U. California, Berkeley

Justin SkobleJustin Skoble, U. California, Berkeley, U. California, Berkeley

Listeria monocytogenesListeria monocytogenes strains strains

Dyche MullinsDyche Mullins,, U. California, SF U. California, SF

Tim MitchisonTim Mitchison, Harvard, Harvard

Narat John EungdamrongNarat John Eungdamrong, Harvard, Harvard

Frank GertlerFrank Gertler, MIT, MIT

Reconstitution & ExtractsReconstitution & Extracts

Peter DevreotesPeter Devreotes, Johns Hopkins, Johns Hopkins

John HammerJohn Hammer, NIH, NIH

Dictyostelium discoideumDictyostelium discoideum strains strains

Tom MasonTom Mason, CalTech, CalTech

Denis WirtzDenis Wirtz, Johns Hopkins, Johns Hopkins

Jim HardenJim Harden, Johns Hopkins, Johns Hopkins

Polymer PhysicsPolymer Physics

OutlineOutline

BiologyBiology: actin-based cell motility: actin-based cell motility TechnologyTechnology: laser-tracking and : laser-tracking and

microrheologymicrorheology Nanometer-scale steppingNanometer-scale stepping

– Complexity of Complexity of ListeriaListeria motility motility Force-velocity relationshipForce-velocity relationship

F-actinF-actin (all cells; muscle)(all cells; muscle)200-4,000 G-actin subunits200-4,000 G-actin subunits

notnot covalently associated covalently associated

G-actinG-actin(monomer)(monomer)

~5.4 nm~5.4 nm

plus-endplus-end

Svitkina et al. 1997Svitkina et al. 1997

Cell structure determined by Cell structure determined by cytoskeleton:cytoskeleton:

cytoskeletoncytoskeleton = = network of cross-linked filamentsnetwork of cross-linked filaments

Dynamics of actin (GDynamics of actin (GF)F)

plus-endplus-end

==

CellCellboundaryboundary

Target cellTarget cell

MDCK columnar cellsMDCK columnar cells

Life Cycle of Life Cycle of Listeria monocytogenesListeria monocytogenes----penetration into adjacent cell (MDCK cells)penetration into adjacent cell (MDCK cells)

Robbins et al. 1999 J Cell Biol 146, 1333-49Robbins et al. 1999 J Cell Biol 146, 1333-49

listeriolysin

hostactin

divisionpropulsion

spread

Similar BiochemistrySimilar Biochemistry

Cells (WASp)Cells (WASp)Listeria (ActA)Listeria (ActA)

OutlineOutline

BiologyBiology: actin-based cell motility: actin-based cell motility TechnologyTechnology: laser-tracking and : laser-tracking and

microrheologymicrorheology Nanometer-scale steppingNanometer-scale stepping

– Complexity of Complexity of ListeriaListeria motility motility Force-velocity relationshipForce-velocity relationship

Wiggles reveal mechanical Wiggles reveal mechanical (viscoelasticity)(viscoelasticity)

environment around a particle environment around a particle

For tracer particlesFor tracer particles (low vol fxn, larger than pores)(low vol fxn, larger than pores)::

big wiggles = soft/thinbig wiggles = soft/thin small wiggles = hard/thicksmall wiggles = hard/thick

Time-scale importantTime-scale important:: liquids liquids differentdifferent from solids from solids

*Theory: Mason & Weitz, 1995; Diffusing-wave spectroscopy (DWS)*Theory: Mason & Weitz, 1995; Diffusing-wave spectroscopy (DWS)

For General Viscoelastic For General Viscoelastic MaterialsMaterials

WigglesWiggles2D2D==RR22(())aa=particle radius=particle radius

Better Approximation:Better Approximation:

(( )u=u=

dd ln G ln Gdd(u)(u)dd ln u ln u

22

2k2kBBTT33a a RR22(())

GGdd(() )

))dd ln ln RR22(())dd ln ln 1 +1 +

2k2kBBTT

33a a RR22(())GGdd(() )

Rough Approximation:Rough Approximation:

dd ln G ln Gdd(u)(u)dd ln u ln u

ln u+w

u-w d ln u

G* = GG* = Gdd(w) exp(w) exp[[ii(())]]

Laser-Tracking Microrheology (LTM)Laser-Tracking Microrheology (LTM)Mason et al. 1997 Phys Rev Lett 79, 3282-85Mason et al. 1997 Phys Rev Lett 79, 3282-85

Laser-Tracking InstrumentLaser-Tracking Instrument

x =x =((aa++bb)-()-(cc++dd))aa++bb++cc++dd

y =y =((bb++cc)-()-(aa++dd))aa++bb++cc++dd

No optical forcesNo optical forces (<0.1mW) (<0.1mW) --not an optical tweezers --not an optical tweezers

High resolutionHigh resolution (latex beads, lipid droplets)(latex beads, lipid droplets)

~0.2 nm spatial (ms)~0.2 nm spatial (ms) ~20 ~20 sec temporalsec temporal

Spatial ResolutionSpatial ResolutionResolutionResolution (latex beads, lipid droplets)(latex beads, lipid droplets)

~0.3 nm spatial (ms)~0.3 nm spatial (ms) ~20 ~20 sec temporalsec temporal

Proof-of-Proof-of-Principle:Principle:

LTM of 3% PEO LTM of 3% PEO

•Very accurate over 3.5 decadesVery accurate over 3.5 decades of moduli and 4.5 decades of of moduli and 4.5 decades of frequency (<15% error) frequency (<15% error) Mechanical rheometry (strain-controlled cone & plate)Mechanical rheometry (strain-controlled cone & plate) Diffusing wave spectroscopy (multiply-scattered light) Diffusing wave spectroscopy (multiply-scattered light)

•Phosphor latency of NewviconPhosphor latency of Newvicon video introduces major phase video introduces major phase shift error shift error

Mason et al. 1997, Phys Rev Lett 79, 3282-5

Non-invasive Non-invasive measurements:measurements:

LTM in COS7 cellsLTM in COS7 cells (not motile) (not motile)

Yamada Yamada et alet al. 2000, . 2000, Biophys J. 78Biophys J. 78, 1736-47, 1736-47

• Natural “granules” (lipid droplets) ~300 nmNatural “granules” (lipid droplets) ~300 nm -- spherical, rigid, and very refractile -- spherical, rigid, and very refractile

• Laser-tracking sensitivity gives non-invasive,Laser-tracking sensitivity gives non-invasive,in situin situ estimate of particle size estimate of particle size (Mie-like)(Mie-like)

• Fast (3-30s, including calibration by PZT)Fast (3-30s, including calibration by PZT)

LL

ERER

oPoP

LamellaeLamellae (F-actin; 820, 28 (F-actin; 820, 28))

Endoplasmic ReticulumEndoplasmic Reticulum (vimentin; 330, 45 (vimentin; 330, 45))

Other perinuclearOther perinuclear (~50, ~90 (~50, ~90))

F-actin, entangledF-actin, entangled (80 (80 M; 11, 23M; 11, 23))

Moduli Values: GModuli Values: Gdd & & at 10 rad/s; Units: dyne/cm at 10 rad/s; Units: dyne/cm22

Kuo Lab: Current Model Kuo Lab: Current Model SystemsSystems

How do proteins generate force?How do proteins generate force?– Listeria monocytogenesListeria monocytogenes – food borne pathogen that – food borne pathogen that

spreads by “hijacking” host cell’s actin-based motilityspreads by “hijacking” host cell’s actin-based motility– motility can be reconstituted using only purified – motility can be reconstituted using only purified proteinsproteins

How do cells respond to and generate forces?How do cells respond to and generate forces?– Dictyostelium discoideumDictyostelium discoideum

Cell division (collab: D. Robinson)Cell division (collab: D. Robinson) PhagocytosisPhagocytosis Chemotaxis (collab: P. Devreotes)Chemotaxis (collab: P. Devreotes)

– Animal cells: adhesion, spreading, particle uptakeAnimal cells: adhesion, spreading, particle uptake(collab: L. Romer, C. Chen, K. Leong)(collab: L. Romer, C. Chen, K. Leong)

How do cells maintain tissue integrity?How do cells maintain tissue integrity?– Mouse keratinocytesMouse keratinocytes (collab: P. Coulombe) (collab: P. Coulombe)

Validity of microrheology assumptionsValidity of microrheology assumptions– Continuum? Ergodic?Continuum? Ergodic?

ListeriaListeria in COS7 cells in COS7 cells

Yamada Yamada et alet al. 2000, . 2000, Biophys J. 78Biophys J. 78, 1736-47, 1736-47

““Classic” Brownian ratchetClassic” Brownian ratchet

Because of staggered filaments inBecause of staggered filaments inF-actin, the intercalation distance isF-actin, the intercalation distance is=2.7nm (G-actin is 5.4nm).=2.7nm (G-actin is 5.4nm).

PredictionPredictionAt At //velocityvelocity),),Brownian fluctuations Brownian fluctuations

(Peskin (Peskin et alet al, 1993 Biophys J 65:316-324), 1993 Biophys J 65:316-324)

PredictionPrediction: If flexing filament is bound, binding : If flexing filament is bound, binding must be flexible enough to allow intercalation must be flexible enough to allow intercalation (>2.7nm).(>2.7nm).

““Elastic” Brownian ratchetElastic” Brownian ratchet(Mogilner and Oster, 1996 Biophys J 71:3030-45)

Wiggles too small; Steps during Wiggles too small; Steps during motilitymotility

2.22.2 7.67.6 1313 18.418.4 23.823.8 29.229.2 34.634.6 4040 45.445.4 50.850.8 56.256.2 61.661.6 6767 72.472.4 77.877.8-8.1-8.1

-2.7-2.7

2.72.7

Parallel (nm)Parallel (nm)

Pe

rp (

nm

)P

erp

(n

m)

INSIDEINSIDECELLSCELLS

RECONSTITUTEDRECONSTITUTEDEXTRACTSEXTRACTS

((MethylcelluloseMethylcellulose))

2 2 mm

Wit

hin

livi

ng h

ost c

ell

Wit

hin

livi

ng h

ost c

ell

Kuo & McGrath (2000) Nature 407, 1026-9Kuo & McGrath (2000) Nature 407, 1026-9

Speed controlled by duration of Speed controlled by duration of pausespauses

• Despite presence of half-steps, Despite presence of half-steps, the the average distanceaverage distance between between pauses is pauses is constant with speedconstant with speed(5.2(5.21.1 nm, n>650)1.1 nm, n>650)

• Duration of pauses increases as Duration of pauses increases as bacteria slow (power law= -1)bacteria slow (power law= -1)

Reconstituted extracts + methylcelluloseReconstituted extracts + methylcellulose

Steps should Steps should notnot be be observable!observable!

Hundreds of filaments shouldHundreds of filaments should not be molecularly coordinated not be molecularly coordinated nor molecularly aligned! nor molecularly aligned!

One filament is too soft, particularly if the end is fluctuatingOne filament is too soft, particularly if the end is fluctuatingmonomer dimensions to allow monomer intercalation.monomer dimensions to allow monomer intercalation.

Models that generate Models that generate “steps”“steps”

#Tethering Filaments:#Tethering Filaments:

OneOne (Kuo & McGrath;(Kuo & McGrath;

Dickinson & Purich) Dickinson & Purich)

FewFew (Mogilner & Oster) (Mogilner & Oster)

AllAll (Mahadevan) (Mahadevan)

Models that generate Models that generate “steps”“steps”

#Tethering Filaments:#Tethering Filaments:

OneOne (Kuo & McGrath;(Kuo & McGrath;

Dickinson & Purich) Dickinson & Purich)

FewFew (Mogilner & Oster) (Mogilner & Oster)

AllAll (Mahadevan) (Mahadevan)

Models that generate Models that generate “steps”“steps”

#Tethering Filaments:#Tethering Filaments:

OneOne (Kuo & McGrath;(Kuo & McGrath;

Dickinson & Purich) Dickinson & Purich)

FewFew (Mogilner & Oster) (Mogilner & Oster)

AllAll (Mahadevan) (Mahadevan) Spatial periodicity of systemSpatial periodicity of system

Biochemical Complexity:Biochemical Complexity:Two systems activated/recruited by Two systems activated/recruited by

ActAActA

ARP2/3ARP2/3 (Actin-Related Protein) (Actin-Related Protein)

Nucleates F-actinNucleates F-actin Dendritic networksDendritic networks Lamellapodia-likeLamellapodia-like

VASPVASP (Vasodilator-Activated Serine Phosphoprotein) (Vasodilator-Activated Serine Phosphoprotein)

Delivers profilin-actin (ATP)Delivers profilin-actin (ATP) Protect barbed (+) ends?Protect barbed (+) ends? Straighten actin filaments Straighten actin filaments (debranch?)(debranch?)

Filopodia-likeFilopodia-like

ARP2/3ARP2/3 VASPVASP

Removing VASPRemoving VASPMutant ActA (GGG) that cannot bind VASPMutant ActA (GGG) that cannot bind VASPExtract lacking VASP (MVExtract lacking VASP (MVD7D7 cell line) cell line)

Effects of removing VASP:Effects of removing VASP: Slower speedsSlower speeds Less directional persistenceLess directional persistence Consistent with Consistent with multiple (few) tethersmultiple (few) tethers without VASP without VASP

Biochemical Complexity:Biochemical Complexity:Two systems activated/recruited by Two systems activated/recruited by

ActAActA

ARP2/3ARP2/3 VASPVASP

Reduced SystemReduced System (no recycling)(no recycling):: ActA (on beads)ActA (on beads) ARP2/3ARP2/3 G-actinG-actin Capping ProteinCapping Protein

Motility with Subset of Motility with Subset of Proteins:Proteins:

ARP2/3, actin, capping proteinARP2/3, actin, capping protein

Stepsizes not regular, but some Stepsizes not regular, but some stretches appear very regularstretches appear very regular~3nm steps appear often ~3nm steps appear often

Pure proteins Pure proteins veryvery different different from extractfrom extract

--Concentration of proteins?--Concentration of proteins?

Models that generate Models that generate “steps”“steps”

#Tethering Filaments:#Tethering Filaments:

OneOne (Kuo & McGrath;(Kuo & McGrath;

Dickinson & Purich) Dickinson & Purich)

FewFew (Mogilner & Oster) (Mogilner & Oster)

AllAll (Mahadevan) (Mahadevan) Spatial periodicity of systemSpatial periodicity of system

JerkyJerkymotion?motion?

Stretches ofStretches ofregularity?regularity?

For General Viscoelastic For General Viscoelastic MaterialsMaterials

WigglesWiggles2D2D==RR22(())aa=particle radius=particle radius

Better Approximation:Better Approximation:

(( )u=u=

dd ln G ln Gdd(u)(u)dd ln u ln u

22

2k2kBBTT33a a RR22(())

GGdd(() )

))dd ln ln RR22(())dd ln ln 1 +1 +

2k2kBBTT

33a a RR22(())GGdd(() )

Rough Approximation:Rough Approximation:

dd ln G ln Gdd(u)(u)dd ln u ln u

ln u+w

u-w d ln u

G*(w) = GG*(w) = Gdd(w) exp(w) exp[[ii(())] ; |] ; |GG*| = *| = GGdd

ListeriaListeriatailtail

bead bead (0.5 (0.5 m)m)

Strategy to measure Strategy to measure forcesforces

-- use methylcellulose-- use methylcellulose

ChallengesChallenges::• Interfere with biochemistry?Interfere with biochemistry?• Quantify moduli (hence FQuantify moduli (hence Fdragdrag)?)?

HeterogeneityHeterogeneity too too thickthick to pipet: dissolve to pipet: dissolve in situin situ 15 min15 min

==> ==> Local MeasurementLocal Measurement (tracer particles) (tracer particles)

( (Laser-Tracking MicrorheologyLaser-Tracking Microrheology))

- NOT - NOT --

Quantifying methylcellulose ‘load’Quantifying methylcellulose ‘load’on motilityon motility

Use Use Laser-Tracking Laser-Tracking MicrorheologyMicrorheology(LTM) to acquire (LTM) to acquire completecomplete viscoelastic spectra, viscoelastic spectra, despite despite heterogeneity (not at heterogeneity (not at equilibrium).equilibrium).

8 pN8 pN45 nm/s45 nm/s

80 pN80 pN7.3 nm/s7.3 nm/s

FFdragdrag=12(1.4)=12(1.4)aa22|G*(|G*()|, )|, =v/2a =v/2a

G G c c3.33.3

RNA polymeraseRNA polymerase (Wang (Wang et al.et al. 1998) 1998)

ListeriaListeria velocity with methylcellulose velocity with methylcellulose ‘load’‘load’

Kinesin Kinesin (Visscher (Visscher et al.et al. 1999) 1999)

Skeletal Muscle, frogSkeletal Muscle, frog (Hill 1937) (Hill 1937)

cc=4.5=4.5

Skeletal: Skeletal: cc=1.3-4=1.3-4Cardiac: Cardiac: cc=3-6.1=3-6.1

Why biphasic relationship?Why biphasic relationship?

1.1. Kinetics of “working” Kinetics of “working” vs. “attached” filamentsvs. “attached” filaments(Mogilner & Oster, 2003)(Mogilner & Oster, 2003)

2.2. Biochemistry (VASP?)Biochemistry (VASP?)

More actin in tail with More actin in tail with loadingloading

Mogilner & Oster, 2003

McGrath et al, 2003

Pure Proteins Stronger than Pure Proteins Stronger than Bovine Brain ExtractBovine Brain Extract

Pure Proteins Pure Proteins (no recycling)(no recycling)::ARP2/3ARP2/3ActinActinCapping ProteinCapping Protein

ProblemProblem: Agarose does not obey : Agarose does not obey Cox-Merz rule.Cox-Merz rule.

Microneedle MeasurementsMicroneedle MeasurementsMarcy Marcy et al.et al. 2004 2004

Biochemical DifferencesBiochemical DifferencesUsing purified proteinsUsing purified proteinsWASP stimulation (not ActA)WASP stimulation (not ActA)

ComparisonComparison::Force-velocity relationship very Force-velocity relationship very gentle (not biphasic)gentle (not biphasic)Tail wall “thickens” with load Tail wall “thickens” with load (similar to fluor.)(similar to fluor.)

SummarySummary

BiologyBiology: actin-based cell motility: actin-based cell motility TechnologyTechnology: laser-tracking and : laser-tracking and

microrheologymicrorheology Nanometer-scale steppingNanometer-scale stepping

– Complexity of Complexity of ListeriaListeria motility motility Force-velocity relationshipForce-velocity relationship

Actin-based motility of Actin-based motility of pathogenspathogens

-- -- Listeria monocytogenes & Shigella flexneriListeria monocytogenes & Shigella flexneri

Listeria monocytogenesListeria monocytogenes:: food-borne infectionsfood-borne infectionsShigella flexneriShigella flexneri:: bacillary dysentery bacillary dysenteryRickettsiae conoriiRickettsiae conorii & & R. rickettsiaeR. rickettsiae:: Rocky Mountain spotted feversRocky Mountain spotted feversVacciniaVaccinia virus: virus: related to smallpox virusrelated to smallpox virus

listeriolysin

hostactin

divisionpropulsion

spread

??

Listeria Outbreak, 2002Listeria Outbreak, 2002

40 cases in Northeast US, including 7 40 cases in Northeast US, including 7 deathsdeaths

10/9/02: Recall of 0.3 million pounds of 10/9/02: Recall of 0.3 million pounds of cooked poultry deli meats (Pilgrim’s cooked poultry deli meats (Pilgrim’s Pride/Wampler)Pride/Wampler)

10/14/02: Recall of additional 27.4 10/14/02: Recall of additional 27.4 million pounds (Pilgrim’s million pounds (Pilgrim’s Pride/Wampler);Pride/Wampler); 6% of total turkey production 6% of total turkey production

== == Largest recall in USDA historyLargest recall in USDA history == ==

Dendritic Nucleation Dendritic Nucleation (Arp2/3)(Arp2/3)

Biochemical Complexity:Biochemical Complexity:Two systems activated/recruited by Two systems activated/recruited by

ActAActA

ARP2/3ARP2/3 (Actin-Related Protein) (Actin-Related Protein)

Nucleates F-actinNucleates F-actin Dendritic networksDendritic networks Lamellapodia-likeLamellapodia-like

VASPVASP (Vasodilator-Activated Serine Phosphoprotein) (Vasodilator-Activated Serine Phosphoprotein)

Delivers profilin-actin (ATP)Delivers profilin-actin (ATP) Protect barbed (+) ends?Protect barbed (+) ends? Straighten actin filaments Straighten actin filaments (debranch?)(debranch?)

Filopodia-likeFilopodia-like

ARP2/3ARP2/3 VASPVASP

Reduced SystemReduced System (no recycling)(no recycling):: ActA (on beads)ActA (on beads) ARP2/3ARP2/3 G-actinG-actin Capping ProteinCapping Protein

““Classic” Brownian ratchetClassic” Brownian ratchet(Peskin (Peskin et alet al, 1993 Biophys J 65:316-324), 1993 Biophys J 65:316-324)

Paradox: How can polymerization push?Paradox: How can polymerization push?

ListeriaListeriatailtail

bead bead (0.5 (0.5 m)m)

Strategy to measure Strategy to measure forcesforces

-- use methylcellulose -- use methylcellulose

ChallengesChallenges::• Interfere with biochemistry?Interfere with biochemistry?• Quantify moduli (hence FQuantify moduli (hence Fdragdrag)?)?

Methylcellulose does Methylcellulose does notnot affect affect biochemistrybiochemistry

VCAVCA (C-term of WASp) (C-term of WASp) activates activates ARP2/3 ARP2/31.5% MCL1.5% MCL is highest methyl is highest methyl cellulose concentration cellulose concentration

Methyl cellulose has no effect Methyl cellulose has no effect on actin alone (not shown) on actin alone (not shown) and ARP2/3-induced actin and ARP2/3-induced actin polymerization kinetics.polymerization kinetics.

More rigorous model More rigorous model (Mogilner (Mogilner & Oster)& Oster)

Two classes of actin filaments:Two classes of actin filaments:• AttachedAttached (strain-dependent rate of dissociation) (strain-dependent rate of dissociation)• WorkingWorking (elastic Brownian ratchet; not attached) (elastic Brownian ratchet; not attached)

Microneedle MeasurementsMicroneedle MeasurementsMarcy Marcy et al.et al. 2004 2004

Solvent Effects on Solvent Effects on MethylcelluloseMethylcellulose