Probing polymerization forces using

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Probing polymerization forces using Actin
propelled lipid vesicles
Arpita Upadhyaya Department of Physics Massachuset
ts Institute of Technology
Biocomplexity Workshop III, 2002 University of
Notre Dame
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Cells in motion
How do cells move around ?
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Crawling of most cells is based on actin
keratocyte
labeled actin
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Actin Polymerization
Spontaneous addition of monomers to produce
polymer filament.
Plus end
Minus end
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The polymerization motor
How can polymerization push ?
Thermal fluctuations are important
Brownian ratchet model Peskin, Odell and Oster
Elastic Brownian ratchet
Oster and Mogilner
However, a single filament is too weak
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Experimental systems to explore force generation
by actin polymerization
- Biochemically very complex - Biophysical
properties are hard to quantify
Eukaryotic cells(e.g neutrophils)
- Biochemically well defined - Biophysical
properties are easier to measure
Prokaryotic pathogens(e.g Listeria monocytogenes)
Loisel, Boujemaa, Pantaloni Carlier. Nature
401, 613 (1999).Cameron, Giardini, Soo
Theriot, Nature Rev. Mol. Cell Biol. 1, 110
(2000).
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The bacterial pathogen Listeria
5 ?m
(Julie Theriot)
5 ?m
Listeria monocytogenes propels itself through the
cytoplasm of a host cell by polymerizing actin in
its wake
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Motility in-vitro ActA Arp2/3
complex Actin Cytoplasmic extract
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Listeria
Lamellipodium
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The spherical Listeria ActA coated beads
(Cameron et al.)
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The spherical Listeria ActA coated beads
(Cameron et al. van Oudenaarden et al.)
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Experimental systems to explore force generation
by actin polymerization
- Biochemically very complex - Biophysical
properties are hard to quantify
Eukaryotic cells(e.g neutrophils)
- Biochemically well defined - Biophysical
properties are easier to measure
Prokaryotic pathogens(e.g Listeria monocytogenes)

• - Biochemically well defined
• Biophysical properties are easier to measure
• Biochemical and biophysical properties can
• be changed

ActA coated beads
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Why lipid vesicles

• Lipid vesicles deform under external loads
• Shapes can be used to measure forces
• Fluid under shear, but can bend and stretch

Microtubules polymerizing inside vesicles
(Fygenson et al.)
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Our biophysical model system Synthetic
phospholipid vesicles coated with ActA
tethers
vesicles
Fluorescence (red actin, green lipids)
DIC
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Actin driven self-organization of lipid vesicles
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Branching of tethers
Vesicles can make sharp turns
C
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ActA - vesicles as biophysical model systems for
force generation by actin

• Close similarity to real cells
• Tunable size
• Tunable lipid and membrane protein composition
• Tunable physical properties (e.g. elastic
  moduli)
• Controllable biochemistry

But most importantly, Being flexible, they
enable a quantitative measurement of the forces
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actinlipid
lipid
actin
5 ?m
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Spontaneous polarization of ActA
2 ?m
Phase contrast
Fluorescent ActA (cys-mutant)
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Vesicle deformation Energetics
A
Ao
V
Vo
actin pressure
free vesicle (rest area Ao, rest
volume Vo)
vesicle deformed by actin (area A gt Ao, volume V
lt Vo)
0
membrane stretching
osmotic pressure
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Polymerization induced stresses
Osmotic Pressure
Global, outward
Stress from stretching
Local, inward
Tension
Stress depends on the difference in curvatures
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Actin can pull and push
squeezing forces
retractile forces
S
T
S
2 ?m
Actin stress
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How to calculate deformation of a vesicle ?
membrane displacement
polymerization velocity
stresses (forces/area)
global pressure
local stretching depends on local curvatures
?1 ?2
Parameters Stretching modulus K
Initial Concentation Co
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Probing polymerization forces
0
4
140
nN/?m2
0
200
actin pressure (nN/?m2)
240
-4
T
S
S
coordinate along membrane interface
280
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Stepping motion of vesicles
5 ?m
Distance (?m)
Slow phase alternates with fast phase.
Time (s)
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Actin must bind to the vesicle
Vtrailing
?/min
?/min
Vleading
Aspect ratio
Time (s)
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Stepping of a model vesicle
4
nN/?m2
0
-4
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Actin driven motion conclusions

• Actin exerts retractile and propulsive forces
  on vesicles.
• Flexible vesicles exhibit stepping motility.
• Actin is strongly bound to membrane.
• Importance of membrane mechanical properties in
  determining forces exerted by actin relevance
  to real cells.
• Mimic the biochemistry of the lamellipodium

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ACKNOWLEDGMENTS
Alexander van Oudenaarden Jeff Chabot Albina
Andreeva
Funding Dupont/MIT Alliance Pappalardo
Foundation