Establishing direction during chemotaxis in eukaryotic cells: What can theoretical models tell us?

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Establishing direction during chemotaxis in
eukaryotic cells What can theoretical models
tell us?
Wouter-Jan Rappel UCSD
Collaborators Herbert Levine and William F.
Loomis, UCSD Peter J.
Thomas, The Salk Institute
Supported by NSF
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Chemotaxis

• Ability to respond to spatial and temporal
  gradients of chemoattractants/repellants
• Present in many eukaryotic cell types
• Gradients determine direction of motion

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Examples

• Wound healing
• Embryogenesis
• Neuronal patterning
• Angiogenesis

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Neutrophil chasing a bacterium (Staphylococcus
aureus) Movie made by David Rogers, taken from
the website of Tom Stossel (expmed.bwh.harvard.edu
)
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Chemotaxis in Dictyostelium Discoideum

• In Dicty, cells display strong chemotactic
  response to cAMP
• Dicty cells move up the gradient

From S. Lee, Firtel lab, UCSD
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What Is Dicty?

• Unicellular amoeba ( 10 mm).
• Live as separate cells on forest floor feed on
  bacteria.
• Upon starvation cells interact by chemical
  signals, adhesion, etc. and aggregate (50,000
  cells).
• Differentiate into 20 stalk and 80 spore cells.
• Form slug ( mm) and fruiting body.

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Lifecycle
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Why study chemotaxis in Dicty?
Why study anything in Dicty?

• Short life cycle (24 hours).
• Easy to grow.
• Many mutants developed.
• Exhibits many important biological processes.
  

• Genetic manipulations have revealed large part of
  the architecture of the signaling network.
• Library of strains with GFP-fused proteins.
• These strains can be used in subcellular
  fluoresence microscopy.

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Cells are chemotactic to cAMP

• cAMP binds to cell membrane via receptor CAR1
  (5,000 molecules).

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cAMP waves in developing populations
Every 6-8 minutes Cells move in first half of wave
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Asymmetric cAMP stimulus
Recent experiments using GFP-tagged PH
(Pleckstrin Homology) domain proteins
Frames every 2 s Three pulses from right, 6 s
duration
http//www.med.jhu.edu/devreotes
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Uniform stimulus
Response to a uniform increase in
chemoattractant
concentration. Frames were taken every 2 seconds.
The chemoattractant was added just before the
cell goes out of focus. From C.A. Parent and P.N.
Devreotes, Johns Hopkins.
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C.A. Parent et al., Cell 95, 81 (1998)
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Relay model for Dicty
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Theoretical modeling of signaling
Asymmetry is established in very short time
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We propose
Inhibitory process delivered via an intracellular
messenger
This messenger diffuses in the interior of the
cell and competes with the external signal
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cGMP is a good candidate

• No direct evidence against cGMP
• Produced rapidly after cAMP stimulus

Mutant data supports role of cGMP in chemotaxis
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Relay model
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Our model
Membrane can be in three states Quiescent,
Activated or Inhibited The transition rates
between these states are dependent on the
extracellular cAMP concentration and the
intracellular cGMP concentration The excited
state of the membrane produces the localization
of PH domain proteins and subsequent downstream
events cAMP and cGMP diffuse in the exterior and
interior repectively Cells are treated as two
dimensional ellipsoids
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Membrane dynamics
Quiescent
Inhibited
Activated
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Full model
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Uniform pulse of cAMP
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Asymmetric pulse
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Asymmetric pulse, cont.
Amplification ratio AR
Here AR5
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Model in cartoon form
cAMP
cAMP
cAMP
cAMP
cAMP
cAMP
a
i
cGMP
i
cAMP
a
cAMP
cGMP
i
cGMP
a
i
i
cGMP
a
cAMP
cGMP
cGMP
cGMP
i
cAMP
a
i
cGMP
cAMP
cGMP
a
cGMP
cGMP
cAMP
cGMP
i
a
cGMP
i
cAMP
a
cGMP
i
cGMP
a
i
cAMP
a
cAMP
i
cAMP
cAMP
cAMP
cAMP
cAMP
q
cAMP
cGMP
i
a
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Pulse from the left (front) followed by pulse
fromthe right (back)
Predictions
We propose the following experiment
This will give insight in the time scales of the
inhibition and the activation
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Timing experiment uses laminar flow
cAMP
Buffer
cAMP
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Result from the numerical model
Time delay (s)
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Predictions, cont.
cGMP mutants should have radically altered PH
domain protein localization patterns These
mutants should not chemotax properly
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Some more remarks
Our model adresses first few seconds Does not
include cAMP production, PH domain protein
localization, cAMP/cGMP adaptation, establishment
of polarity Cannot account for behavior in long
lasting spatial gradients
Our model suggests that a spatio-temporal signal
is needed for directional sensing Directional
sensing absent in static spatial gradients?
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Summary

• Model can produce significant asymmetry within a
  few seconds
• Requires rapidly diffusing internal messenger
• Likely candidate cGMP (mutant data)
• Specific predictions can verify model

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Future work

• Perform proposed dual injection experiment
• Verify prediction for mutant experiments
• Extend model past initial response (include
  adaptation)
• Develop different numerical techniques
  (phase-field method)

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Sensitivity to parameter values
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Response to pulse from micro capillary
cAMP micro capillary near right upper corner.
cells lacking actin filament formation. Micro
capillary moves around
Images taken every 5 seconds. From C.A. Parent
and P.N. Devreotes.
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Asymmetric response of PH domain proteins also
found in Neutrophils
G. Servant et al., Science 2000
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Experimental set-up in Eberhard Bodenschatzs lab
S.K.W. Dertinger et al. Anal. Chem. 2001, 73, 1240