P1253037259eYoHA

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Distance Detection Through the MAPK Pathway
Noa Rappaport
17/5/05
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Mating
Background
.
.
.
.
?
a
shmoo
mating
Bardwell, 2005
3
The Question
How do cells measure distance ?
?
.
.
4
Possible strategy I simple diffusion
1D
3D
t20s
t10s
t2s
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Superimposed intracellular switch ?
Greaded response
OR
Sharp switch
sst?
nH 1
Huang Ferrell, 1996
Poritz et. al., 2001
In Oocytes
Pheromone response (After a very long time 6h)
So, what do we have ?
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Initial Reponse Second option
Experimental Observations
n6
Fus3
Kss1
Theoretical curve
Theoretical curve
Experimental curve
Experimental curve
Normalized MAPK level
Normalized MAPK level
6.8
0.2
6.8
0.2
? Factor conc. (nM)
? Factor conc. (nM)
Conclusion diffusion alone cannot measure
distance, where does the shift in kd come from ?
Strong amplification, but No switch !
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Going back to extracellular diffusion will
protease help ?
Activation
Activation
!
distance
distance
Fast protease degradation
Strong dependence on protease concentrationgt
cell number !
Conclusion cannot measure distance
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Possible role for endocytosis
Endocytosis defective mutant
Without ? factor
With ? factor
Schandell Jenness, 1994
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Possible role for endocytosis
Experimental Observations
of ? factor receptors/cell molecules per cell at 107cells/ml ? Factor concentration
104 6104 1nM
Experiment (bar1? strains)
Add ? factor
Add ? factor boost
Measure MAPK activity
Results
0.09nM
0.2nM
6nM
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Endocytosis causes an apparent smaller kd
k1
d1
d0
R
Ra
Ra
R
k-1
p
M
M
d1gtgt d0
QSSA
Can endocytosis explain threshold ?
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Simulations constant ? factor concentration
?
?
?
?
?
cell
Effective shell
Assumes

• Each cell feels" a uniform concentration
• The concentration changes in a uniform way
• The cells are spread uniformly
• The receptor is spread in a uniform way across
  the cell
• The amount of receptor does not rise along the
  process (constant production rate)

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Are the approximations valid ?
of source cells can be translated into
effective L
pulses
.
.
Practically uniform distribution of ?
D30?m2/s
Rcell5?m
.
.
In steady state equal activation to all
constant production
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Low ? concentration (0.06 nM)
Time dependence simulations
High ? concentration (60 nM)
R
R
?
?
R-?
R-?
Activation level
Activation level
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? factor half life
Higher concentrations steep increase
? factor t½ (sec)
Low initial conc. constant
Initial ? factor conc. (nM)
Prediction I Shorter activation duration for
higher alpha factor concentrations
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Endocytosis generates a threshold at later times
Effective nh
Activation level (mol.)
Initial ? factor conc. (nM)
Time (sec)
Prediction II Activation becomes switch like
with time
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Duration
Experimental Observations
0.6 nM
600 nM
6 nM
Normalized MAPK activity
Time (min)
High a factor concentrations persistent MAPK
activity
Normalized MAPK activity
Low a factor concentrations transient MAPK
activity
Time (min)
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Gene Expression
Experimental Observations
5 minues
90 minues
Response becomes threshold like with time
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? factor is secreted in pulses? Maybe

• ? factor has a short half life

mRNA half life of 2.5-4 minutes

• ? factor is cell cycle regulated

MFA1
Size-based Synchronization
? factor-based Synchronization
Cdc15-based Synchronization
Periodicity score 1.44
MFA2
? factor-based Synchronization
Cdc15-based Synchronization
Size-based Synchronization
Periodicity score 3.364
Data from Spellman et al., (1998).  Comprehensive
Identification of Cell Cycle-regulated Genes of
the Yeast Saccharomyces cerevisiae by Microarray
Hybridization.  Molecular Biology of the Cell 9,
3273-3297.
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Future work

• Extension of the model
• 3D analysis
• Use of endocytosis defective mutants
• Verify in WT strains
• Single cell analysis in physiological conditions
• Generation of gradient?

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Thanks

• Offer
• Roy
• Naama
• Everyone else !

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Diffusion Models-1D
.
.
.
.
L
a
?
Source
Sink
Pulse-like
Constant
Strength
Distance
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Parameters

• cell_volume 555 in micrometer3
• R_init 104 molecules
• shell_volume 105 in micrometer3
• kd nM2Mol(6.8)shell_volume
• k_on 520/shell_volume/R_init
• k_off kdk_on
• total_activated_molecule 102
• kd_activation R_init0.1/6.8
• d_1 1/180
• d_2 d_11000
• d_0d_1/30
• d_3 d_1/100
• alpha_conc 0.06 nM
• pd_0R_init
• complex_init 0
• alpha_range logspace(-2,3,100)
• alpha_range logspace(-2,7,1000)