Vav Deficient Macrophages:
The Roles of Rac and Rho in
Cell Migration
Vav-Vav-voom, let’s Rac ’n’ Rho!
Julia Sero
Ty Thomson
12/10/2002
Models of Macrophage Migration
• Hematopoietic cells migrate during
development and immune response
• Macrophage migration mechanism is not
well understood
• Genetic knockout is a molecular biological
tool to investigate gene product functions
• Modeling is an engineering tool to help
direct research and verify hypotheses
Vav- Cell Phenotype
• Vav: guanine nucleotide exchange factor and
adaptor protein (3 isoforms)
– Vav1>Vav3>>Vav2
– Activates Rho GTPases (Rac, Rho, Cdc42)
– Involved in signal transduction from integrins and
other receptors via multiple pathways
• Vav-deficient macrophages show migration
defect
– Vav1-/-;Vav3-/- extend multiple lamellipodia but do not
migrate in wound healing assay
– Short, narrow lamellipodia
– Fail to translocate nuclei or retract tails
Movies:
Wild type:
Double knock out:
Integrin
Vav pathways
Src
Syk
Active Vav
F-actin
Rac
MAPK/ERK
pathway
Calpain
Activation
Rho
ROK
Myosin
Phosphorylation
F-actin
Hypothesis
• Local signals mediated by Vav complexes
crucial for downstream effects
• Rac versus Rho type adhesions:
– Dynamic and propulsive (calpain)
– Stable and anchoring (myosin contraction)
• Balance of signal outputs is necessary for
efficient migration
• DKO cells have an imbalance between focal
adhesion turnover and intracellular
contraction due to loss of Vav
MAPK Pathway
(Modified from Bhalla et al., 2002)
Myosin Activation Pathway
Active Vav
GDP-Rho
GTP-Rho
GTP-Rho/
ROK
ROK
MLC
MLC*
MBS
GTP-Rho/
MBS
Major Model Assumptions
• Vav1, Vav2 and Vav3 all have the same
activity
• Localized signaling can studied, and the
effects summed to account for overall
behavior
Nuclear MAPK * Concentration Profile
Normalized Active Calpain Levels
Normalized Activated Myosin Light
Chain Concentration
Differential of Contraction and
Focal Adhesion Turnover
Differential of Contraction and
Focal Adhesion Turnover
Physiological Range?
Pathological Range?
Summary of Observations
• Calpain production and activation only
significant for active Vav concentrations of
greater than ~1nM
• Myosin activation significant for Vav
concentrations of greater than ~0.1nM
• Postulated wild type active Vav concentration at
about 10-100nM
• If [Vav2]/[Vav1+Vav3] = (1/10 to 1/100), then
DKO could fall in region of myosin activation but
low calpain activation
Model Predictions
• Normal active Vav concentrations in the
range of 10-100nM, while active Vav2 in
DKO concentration about 0.4-4nM
• Vav DKO macrophage phenotype may be
as result of imbalance between focal
adhesion turnover and myosin mediated
contraction
Other Considerations
• Vav also interacts with Cdc42, which is thought
to be important for cell polarity
• Rac and Rho are also involved in F-actin
polymerization
• Time delay observed between Rac and Rho
activation and maturation of focal contacts
• Signaling upstream of Vav has been ignored
• Other pathways downstream of Vav affected
• Concentrations of Rac, Rho, ROK, MLC, MBS
all assumed to be 0.2uM
Proposed Experiments
• Observe DKO phenotype on other substrates in
response to different signals
• Measure biochemical interactions between
species (kinetics) in vitro
• Assay for myosin phosphorylation and active
calpain
• Tension force assay to determine if DKOs lack
propulsive traction forces at leading edge
• Measure half life of focal adhesions (GFPtagged proteins, confocal microscopy)
Thank you!
• Joan Brugge, Amy Hall, and the Brugge
lab (Dept. of Cell Biology, Harvard Medical
School)
• Reshma Shetty
• Ali Khademhosseini
• Doug Lauffenburger
References
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