Vav Deficient Macrophages: The Roles of Rac and Rho in

Cell Migration

Vav-Vav-voom, let’s Rac ’n’ Rho!

Julia SeroTy 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:

Vav pathways

Active Vav

Rac

ROK

Rho

MAPK/ERK pathway

Calpain Activation

Myosin Phosphorylation

Integrin

Src

Syk

F-actin 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 PathwayActive Vav

MLC*MLC

GTP-Rho/ MBSMBS

GTP-Rho/ ROK

ROK

GTP-RhoGDP-Rho

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 (GFP-

tagged 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

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