Many disorders result from inappropriate cell migration, ranging from cancer metastasis to inflammatory diseases such as rheumatoid arthritis. The aberrant migration of tumor cells and leukocytes in these diseases is thought to be due to misregulated chemotactic pathways whose misregulation cause normally benign signaling from cell surface receptors (chemoattractant receptors) to induce potent migratory phenotypes. Recent studies have shown that the cofilin pathway is an essential effector of these misregulated pathways; however, the precise role of cofilin in chemotaxis is controversial. Several studies suggest cofilin is directly involved in chemotaxis and is essential for initiating directional cell migration in response to signaling from chemoattractant receptors. In contrast, other studies indicate cofilin only plays an indirect role by recycling old actin filaments, thereb promoting a dynamic actin cytoskeleton that facilitates cell migration. The different methodologies used to study cofilin have yielded contradictory results. Activation or inhibition of cofilin by genetic perturbations, such as overexpression or ablation of gene expression by RNAi, both result in stabilization of the actin cytoskeleton and inhibition of cell migration. Genetic perturbations modulate signaling in the entire cell over the course of days, and it is likely that these approaches are inadequate for deciphering the molecular logic of the cofilin pathway, which is regulated at the subcellular level on the timescale of seconds. The major goal of this proposal is to develop the capability to manipulate cofilin signaling with high spatial and temporal precision in living cells via the construction of cofilin and LIM kinase (inhibitory regulator of cofilin) analogues that are controlled by light. In contrast to genetic perturbations, photoactivation can manipulate signaling rapidly (~1 s) and locally (~ 1 ?m2).
We aim to use these analogues to study cofilin's role in facilitating tumor cell chemotaxis during cancer metastasis. Photoactivation of cofilin, and activation or inhibition of LIM kinase, will enable us o test different models of signaling from chemoattractant receptors through cofilin to directional migration.
In a variety of diseases such as cancer and rheumatoid arthritis, cells in the body enter tissues inappropriately and cause damage, leading to potential disability or death. The work proposed in this grant will develop the tools necessary to enable researchers to understand how these cells are able to leave their normal environment and enter new tissues. An initial application of these tools will help understand how and why cancer cells spread from the initial tumor to distant sites in the body to form secondary tumors that are the cause of 90% of deaths due to cancer.
