Remodeling of actin filaments is essential for normal cell processes such as wound healing, cell division, and embryonic development. However alterations in the actin cytoskeleton are also associated with serious diseases such as metastatic cancer and cardiomyopathy. Understanding the fundamental mechanisms that govern actin-based cell motility is an important unsolved problem in biology that is critical for developing therapeutics that target alterations in actin dynamics and organization. In order to understand the molecular mechanisms that regulate the assembly of actin filaments, we are focusing on palladin, which we have recently shown can enhance actin polymerization. Normal embryonic development requires palladin and overexpression of palladin is correlated with invasive motility associated with metastatic cancer, however the precise role of palladin in cell motility has yet to be determined. Our current model suggests that palladin promotes actin nucleation and stabilizes actin filaments and that these functions are critical for regulating actin dynamics and organization within the cell. In the proposed studies we will rigorously test this model, determining whether palladin is key for actin-based cell motility and exploring the critical interactions involved to unravel the mechanism.
In Aim 1 of this proposal, we will analyze the role of palladin in nucleation of G-actin by testing different nucleation mechanisms.
In Aim 2, we will determine the effect of palladin-induced polymerization on actin network architecture.
In Aim 3, we will complement our quantitative assays of in vitro actin kinetics with in vivo monitoring of actin-based motility driving the formation of Listeria comet tails. This combination of experiments will allow us to address the question of how palladin contributes to actin-based actin dynamics and organization; and moreover how this role changes with increased expression of palladin.
A major challenge has been to understand how remodeling of the actin cytoskeleton is regulated in normal cell motility and by what means this is altered in diseases such as metastatic cancer and cardiomyopathy. Palladin is a key regulator of actin that has been shown to be essential for life and for normal mammalian embryology, but is also highly overexpressed in the most metastatic populations of cancer cells, suggesting that a clearer understanding of palladin's molecular function may provide insight into mechanisms for controlling the inappropriate motility of invasive cancer cells. Therefore we will use biochemical, structural, and cellular approaches to determine the mechanism by which palladin modulates the cytoskeletal architecture.