Shashank Shekhar - US grants

ABSTRACT Cellular actin dynamics are essential in a number of key processes such as cell migration, wound healing, cell division and endocytosis. Physiological actin dynamics arises from a complex interplay between protein machineries that influence either the assembly of new actin structures or the disassembly of existing actin structures. Over the last few decades, a plethora of proteins regulating actin dynamics in cells have been identified and individually characterized. However, we still do not fully understand how these proteins work together in multiprotein ecosystems and how they give rise to emergent behavior that cannot be predicted simply by adding their known individual activities. Over the last few years, I have discovered several such multicomponent activities. I showed that an enhancer (formin) and a blocker (capping protein) of actin growth can simultaneously bind the same site on an actin filament, in the process initiating their own dynamic exchanges at filament ends. I also accomplished the first direct microscopic demonstration of a long-predicted but never observed acceleration of pointed-end depolymerization of actin filaments by cyclase-associated protein (CAP) and cofilin. Over the next five years, our goal is to uncover how multicomponent biochemical signals and mechanical signals get integrated at the scale of individual actin filaments. We will build on our ground-breaking discoveries by investigating other proteins that we have identified, which either directly bind filament ends or via other end-binding proteins. We will also investigate how mechanical forces alter biochemical interactions of actin binding proteins with actin filaments. To do this, we will employ a unique combination of microfluidics-assisted (mf-TIRF) and multispectral single molecule imaging that I have pioneered over the last few years. We will combine biochemical and biophysical experiments with mathematical modelling. My vision is that a better understanding of molecular mechanisms underlying actin dynamics will pave the way for development of new therapies to combat human ailments like Amyotrophic Lateral Sclerosis (ALS), metastatic cancer, neurological (e.g. Alzheimer's disease and Parkinson's diseases) and developmental disorders (e.g. limb deformities) that are caused due to abnormalities related to actin dynamics.