CAREER: Elucidating VopF-Mediated Pointed-End Actin Polymerization Mechanism and Leveraging it to Discover Novel Pointed-End Elongators

Actin filaments are essential components of the cellular skeleton (the cytoskeleton), a dynamic network of protein polymers that controls cell shape, enables muscle contraction, and drives cell division and cell motility. For over four decades, these filaments were thought to grow almost exclusively from their “barbed” ends while disassembling from the opposite, “pointed” ends. This CAREER project investigates a newly discovered mechanism of actin assembly that challenges this long-standing paradigm, and seeks to determine how certain proteins, including the bacterial protein VopF, can instead drive filament growth from pointed ends. Understanding this unconventional mechanism is important for revealing how living cells organize and remodel their internal architecture, especially in muscle cells, where filament growth is thought to occur from pointed ends although the underlying mechanism has remained unknown. Because defects in actin regulation contribute to diseases such as cardiomyopathies and infectious diseases in which bacterial pathogens hijack the host-cell actin cytoskeleton, this work will inform new biotechnology and biomedical approaches for understanding and restoring cellular function. The project also supports STEM education by training students at the interface of biology, physics, and chemistry through interdisciplinary research, outreach programs for school students, and new university courses introducing students to quantitative approaches in biology. The goal of this research is to define the molecular rules governing pointed-end actin polymerization by combining single-molecule biophysics, protein biochemistry, live-cell imaging, and modeling. VopF was recently identified as the first known protein capable of processive pointed-end actin elongation. The first aim will identify the minimal domain requirements for VopF-mediated pointed-end elongation by determining how its actin-binding domains (including WH2 and proline-rich domains) recruit and assemble actin monomers. The second aim will determine how endogenous pointed-end binding proteins regulate VopF-mediated elongation through the formation of multicomponent regulatory complexes. The third aim will investigate how mechanical force influences VopF’s pointed-end elongation. Findings from in vitro experiments will be validated through live-cell imaging experiments to bridge the gap between biochemical mechanisms and cellular function. The project is expected to establish a mechanistic framework for pointed-end actin assembly, define new principles of cytoskeletal regulation, and facilitate the identification of endogenous mammalian pointed-end polymerases. The work will also advance biotechnology through the development of quantitative single-molecule toolkits for studying and engineering dynamic protein assemblies. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria. NSF Award ID: 2543825 | Program: 01002627DB NSF RESEARCH & RELATED ACTIVIT,01003031DB NSF RESEARCH & RELATED ACTIVIT | Principal Investigator: Shashank Shekhar | Institution: Emory University, ATLANTA, GA | Award Amount: $1,103,118 View on NSF Award Search: https://www.nsf.gov/awardsearch/show-award/?AWD_ID=2543825 View on Research.gov: https://www.research.gov/awardapi-service/v1/awards/2543825.html