The actin cytoskeleton is essential for cell shape and migration, and is implicated in illnesses including cancer and various infectious diseases. Nevertheless, the molecular mechanisms that regulate actin dynamics at the cell cortex to control shape and migration, how actin dynamics are coupled with force generation, and how the mechanisms of actin regulation differ in cells such as fibroblasts and neurons, are not well understood. Actin is also present in the nucleus, where it participates in nuclear shape, transcription, chromatin movement and chromatin remodeling. However, it is poorly understood how the nuclear-cytosolic distribution of cytoskeletal proteins is regulated, whether actin acts as a monomer or polymer in the nucleus, and how it performs its nuclear functions. We will test the overall hypothesis that the Arp2/3 complex and its nucleation promoting factors (NPFs), key regulators of actin nucleation and organization, play important roles in regulating both cytoplasmic and nuclear functions of actin. We will answer three important questions. How does the Arp2/3 catalytic cycle contribute to cell migration in fibroblasts and neurite outgrowth in neurons? How do NPFs influence actin polymerization at the cell cortex, or transcription in the nucleus, to control neurite formation and differentiation of neurons? How are the nuclear-cytosolic distributions of Arp2/3 and actin regulated, and what nuclear functions do they perform? We propose the following aims: (1) We will determine the role of the Arp2/3 catalytic cycle in cell migration and shape by combining biochemical and biophysical approaches to elucidate how Arp2/3 acts in actin dynamics and force generation in vitro, with cell biological approaches to elucidate Arp2/3 functions in fibroblasts and neurons. These studies will reveal how Arp2/3 contributes to cortical actin dynamics in shape and migration of different cell types. (2) We will examine the regulation of an NPF called JMY and its function in neurons, testing the hypotheses that it negatively regulates neurite formation by directly impacting cortical actin and/or by modulating transcription during neuronal differentiation. These studies will reveal new features of NPF regulation, and new roles for NPFs in regulating cortical and nuclear actin to control cell shape and differentiation. (3) We will use the baculovirus AcMNPV as a tool to study nuclear actin regulation and function. We will examine how AcMNPV induces nuclear actin accumulation by manipulating nuclear transport pathways, and whether nuclear actin regulates viral and host transcription. We will also test the hypotheses that nuclear actin organizes viral replication structures and drives viral motility and egress from the nucleus. These studies will reveal new functions for nuclear actin. Because we will uncover basic mechanisms of actin regulation in cell shape, migration and differentiation, and roles for nuclear actin in normal and infected cells, our studies may result in new approaches for diagnosing and treating disease.
The proposed work seeks to answer important outstanding questions about the function and regulation of the actin cytoskeleton, a cellular system that plays a critical role in processes including cell shape, migration, division, and intracellular membrane transport. The actin cytoskeleton is also a crucial contributor to pathogenesis, for example in inflammation, cardiovascular disease, cancer metastasis, and microbial infection. Therefore, understanding the mechanisms that regulate the functions of the actin cytoskeleton may lead to new approaches for diagnosing and treating human disease.
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