PROJECT SUMMARY/ABSTRACT Transport of nucleic acids and proteins from the nucleus to the cytoplasm is essential for nearly all cellular processes, and when mis-regulated, is associated with diseases, tumor formation/growth, and cancer progression. Canonically, this indispensable process has been thought to occur exclusively via Nuclear Pore Complexes, which span the nuclear envelope’s double membranes and provide a critical regulatory step in what exits (and enters) the nucleus. Recently, Nuclear Envelope (NE-) budding was shown to provide an alternative pathway for nuclear exit, particularly for large ribonucleoprotein (RNP) complexes that would otherwise need to unfold/remodel to fit through the pores. In this pathway, large macromolecule complexes are encapsulated by the inner nuclear membrane, cross the perinuclear space, fuse with the outer nuclear membrane, and are released into the cytoplasm, a mechanism strikingly similar to herpesvirus nuclear egress. Thus, NE-budding elegantly allows for large RNP complexes to exit the nucleus together and be delivered as a package for specific cellular functions. Despite its clear biological importance and clinical relevance, very little is yet known about the regulatory or structural machineries that allow NE-budding to occur in any system. Recently, we found that the Wiskott Aldrich Syndrome family actin nucleation protein, WASH, its four subunit regulatory complex (SHRC), and Arp2/3 are necessary for NE-budding. Using WASH/SHRC as a new entry point, in tandem with strategies to discover novel genes/proteins involved in this process, our long-term goal is to understand the molecular and cellular mechanics that govern NE-budding. The specific aims of this proposal are to determine the mechanism(s) of WASH/SHRC function in NE-budding, and to identify/analyze the infrastructural components/machineries governing the dynamic NE-budding process using a combination of genetic, biochemical, cell biological, time-lapse live imaging, and super- resolution/EM microscopy approaches. Drosophila provides an excellent, genetically amenable, organism for studying this conserved process due to its amenability for imaging and the wealth of cutting edge cell/molecular techniques and reagents. The information gathered in these studies will help to elucidate the mechanisms governing this exciting new nuclear export pathway in normal development or when mis-regulated in disease conditions, and may inform the study of herpesvirus nuclear egress as well.

PROJECT NARRATIVE The transport of molecular complexes from the nucleus to the cytoplasm is essential for most developmental processes and has been thought to occur only through specific openings in the nuclear envelope/membrane. A new alternate export pathway was recently described wherein large molecular complexes exit the nucleus by ‘budding’ through the nuclear envelope/membrane, a process that we find requires the highly-conserved WASH subfamily of Wiskott-Aldrich Syndrome (a disease with clinical symptoms including immune deficiency and cancer) proteins. These studies are expected to provide new insight into the rules governing this new nuclear export pathway in normal cellular processes, the consequences of inappropriate regulation leading to disease conditions such as laminopathies, neurological disorders, aging, herpesvirus infections, and/or cancer onset/progression, as well as provide a fresh basis for new potential therapeutic developments.