Modulation of the actin cytoskeleton by the nebulin domain protein Lasp

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The overall objective of my NSERC research program is to understand the modulation and regulation of the actin cytoskeleton during development. The properties of filamentous actin, that is assembly, disassembly, stabilization, and bundling, are regulated by a wide range of actin binding proteins, resulting in many different structures in differentiated cells. Here we propose to investigate how Lasp, the single Drosophila member of the actin-binding nebulin domain protein family, regulates the actin cytoskeleton during spermatogenesis. Actin cones are prominent structures in testes, which migrate along the axoneme to separate spermatids previously in a syncytium into 64 individual sperm. This process is called spermatid individualization. We could show that actin cone migration is incomplete in Lasp mutants, resulting in defective sperm. In addition, Lasp plays a role in anchoring the stem cell niche in testis. Thus, we propose to analyze how Lasp regulates actin filaments during actin cone migration in Drosophila, and how it anchors the stem cell niche. Aims: (1) Characterize actin cone structure in Lasp mutants. We will first analyze actin cone size and migration speed in Lasp mutants and upon Lasp overexpression in isolated cysts. We will likewise study the localization of other actin regulatory proteins such as CapZ, Arp2/3, profilin, actinin, and villin in Lasp mutants. We will then test by cytochalasin D staining and injection of fluorescent G actin into testes if barbed ends of actin filaments are affected in Lasp mutants. We will also analyze actin cone ultrastructure in wild type and Lasp mutants by transmission electron microscopy. (2) Analyze actin turnover in actin cones by live imaging. We will employ fluorescence recovery after photobleaching (FRAP) to assess actin filament turnover. We will bleach GFP-actin and assess the recovery rate of filamentous actin in Lasp mutant versus wild type in different areas of the actin cone to determine if actin turnover is affected. As a negative control, we will test recovery after addition of cytochalasin, which blocks monomer addition and should therefore slow the recovery rate, as well as in a viable CapZ mutant, a positive control, which should increase the recovery rate. These experiments will provide information on turnover and assembly of actin cones in wild type and Lasp mutants. (3) Investigate the function of Lasp protein domains and interacting proteins. We will use a triple-tagged Lasp transgene to biochemically isolate proteins interacting with the Lasp LIM, nebulin, and SH3 domains in vivo. Interacting proteins will then be identified by mass spectrometry. In parallel, we will determine the localization of domain-specific transgenes in actin cones and their ability to rescue defects in spermatid individualization. These approaches will identify the domains and protein partners required for actin cone migration, but also for anchoring the male stem cell niche, as these constructs will be expressed in both germ cells and hub cells. Impact: Our work on Lasp will lead to a better understanding of spermatogenesis and actin cone migration. In addition, our basic cell biological studies on Lasp will shed light on the mechanism of nebulin domain function in spermatogenesis. We believe our results will extend to other nebulin family members in vertebrates, revealing how nebulin domains regulate actin filaments in different contexts. Our results will therefore provide insight into the dynamic regulation of the actin cytoskeleton, which allows morphogenesis and by extension development to proceed properly. This research program will provide outstanding training opportunities for graduate and undergraduate students in all aspects of cell biology, genetics, and biochemistry.