Edward M. Bonder - US grants

The ability of cells to properly change shape, migrate and divide, within the embryo, can determine the success or failure of embryonic development. Many of the motile events during morphogenesis are directed by the cell's actin cytoskeleton. The actin cytoskeleton of eukaryotic cells is a highly dynamic assembly of assorted structures. Fortunately, there exists a functional universality linking the actin regulatory proteins from broadly diverse cell types. The specific goals of this project are: a. Characterize the effects of sea urchin egg llOK, 95K, 50K, and 13K (profilin?) actin binding proteins on actin filament assembly and structure. In particular, the modulatory effects of second messengers such as Ca , pH, phospholipid and the nucleotide bound to the actin subunit will be analysed; b. Investigate the mechanism of Ca -sensitive regulation of egg spectrin-actin crosslinking: c. Generate and characterize polyclonal and/or monoclonal antibodies that monospecifically crossreact with the isolated and characterized actin binding proteins; d. Employ the anti-actin binding protein antibodies for immunocytochemical studies determining the spatio-temporal association of the regulatory proteins with the various actin cytoskeleton domains in oocytes, eggs and embryos; e. Examine the synthesis of the actin binding proteins during oogenesis and embryogenesis using in situ labeling and immunoprecipitation and/or autoradiography. Successful completion of the above aims will provide the necessary correlations between the in vitro protein interactions and the in vivo actin cytoskeletal localization and synthesis to generate hypotheses describing the potential molecular mechanisms modulating the actin cytoskeleton. Additionally, the production of anti-actin binding protein antibodies will provide the necessary probes to test the hypothetical molecules mechanisms in vivo and to begin examining differential expression of the actin binding proteins during embryogenesis.

A variety of diseases of the cardio-vasculature including, spherical elliptocytosis, atherosclerosis, clotting disorders, and hypertension have been linked to structural alteration and dysfunction of the actin cytoskeleton and acto-myosin activity. Relevant to this application are the platelet mediated responses during clotting where these cells are structurally transformed, undergo degranulation, and extensively aggregate to participate in clot formation. Essential to these events is the restructuring of the actin cytoskeleton and its participation in exocytosis of intracellular granules. In the present application, we employ sea urchin coelomocytes as a model for platelet activation since, like platelets, they function in clot formation, undergo structural transformation reliant upon the actin cytoskeleton, and exhibit degranulation. However, unlike platelets, these cells are excellent models for high resolution light microscopic studies and correlative biochemical analyses. The planned training program will focus on experiments designed to define the associated of a 110 kDa unconventional myosin with intracellular vesicles and to determine unconventional myosin's role in vesicle motility and cytoskeletal reorganization. Within the scope of the project, the participants will be trained in a variety of methodologies including, but not limited to, protein fractionation, light and electron microscopy, fluorescence analog cytochemistry, immunocytochemistry, biophysics, and molecular biology. Results of ,D studies will expand our knowledge of how extracellular signals lead to cytoskeletal transformation, vesicle motility, and degranulation. Since, structural and regulatory pathways within the actin cytoskeleton appear to be highly conserved across diverse species, defining the mechanisms at work in coelomocyte activation will without doubt impact our understanding of equivalent events in mammalian platelets.

An award is made to Rutgers University – Newark to acquire a Zeiss LSM980 Laser Scanning Confocal Microscope with Airyscan 2. The Zeiss LSM 980 will be integrated into the Advanced Imaging Core Facility (AICF), of the Department of Biological Sciences (DBS), a core university facility that serves Rutgers and institutions of higher education in Newark. Installation of the microscope provides critically needed instrumentation that will have an unquestionable positive impact at Rutgers-Newark as an anchor institution with broad mission to improve and advance STEM education, training, and outreach. Acquisition of the Zeiss LSM 980 will initiate improvement in the undergraduate curriculum in Biology by stimulating development of a six-week long biological imaging laboratory series in a core biology course that enrolls 600 students annually. Using in-person and virtual teaching/learning strategies, students will be able to have an exhilarating first-hand experience at multi-dimensional imaging. DBS will add hands-on laser confocal imaging to a capstone upper division laboratory course in microscopy. To reach a broader undergraduate and graduate student audience, the leadership team will work with NSF-LSAMP, NIH-G-RISE, and Rutgers-NASA Space Grant Consortium programs at Rutgers-Newark to introduce multi-dimensional imaging using the Zeiss LSM 980. It is anticipated that this approach will encourage increased undergraduate participation in research, particularly URM students, and encourage pursuit of STEM careers. STEM faculty at Rutgers-Newark have a long-standing tradition of partnering with American Chemical Society Project SEED and Newark Public Schools to provide urban high school students with hands-on laboratory experience. Microscopy is often a key part of that experience. The leadership team is invested in bringing the wonder of fluorescence imaging to local high school students through the 4-week summer immersion program RUN-IMAGE and the academic year program Aim High NPS-NorthStar Academy. Each of these programs will benefit from both hands-on and virtual imaging relying upon the Zeiss LSM 980 and other microscopes in the AICF. The addition of the Zeiss LSM 980 to the AICF will have societal impacts on multiple fronts. Introducing advanced imaging to students at all levels will improve science literacy and build an understanding of the process of scientific discovery and application. This will have a future positive impact as US citizens are asked to make important societal decisions dependent upon an understanding of science. Further, the Zeiss LSM 980 will strengthen the ability of Rutgers-Newark to hire and retain top-shelf faculty whose research and teaching skills will add to education, training, and outreach at Rutgers-Newark. The LSM 980 will provide new faculty in the biological science with an essential modern-day research instrument that is vital for success in obtaining extramural grant funding and in publishing peer-reviewed work.

The Zeiss LSM 980 with Airyscan 2 provides a state-of-the-art microscopy workstation that provides multi-dimensional fluorescence imaging as well as super-resolution research capacity. The microscope will support fundamental research examining the mechanisms of cell signaling and membrane-protein sorting in neural, intestinal epithelial, and bacterial cells. The Zeiss LSM 980 will enable researchers will use novel fluorescent-reporter proteins in conjunction with innovative genetic animal and in vitro cell/organoid models to follow the dynamics and regulatory function of small GTPases and transcription factors in regulating cell and tissue differentiation and maintenance. Super-resolution fluorescence imaging and photo-manipulation will be used to discover novel protein-membrane and protein-protein interactions in the assembly of macromolecular complexes that regulate lipid transport, assembly of cell surface receptors, and intracellular sorting of signaling complexes in bacterial membranes, during myelin formation in peripheral nervous system, and embryonic development in mammalian central. The Zeiss LSM980 will be used in state-of-the-art optogenetic experimentation on the important role of cytoskeleton-chromatin interactions on gene expression and epigenetics. Additionally, the microscope will be used to provide multi-dimensional fluorescence imaging of cell signaling and remodeling pathways activated in response to tissue and cell injury from trauma including, impact, stretch, radiation, and chemical insult. All the research projects are fully integrated with STEM education/training of high school, undergraduate, graduate students and postdoctoral students serving as the foundation for improved STEM literacy and future leadership.

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.