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The Section on Membrane and Cellular Biophysics studies molecules, membranes, viruses, organelles, and cells to understand viral and parasite infection, exocytosis, and apoptosis. Pathological processes and our defenses against them fundamentally depend on membrane fusion, fission, and poration. The section comprises two groups. One concentrates on elucidating structural intermediates in biological membrane fusion, fission, and pore formation and their energetic requirements. The other group focuses on characterizing the mechanisms of exocytosis
Leonid Chernomordik and his group study the role of lipids and the structure of lipid-involving intermediates in membrane fusion. The researchers are continuing to probe fusion along the lines of their stalk-pore hypothesis, with particular emphasis on the mechanisms by which specialized fusogenic proteins mediate formation of the lipid-involving intermediates. To achieve their goal, Dr. Chernomordik and colleagues have dissected fusion reactions mediated by influenza virus hemagglutinin and baculovirus gp64 into distinct stages by using different reversible inhibitors. The emerging pathway includes a local hemifusion intermediate that forms within a ring of activated fusion proteins. The investigators are working now on the identification of specific mechanisms by which conformational changes in fusogenic proteins are coupled with membrane rearrangements.
Dr. Leonid Margolis’s work addresses a major challenge of modern cell biology: to understand the effect of cell microenvironment on cell function. Cell suspensions and monolayer cultures are poor representatives of life, and it is difficult to image cells in this microenvironment. To remedy these problems, Margolis and his colleagues have been developing new systems of culturing three-dimensional tissues. Ongoing projects include HIV pathogenesis in human lymphoid tissue ex vivo. The researchers found that lymphoid tissue in vitro, without humoral or cellular interactions with the host, is sufficient for infection with different types of HIV-1 isolates, dissemination of virus throughout the tissue, depletion of CD4+ T cells, release of virus into the media, lymphocyte apoptosis, and a functional immune response. The results show that human HIV variants that use CXCR4 as their coreceptor and are typical for late stages of HIV disease are more pathogenic than HIV variants using CCR5, which are typical for early stages of the disease. Using isogenic viral chimera, the investigators proved a causative relationship between CXCR4 usage and viral ability to deplete CD4+ T cells severely and immunosuppress lymphoid tissues ex vivo. Recently, the laboratory discovered that the reason that CCR5-using viruses are less pathogenic than CXCR4-using viruses is that the former find fewer cognate CD4+ T cell targets in tissues than do the latter.
With biological and methodological applications in mind, Dr. Andreas Chrambach and his group pursue the improvement of analytic and preparative electrophoresis. The research recognizes that the present emphasis on an inventory of gene products will eventually decline as interest begins to focus on an inventory of the post-translational forms of macromolecules, intermolecular complexes, and subcellular particles that constitute the building blocks of biological reality. Dr. Chrambach and his group are developing methods that will respond to that new interest.
Dr. Alfred Yergey and his colleagues apply knowledge of the physical chemistry of gas phase ions to basic research in structural biology. In their project on hydration, they are using equilibrium ion-molecule chemistry to characterize and understand the energetic basis of the interactions between water and its biological solutes. In its project on protein characterization, the section is collaborating with investigators in need of information on molecular weight and molecular weight distribution, identity, and the nature and extent of post-translational modifications of proteins. The demands of these problems require performance at state-of-the-art levels of mass spectrometric measurement coupled with development of new approaches to address issues of sensitivity and specificity. The range of the applied research extends to mapping of picomolar quantities of peptides extracted from proteins digested in situ from electrophoretically separated proteins, obtaining partial peptide sequences at picomolar sensitivities to facilitate the construction of nucleotide probes, and mapping epitopes of femotomolar quantities of proteins isolated by noncovalent interactions with antibodies.
The NASA/NIH Center for Three-Dimensional Tissue Culture, directed by Joshua Zimmerberg with deputy directors Leonid Margolis and Paul Blank, is a pan–NIH facility within LCMB. The center provides NIH researchers with an opportunity to develop new model systems for diseases whose pathology cannot be reproduced by merely growing the right cells in monolayer culture. Several NASA-designed rotating wall vessels (which culture cells under minimal shear forces in a well-oxygenated medium under conditions that mimic microgravity) are made available together with experienced technicians to test tissues, primary cell cultures, and cell lines under low-shear fluid conditions that appear to facilitate cell-cell interactions and promote differentiation. To facilitate research, extensive consultations with a group seminar determine the applicability of center resources to the aims of the interested investigator. Second, investigators and staff design pilot projects. Third, principal investigators with successful pilot projects can apply for center funding for salary, equipment, and consumables. Given that the scientific concern for tissue culture is common to all Institutes and that the development of tissue culture needs active interinstitute collaboration, the NASA/NIH Center hopes to facilitate such collaborations and provide access to state-of-the-art imaging modalities to determine structure/function relationships between cells of developed tissues.