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Program in Physical Biology

Director: Joshua Zimmerberg, MD, PhD

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The Program in Physical Biology (PPB), led by Joshua Zimmerberg, uses systems ranging in complexity from channel internal-surface physics to HIV pathophysiology in human tissue, to investigate the physicochemical basis of molecular, physiological, and pathological processes and interactions. The PBB develops novel, non-invasive technologies to probe the processes' physical and chemical parameters. The research focuses on the physical chemistry of surface forces, DNA-protein interactions, polymer organic chemistry, membrane biochemistry, pore-forming antibiotics, electrophysiology, cell biology, parasitology, immunology, tissue culture, laser micro-dissection, and virology. Diseases of special interest include macular degeneration, diabetes, malaria, dengue, and HIV.

The Section on Molecular Transport, led by Sergey Bezrukov, focuses on transporter-facilitated transport of metabolites and other large solutes across cell and organelle membranes. In the Human Genome there are 43 distinct families of transport systems that comprise more than 300 isoforms of individual solute carriers. Although the majority of the transport systems are responsible for uptake of specific substrates, a substantial number of transporters are used for uptake of the same solute, and overlapping expression of multiple isoforms often exists in the same cell type. Thus, the question arises as to why there are so many transporter isoforms. Though this variety of isoforms may seem redundant and, in principle, could be explained by the lack of strong evolutionary pressures to decrease the size of the genome, the Section's analysis of transporter optimization offers a different interpretation. It has been shown that transporter efficiency is fine-tuned to specific ranges of substrate concentration. Thus, different isoforms might be tailored accordingly, adjusting their amino acid composition for the optimal strength of substrate/transporter interactions and the transition rates between different conformations, with one gene coding for a uniporter protein that functions most efficiently at high solute concentrations and another gene coding for one that is most efficient at low concentrations.

The Section on Medical Biophysics, led by Robert Bonner, develops new optical technologies in order to characterize or modify early stressors that drive chronic diseases and for use in developing effective disease prevention strategies. Through integrated analysis of multispectral, multimodal clinical retinal imaging, the group maps distributions and dynamics of retinal photochemicals and relates them to cellular dysfunction and early disease progression. Applying these new methods in clinical studies, the Section seeks to test the hypothesis that spectral shifts in retinal irradiance can reduce imbalances among retinal photochemical pathways and that chronic photochemical imbalances drive early age-related and Stargardt's maculopathies, which could be reduced or prevented by appropriate external filters (e.g., spectral sunglasses). The Section's noninvasive molecular mapping methods might facilitate characterization of early retinal disease states, including more readily reversible "preclinical" disease, and the effects of benign, low-cost preventions strategies. The group is also adapting its prior invention of laser capture microdissection into simpler systems more easily integrated with clinical pathology and multiplex molecular analysis of specific cells and organelles extracted from complex tissue.

Recent studies performed by the Section on Membrane Biology, led by Leonid Chernomordik, is to understand how proteins drive membrane fusion in important cell biology processes. The starting point in the Section's analysis is a consideration of the physical factors that determine the tendency of the membrane bilayers to change their topology. The analysis of the molecular mechanisms of diverse membrane rearrangements will likely bring about new ways of controlling them and clarify the generality of emerging mechanistic insights. In one of its recent projects, the Section focused on fusion mediated by the influenza virus hemagglutinin. Fusion between the viral envelope and the membrane of acidified endosome delivers viral RNA into cytosol. In the initial conformation of hemagglutinin (HA), the transmembrane protein HA2 (HA's fusogenic subunit) is locked in a metastable conformation by the receptor-binding HA1 subunit of HA. The unexpected finding that the final conformation of the HA2 ectodomain mediates fusion between lipid bilayers and between biological membranes suggests that the fusion process is driven by this final conformation rather than by the energy released by protein restructuring into the final form. In another project, the Section explored the late stages of syncytium formation initiated by viral fusogens and found that fusion pore expansion at late stages of cell-to-cell fusion is mediated, directly or indirectly, by intracellular membrane-shaping proteins involved in endocytosis. The work emphasizes an interesting overlap between proteins controlling late stages of cell-to-cell fusion and proteins that drive oppositely directed process of membrane remodeling in budding and fission.

The general goal of the Section on Intercellular Interactions, led by Leonid Margolis, is to understand the mechanisms of sexual transmission of human pathogens, including the human immunodeficiency virus (HIV), to the human genital tract and their tissue pathogenesis and to develop efficient anti-virals. During the past year, the Section studied seminal cytokines, in particular their modulation during HIV-1 infection, revealing the importance of coinfecting herepesviruses which, together with HIV-1, alter the immunological landscape of semen. Herpesviruses are known to promote transmission and to facilitate pathogenesis. Herpesvirus-induced cytokines may serve as another target for the preventive strategy. Recently, the vaginally applied microbicide tenofovir was found to decrease transmission not only of HIV-1 but unexpectedly also of herpes simplex virus. The Section deciphered the mechanism of this effect, a finding that should prove useful for the development of multi-targeted antivirals. The current working hypothesis linking HIV-1 disease with atherosclerosis is that the progression of both diseases is fueled by inappropriate activation of the immune system. To test this hypothesis and to identify which aspects of immunoactivation play a critical role in both pathologies, the Section investigated the activation status of lymphocytes found in atherosclerotic plaques. The Section further developed and standardized the system of ex vivo tissues so as to make it reflect various in vivo aspects of intercellular interactions more faithfully than isolated cells in suspension or monolayers cultures and to broaden its application for the scientific community.

The Section on Cell Biophysics, led by Ralph Nossal, studies of cell behavior that can be linked to underlying physical mechanisms, for which the Section develops and applies methodologies based on mathematical and physical principles. The research also utilizes biochemical and cell biological techniques. Among current projects are (i) constructing a physical model to explain the stochastic nature of coated-vesicle biogenesis during receptor-mediated endocytosis, (ii) determining the mechanical properties of clathrin cages and using that knowledge to gain insight into membrane transformations that occur during vesicle trafficking, (iii) exploring how substrate mechanical properties affect the movements of locomoting eukaryotic cells, and (iv) understanding how certain small molecules interact with microtubules and thereby act as anti-mitotic agents. The Section also develops new experimental methods to characterize these and related phenomena, focusing increasingly on developing an integrated understanding of cellular activities that are coordinated in space and time.

The Section on Macromolecular Recognition and Assembly, headed by Donald Rau, focuses on the nature of forces, structure, and dynamics of biologically important assemblies. The group showed that measured forces differ from those predicted by current theories and interpreted the observed forces to indicate the dominant contribution of water-structuring energetics. The observation that interacting macromolecules tenaciously retain their hydration waters unless the surfaces are complementary has profound implications for recognition reactions. To investigate the role of water in binding, the group measures and correlates changes in binding energies and hydration that accompany recognition reactions of biologically important macromolecules, particularly sequence-specific DNA-protein complexes.

The Section on Membrane and Cellular Biophysics, led by Joshua Zimmerberg, studies membranes, viruses, organelles, cells, and tissues in order to understand the molecular organization of cellular membranes, the physico-chemical mechanisms of membrane remodeling, and the molecular anatomy of tissues, which will lead to a better understanding of viral, parasitic, metabolic, developmental, and neoplastic diseases. This past year, the Section's exocytosis project focused on a new method for the purification of synaptic vesicles, which contain a variety of proteins and lipids that mediate fusion with the pre-synaptic membrane. The Section also undertook two remodeling projects, one in collaboration with Sergey Bezrukov's Section on the optimization of receptors, and a second on the ultrastructural analysis of biological structures. Combining tomography with negative staining can provide three-dimensional images. The Section used methylamine tungstate to fulfill the basic requirements for a negative stain for tomography, namely, that the density and atomic number of the stain are optimal, and that the stain is not degraded with the intensive electron dose needed to collect a full set of tomographic images. Tomograms derived from multiple projections of EM images of the same structure yielded detailed images of single proteins on the surface of influenza A virus.

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