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Program in Physical Biology
The Program in Physical Biology (PPB) uses systems ranging in complexity from van der Waals interactions to the physics of imaging 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, cancer imaging in vivo, virology, macular degeneration, and HIV pathogenesis. This year’s breakthroughs include a new explanation of how dynamin works and discovery of a new regulator of oxidative phosphorylation in mammalian cells.
Peter Basser heads the Section on Tissue Biophysics and Biomimetics, which strives to understand fundamental relationships between function and structure in soft tissues, in “engineered” tissue constructs, and in tissue analogues (e.g., polymer gels). We try to understand fundamental relationships between function and structure in soft tissues, “engineered” tissue constructs, and tissue analogues—how microstructure, hierarchical organization, composition, and material properties of tissue affect biological function and dysfunction. To investigate biological and physical model systems at different time and length scales, we make physical measurements in tandem with analytical and computational models. Primarily, we use water to probe both equilibrium and dynamic interactions among tissue constituents over a wide range of time and length scales. To determine the equilibrium osmo-mechanical properties of well-defined model systems, we vary water content or ionic composition. To probe tissue structure and dynamics, we employ small-angle X-ray scattering (SAXS), small-angle neutron scattering (SANS), static light scattering (SLS), dynamic light scattering (DLS), and nuclear magnetic resonance (NMR) relaxometry. We use mathematical models to understand how changes in tissue microstructure and physical properties affect essential transport processes (e.g., mass, charge, and momentum). The most direct non-invasive method for characterizing these transport processes in vivo is magnetic resonance imaging (MRI), which can characterize normal tissue microstructure and follow its changes in development, degeneration, and aging. Another goal is to translate our new quantitative methodologies from bench to bedside.
The Section on Molecular Transport, led by Sergey Bezrukov, studies channel-facilitated transport of metabolites across cellular and organelle membranes by reconstituting channel-forming proteins into planar phospholipid bilayers. This year the Section concentrated on the physical mechanisms involved in transport regulation. Three particular mechanisms were investigated. First, with a model peptide channel as a single-molecule sensor, it was discovered that partitioning of the prototypical anesthetic halothane into lipid bilayers is dependent on the stress of lipid packing. Second, using a channel aqueous pore as a nano cuvette, an unexpected role of molecular hydration in adamantane-cyclodextrin complexation dynamics was established. Third, the functional significance of electrostatic interactions in regulation of the voltagedependent anion channel of the mitochondrial outer membrane by tubulin was investigated. Understanding these mechanisms at the quantitative level of physical interactions allows the Section to uncover previously unsuspected functional links between different signal transduction pathways.
The Section on Medical Biophysics, led by Robert Bonner, is developing optical technologies to characterize early stages of disease and to monitor responses to therapy in cancer and age-related macular degeneration (AMD). By integrating multispectral, non-invasive, clinical retinal autofluorescence imaging with automated image analysis, we seek to map the distribution of several intrinsic photochemicals implicated in health preservation and early disease. In pilot clinical studies, we are applying our new technology to understanding how changes in retinal spectral irradiance affect the balance between retinal photochemical pathways that, we hypothesize, drive early age-related maculopathy. Our broader goals are to quantify earlier stages of local molecular imbalance throughout the retina and to develop reliable means for classifying and quantifying early disease in order to evaluate the effectiveness of strategies to prevent disease progression. We are also continuing to develop our inventions of expression microdissection and laser capture microdissection for better integration with multiplex molecular analyses of specific cells and organelles extracted from complex tissue.
Recent studies performed by the Section on Membrane Biology, led by Leonid Chernomordik, have concentrated on cell-to-cell fusion and post-mitotic reassembly of nuclear envelope. To examine the mechanisms by which nascent fusion pores expand to yield multinucleated cells in development (for instance, during skeletal muscle formation) and in many diseases, the Section has studied fusion initiated by protein fusogen of influenza virus. While early fusion stages are controlled by the fusogens, pore expansion requires cell metabolism. The actin cytoskeleton that has been often suggested to drive pore expansion has been found to restrict it. The Section has also explored post-mitotic reassembly of intracellular membranes. After each division of eukaryotic cell, the nuclei of the daughter cells surround themselves with membrane envelopes studded with nuclear pores. Changes in the pore functionality and nuclei morphology underlie many hereditary diseases. The Section has uncovered a feedback mechanism that connects the assembly of nuclear pores with an expansion of the envelope, and function of the pores with their lateral distribution.
The Section on Analytical and Functional Biophotonics, led by Amir Gandjbakhche, devises quantitative biophotonics methodologies and associated instrumentation to study biological phenomena at different length scales, from nanoscopic to microscopic. During the past year, the Section (1) has developed a pharmacokinetics model which enables one to quantify the degree of HER2 receptors expression in various types of breast carcinoma using a near-infrared, scanning, time-resolved imaging system; (2) developed theoretical models of photon migration needed to monitor tumor status by pH deep inside tissues; (3) designed an optical polarization camera which can be mounted in a standard tcolposcope to characterize the status of collagen texture in cervix; (4) developed new algorithms to assess vascularity in AIDS-associated Kaposi’s sarcoma; (5) developed numeric models to assess quantitatively the effects of multiple light scattering on two-photon microscopy and fluorescence correlation microscopy; and (6) developed a mathematical model of diffuse optical tomography for brain imaging of war veterans whose heads retain metallic shrapnel and children undergoing hemispherectomy.
The goal of the Section on Intercellular Interactions, led by Leonid Margolis is to investigate the mechanisms of HIV pathogenesis in the context of human tissues, particularly the role of non–HIV-microbes (“co-pathogens”) associated with HIV infection. The section investigates these mechanisms in the framework of a new concept of a dynamic equilibrium between multiple persistent viruses in the human body, which HIV infection unbalances. This leads to infections by new viruses and reactivation of “latent viruses”, in particular of human herpesviruses . By using the SIV/macaque model the section showed that herepesvirus 6 significantly affects lentiviral evolution in vivo, leading to the selection of chemokineresistant SIV variants. Interactions between herepesviruses and HIV-1 in coinfected tissues can be exploited to suppress HIV-1 infection. Towards this goal the section used derivatives of an anti-herpetic drug acyclovir, that was earlier shown by this section to become an HIV RT inhibitor but only in herpesvirus-coinfected tissues. Now these derivatives were further developed to suppress HIV-1 independently of herepesviruses coinfection. To study complex cellular and molecular mechanisms of inter-viral interactions and the effects of these interactions on HIV transmission and pathogenesis, the section developed new experimental models of human tissues infected under controlled laboratory conditions ex vivo.
The Section on Cell Biophysics, led by Ralph Nossal, aims to understand the physical basis for various basic cell processes. During the past year, the Section (1) used quantitative time-resolved microscopy to show that HIV virions move within cervical mucus in discrete steps probably linked to the relaxation of mechanical stress; (2) used atomic force microscopy to investigate conformational fluctuations of clathrin triskelions in solution, showing that the triskelions are flexible in aqueous environments; (3) studied how the presence of high background scattering from an optically dense medium influences the interpretation of data obtained by fluorescence correlation spectroscopy of molecularly crowded solutions; (4) defined the molecular mechanism and binding site of peloruside, the first microtubule-stabilizing drug with a site and mechanism distinct from taxol; and (5) identified tubulin as regulating mitochondrial oxidative phosphorylation by reversibly blocking the VDAC channel-mediated exchange of ATP and ADP across the outer membrane of the mitochondria.
The Section on Molecular Biophysics, led by Adrian Parsegian, has a long-term goal to build a practical physics of biological material. They measure, characterize, and codify the interactions that govern the organization and self-assembly of various of biological molecules. Connected in part with the recent NIH-wide interest in nanotechnology, we are building on our experience with van der Waals fluctuation forces to formulate interactions involving carbon nanotubes not only in their assembly but also and more important as substrates for biopolymers such as DNA. Our undertaking is strengthened by its strong connection with physical theory. Through a series of measurements and analyses of the different kinds of interactions as revealed in vivo, in vitro, and in computation, we are working with DNA assemblies, such as those seen in viral capsids and in vitro, polypeptides and polysaccharides in suspension, and lipid/water liquid-crystals. In these systems, we simultaneously observe the structure of packing as well as measure intermolecular interaction energies. We have also worked with experimentalists who are able to create repulsive as well as attractive charge fluctuation forces, with implications for nanotechnology.
The Section on Macromolecular Recognition and Assembly, headed by Donald Rau, focuses on the coupling of forces, structure, and dynamics of biologically important assemblies. The next challenge in structural biology is to understand the physics of interactions between molecules. The ability to take advantage of an increasing number of available protein and nucleic acid structures will depend critically on establishing the link between structure and energy. A fundamental and quantitative knowledge of intermolecular forces is essential for (1) understanding the interactions among biologically important macromolecules that control cellular function and (2) rationally designing agents that can effectively compete with the interactions associated with disease. We have shown that measured forces differ from those predicted by current theories, and we have interpreted the observed forces as indicating 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, we measure and correlate changes in binding energies and hydration that accompany recognition reactions of biologically important macromolecules, particularly sequence-specific DNA-protein complexes. We observe a strong correlation between retained water and binding energy—stronger binding means less water retained at the DNA-protein interface.
The Section on Membrane and Cellular Biophysics, led by Joshua Zimmerberg, studies membrane mechanics, intracellular molecules, membranes, viruses, organelles, and cells in order to understand viral and parasitic infection, exocytosis, and apoptosis. This year the Section elucidated the mechanism of action of dynamin, the protein engine of endocytosis, demonstrating that proteins act at the scale of 10s of nanometers to act as boundary conditions for morphological changes at the nanometer scale carried out (at a distance) by lipids. Second, they discovered a new method for membrane preparation and showed a difference between cell and membrane cholesterol. Third, they found that fusion of GLUT4 vesicles downstream of Exo-70-mediated tethering is the rate-limiting step in insulin action on adipose cells. Fourth, they we determined the mechanism by which human bone marrow stromal cells (BMSCs) are immunosuppressive and escape cytotoxic lymphocytes (CTLs). They find both Fas and FasL expression by primary BMSCs. Jurkat cells or activated lymphocytes were each killed by BMSCs after 72 h of co-incubation. Fifth, they found that human lymphoid tissue could not be activated in either simulated microgravity of rotation in a NASA-designed bioreactor, or in actual microgravity of the International Space Station.