Program In Physical Biology
Joshua Zimmerberg, MD, PhD, Program Director
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 for the stability of the influenza virus in winter and a new anti-viral agent against HIV.
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). The Section produced a method based on anomalous X-ray scattering to measure the ion distribution around charged biopolymer molecules and built a tissue micro-osmometer that permits continuous monitoring of water uptake of small specimens. It also developed an experimental method to map the elastic properties of tissues and cells and designed and constructed calibration standards for MRI imaging. The Section designs and implements novel quantitative in vivo neuroimaging methods to scan children and adults noninvasively, combined with effective strategies for correcting distortion in diffusion-tensor imaging scans of the human brain. The Section elaborates new quantitative MRI methodologies to probe he microstructure and architectural organization of tissue, with an emphasis on the brain, including a method to measure the axon diameter distribution within nerve fascicles. Another MRI-based approach uses concepts from the theory of porous media to characterize features of gray matter microstructure in order to perform Brodmann parcellation of the cerebral cortex.
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. The Section discovered a functional relationship between the cytoskeleton and the voltage-dependent anion channel (VDAC) of the outer membrane of mitochondria. Nanomolar concentrations of dimeric tubulin induced highly voltage-sensitive reversible blockade of mammalian VDAC channels that were reconstituted into planar phospholipid membranes. Experiments with isolated mitochondria confirmed the Section’s findings. Tubulin added to isolated mitochondria dramatically decreased the availability of ADP to adenine nucleotide translocase and restored the low permeability of the mitochondrial outer membrane found in permeabilized cells. This newly revealed evidence of critical cytoskeletal involvement in cellular metabolism regulation may shed light on the mechanism of action of a group of anti-tumor drugs, such as paclitaxel.
The Section on Medical Biophysics, led by Robert Bonner, develops and applies new optical technologies to critical problems in medicine. To map the distribution of several intrinsic photochemicals implicated in health preservation and early disease, the Section is currently developing multispectral, non-invasive clinical retinal autofluorescence imaging with automated image analysis. The Section’s broader goals are to quantify earlier stages of local molecular imbalance throughout the retina and to develop reliable means to quantify early disease and the effectiveness of strategies to prevent its progression. The Section is further developing laser capture micro-dissection, which it invented, and its newer variant, expression micro-dissection, to improve 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 late stages of cell-to-cell fusion and the mechanisms of drug delivery by cationic cell-penetrating peptides. The work on cell fusion initiated by viral protein fusogens has established that expansion of nascent fusion pores into an open lumen of cell-size diameter requires cell metabolism. The actin cytoskeleton restricts rather than drives pore expansion. The work on cell-penetrating peptides has emphasized the importance of endosomal escape as a critical stage in delivery of the peptide into cytosol.
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) began characterizing various types of breast carcinoma with a near-infrared, scanning, time-resolved imaging system, specifically targeting HER-2 receptors; (2) developed theoretical models of photon migration needed to monitor tumor status by pH deep inside tissues; (3) designed an optical camera with liquid crystal retarder for active polarization imaging of biological texture while improving statistical tools to enhance visualization of structures hidden in biological tissue; (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 twophoton 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.
The goal of the Section on Intercellular Interactions, led by Leonid Margolis, is to understand 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 developed a new anti–HIV strategy based on the unique ability of herpesvirus (HHV) kinases to phosphorylate acyclovir (ACV) to yield an inhibitor of herpesviruses DNA polymerases. We showed that HIV-1 reverse transcriptase incorporates ACV-triphosphate into the nascent HIV-1 DNA chain, leading to the complete termination of reverse transcription. Accordingly, HIV-l proliferation was inhibited in human tissues co-infected with HHVs, which are capable of phosphorylating ACV. The results suggest that ACV may be therapeutically beneficial for various HIV-1–infected patients, given that the majority of humans are already infected with various HHVs, including the low-pathogenic HHV-6 and HHV-7. Clinical trials have been launched to test the therapeutic benefits in 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) studied how HIV penetrates cervical mucus; (2) determined the properties of clathrin triskelions in solution, showing that the lowestenergy conformation has an intrinsic pucker but that the triskelion is sufficiently flexible to change conformation when in a polyhedral clathrin cage; (3) used experimental vibro-fluidized granular models of spatially constrained “molecular” mixtures to reveal that the pressure resulting from the presence of spherical crowders (akin to “depletion forces”) drives a collection of rod-like objects confined to narrow spaces to form aggregates; (4) identified several molecules that preferentially bind to Leishmania tubulin over mammalian tubulin, thus preventing parasite multiplication inside human macrophage cells; and (5) developed an improved technique for the morphological analysis of bacterial biofilms, which reveals new details about biofilm architecture and cellular adhesion.
The Section on Molecular Biophysics, led by Adrian Parsegian, collaborated on a series of measurements of van der Waals dispersion forces and analyzed the pressures required to pack flexible polymers into confined spaces. The Section aims to derive a quantitative measure of molecule assembly as such assembly drives cellular processes. To see the connections between DNA pressure and ejection, the Section has been growing viruses of different lengths for introduction into the same size capsid and then plan to use X-ray diffraction to follow the packing and pressure of DNA within the capsid. The Section also succeeded in following the condensation of triple-helical DNA, which is implicated in the behavior of telomeres. The response to several divalent ions in various tsDNA constructs revealed differences in packing and liquid crystalline tendencies. The Section also extended the Lifshitz theory of van der Waals interactions in lipid multilamellar systems in order to compute forces between bodies with extended interfaces. Of particular importance is the ability to measure repulsive van der Waals forces in systems in which the properties of the medium are intermediate between those of the interacting bodies, thus creating switchable systems.
The Section on Macromolecular Recognition and Assembly, headed by Donald Rau, focuses on elucidating the coupling of the forces, structure, and dynamics of biologically important assemblies. Interacting macromolecules tenaciously retain their hydration waters unless their surfaces are complementary. To investigate the role of water in binding, the Section 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 observed a strong correlation between retained water and binding energy. The Section also devised a novel method for measuring one-dimensional sliding rates and applied it to the linear diffusion of EcoRI, which moves at a rate about one-thousandth of that predicted by simple hydrodynamics, suggesting that DNA-protein charge-charge interactions limit the diffusion rate. DNA-DNA force measurements confirm that arginine and lysine peptides package DNA by employing the same attractive hydration interactions as previously seen for metal ions and the biogenic alkylamines spermidine and spermine.
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. For malaria, the Section studies the mechanisms by which proteases release parasitic cells from erythrocytes, suggesting a protease inhibitor therapy for malaria. Second, the Section discovered that, in enveloped viral disease, lipids forming the envelope of the influenza virus gel at cool temperatures, suggesting a new hypothesis to explain the winter-time occurrence of flu epidemics. Third, the Section found that cholesterol plays double role in viral fusion mediated by the hemagglutinin of influenza: as a lipid-curvature agent that helps hemifusion and as a specific widener of the fusion pore. Fourth, the Section investigates the theoretical effect of protein wetting to determine its role in protein domain formation on cell membranes.