PROGRAM IN PHYSICAL BIOLOGY
By merging the Laboratory of Cellular and Molecular Biophysics (LCMB), the Laboratory of Physical and Structural Biology (LPSB), and the Laboratory of Integrative and Medical Biophysics (LIMB), the Program on 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, noninvasive technologies to probe the processes’ physical and chemical parameters. Researchers focuses on the physical chemistry of surface forces, carbon nanotubes, DNA-protein interactions, gas phase ions, polymer organic chemistry, membrane biochemistry, pore-forming antibiotics, electrophysiology, cell biology, parasitology, immunology, tissue culture, laser microdissection, cancer imaging in vivo, virology, macular degeneration, and HIV pathogenesis.
Peter Basser heads the Section on Tissue Biophysics and Biomimetics, which works to understand fundamental relationships between function and structure in soft tissues, in “engineered” tissue constructs, and in tissue analogues (e.g., polymer gels). Members of the section develop new physical theories, mathematical and computational models, and biomimetic tissue analogues to aid in the design and interpretation of biological experiments. In particular, they developed a method based on anomalous X-ray scattering to measure the ion distribution around charged biopolymer molecules (e.g., hyaluronic acid, proteins); the method is particularly useful in studying the molecules’ interactions with monovalent and divalent counterions. The section designed and built a tissue micro-osmometer that allows continuous monitoring of water uptake of small tissue specimens; developed an experimental method to map the elastic properties of tissues and cells at a microscopic resolution by using an atomic force microscope; and designed and constructed a new generation of tissue phantoms that closely mimic the diffusive properties of biological tissues (e.g., gray matter), which can be used as calibration standards for MRI imaging. The section also develops quantitative in vivo neuroimaging methods to scan pediatric subjects, particularly a processing “pipeline” for analyzing clinical pediatric diffusion tensor MRI (DTI) data; effective strategies for correcting morphological distortions in Echo Planar MRI images and acquiring unbiased quantitative DTI measurements in the human brain; and “radial” diffusion MRI techniques for obtaining high-quality, high-resolution DTI scans of the human brain. The section also continues to develop new quantitative imaging methodologies to probe the structure and organization of normal and diseased tissue, with emphasis on the brain, especially an MRI 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 that cannot be detected with conventional MRI.
Sergey Bezrukov and his colleagues in the Section on Molecular Transport study interactions of low molecular weight compounds with ion channels of different origin. One research goal is to find effective blockers of the channels formed by certain pathogens. Many pore-forming proteins, both bacterial and viral, are considered important virulence factors, making them attractive targets for the discovery of new therapeutic agents. Recently, using structure-inspired drug design, the section demonstrated that aminoalkyl derivatives of beta-cyclodextrin inhibited anthrax lethal toxin (LeTx) action by blocking the transmembrane pore formed by the toxin’s protective antigen (PA) subunit. The section evaluated a series of new beta-cyclodextrin derivatives as potential inhibitors of both anthrax toxins and Staphylococcus aureus alpha-Hemolysin (HL) (the latter is regarded as a major virulence factor in staphylococcal infection) and found that the activity of the derivatives in both cell protection and channel blocking depends on the length and chemical nature of the substituent groups. Several amino acid derivatives of beta-cyclodextrin inhibit the activity of HL and LeTx at low micromolar concentrations and block ion conductance through the pores formed by both HL and PA in artificial lipid membranes, raising the prospect of new effective therapies against various pathogens that use pore-forming proteins.
The Section on Medical Biophysics, led by Robert Bonner, develops and applies new optical technologies to critical problems in medicine. Currently, the section is pursuing improvements in noninvasive clinical retinal autofluorescence imaging to test for the spectral dependence of detrimental photochemistry, of chronic injury in the retina, and of early age-related maculopathy and how normal lens aging (yellowing) and spectrally selective sunglasses modify these processes. In ex vivo studies, the section is further developing laser capture microdissection, which it invented, and its newer variant, expression microdissection, for better integration with a wide range of multiplex molecular analyses of specific cells and organelles extracted from complex tissue. Researchers are seeking to optimize cross-disciplinary interactions within the intramural and extramural communities to advance these technologies and their application to biomedical research, with a particular emphasis on understanding critical molecular events in early disease and using such events to target and evaluate responses to disease prevention strategies.
The Section on Membrane Biology, led by Leonid Chernomordik, studies the mechanistic pathway of membrane fusion. While much is known about proteins that mediate membrane fusion in viral infections and in intracellular protein trafficking, protein fusogens that mediate cell fusion in development had yet to be identified. The section’s recent work, however, revealed the first developmental fusogen, a protein (EFF-1) that forms syncytia in the epidermis of C. elegans. With that, the section focused on the cell fusion that establishes a continuous uterine-vulval tube in C. elegans. Researchers used genetic screening to identify a protein (AFF-1) required for the fusion reaction. Ectopic expression of the protein resulted in fusion between cells that normally do not fuse in C. elegans. Moreover, AFF-1 fused heterologous cells in cultures. Analysis of the functional properties of the first two developmental fusogens EFF-1 and AFF-1, the founding members of a family of nematode fusogens, uncovered a novel homotypic arrangement of the fusion machinery requiring the presence of identical protein fusogens on both fusing membranes; the fusogens might be important for establishing defined boundaries between syncytia and surrounding mononucleate cells.
The Section on Biomedical Stochastic Physics, led by Amir Gandjbakhche, devises quantitative theories, develops methodologies, and designs instrumentation to study biological phenomena whose properties are characterized by elements of randomness in both space and time. The research focuses on developing functional and analytical tools in biophotonics. Over the last year, researchers (1) developed a user-friendly polarization imaging system that simultaneously images cross- and co-polarized light with a quantitative statistical tool, based on Pearson’s correlation coefficient, to enhance image quality and reveal regions of high statistical similarities within the noisy tissue images; (2) designed a time-resolved life-time imaging system for in vivo small-animal studies to map fluorophores’ lifetimes and potentially localize a tumor and monitor its functional status non-invasively in vivo; (3) designed a multimodality imaging technique to assess vascularity in AIDS-associated Kaposi’s sarcoma; (4) designed a novel fiber-optic confocal approach for ultrahigh depth-resolution microscopy beyond the diffraction barrier in the subwavelength nanometric range below 200 nm; (5) quantitatively analyzed the gain obtained from a newly developed two-photon microscope (total emission detection) devised by researchers at the NHLBI; and (6) studied the vasculature network formation in tumor-induced angiogenesis and the role of various vascular endothelial growth factors.
The Section on Intercellular Interactions, led by Leonid Margolis, studies HIV pathogenesis in human lymphoid tissue ex vivo. This culture system, which was developed in the section’s laboratory, supports productive infection with different types of HIV-1 isolates, dissemination of virus throughout the tissue, depletion of CD4+ T cells, release of virus into the medium, lymphocyte apoptosis, and a functional immune response, thus providing a unique way to study HIV tissue pathogenesis. HIV infection is transmitted in lymphoid tissues by CCR5-using HIV-1 (R5); the viruses often later evolve into variants able to use CXCR4 (X4 or R5X4), which are associated with accelerated progression to AIDS. HIV-1 pathogenesis is critically dependent, among other factors, on the site of infection, with gut-associated lymphoid tissue playing an important role in R5/X4 selection, on tissue immune activation, and on co-infecting microbes that interact with HIV. To investigate these aspects of HIV infection in a complex tissue microenvironment, members of the section studied viral pathogenesis in explants of various human tissues infected with HIV-1 alone or in combination with other viruses. They compared R5 and X4 HIV-1 infection of human rectosigmoid and tonsillar tissues and focused on the lymph node immune activation in HIV-1 pathogenesis. They also continued investigations of the effect of non–HIV microbes on HIV infection. The results provide new opportunities for designing strategies to prevent and contain HIV infection through manipulation of the response of human tissues to HIV by mimicking the negative effects of non–HIV microbes on HIV replication.
The Section on Cell Biophysics, led by Ralph Nossal, aims to understand the physical basis for various cell and tissue processes involved in signal transduction, protein trafficking, and cell motility. The section’s long-term goal is to build an arsenal of theoretical and experimental tools to study how such biological activities are coordinated in space and time. During the past year, researchers (1) developed techniques for studying the movement of nanometer-sized particles through dense polymer solutions and cross-linked gels and began to determine how HIV and other viruses involved in sexually transmitted diseases might penetrate cervical mucus; (2) developed methods based on dynamic light scattering and small-angle neutron scattering to determine the properties of clathrin triskelions in solution, showing that the lowest energy conformation has an intrinsic pucker but sufficient flexibility to adjust to steric constraints on assembly into clathrin cages; (3) discovered, by investigating biomimetic models in which all intermolecular interactions other than steric hindrance are suppressed, that the pressure attributable to spherical “crowders” drives a collection of rod-like objects to form aggregates whose sizes and shapes depend on rod and sphere diameters and concentrations and, under certain circumstances, on interactions with the walls of the receptacle by which the system is constrained; (4) cloned the genes for both subunits of Leishmania tubulin in order to produce these materials in bacteria for use in drug screening protocols; and (5) developed methods to probe the mechanical properties of the extracellular polymeric substance present in biofilms and, in Streptococcus mutans, showed that the substance softens and stiffens according to the proton concentration in the surrounding environment.
Adrian Parsegian, in the Section on Molecular Biophysics, uses osmotic stress and X-ray diffraction to measure the molecular forces acting in vitro, in vivo, and within the hard walls of viral capsids. Most recently, the section observed the ejection of DNA from capsids subject to different salt conditions and concluded that expansive pressures can vary up to many tens of atmospheres and lead to initial ejection of DNA. Given that these forces vary with ionic conditions, an unrecognized feature of many viruses is that ionic conditions can spread into the virus and in fact modify the expansive force within. The section is investigating whether the manipulation of such conditions ultimately affects viral infectivity. The section also continues to formulate and compute van der Waals forces that govern molecular assembly. Specifically, during the past year, the section teamed with groups that measure absorption spectra in order to formulate and compute van der Waals forces involving lipids, water, and ions as well as synthetic structures such as carbon nanotubes. By advancing the Lifshitz theory of van der Waals interactions in stratified media such as lipid multilamellar systems, the collaborative group computed forces between bodies with extended interfaces. These bodies can range from the practical—the composite media of electric insulators—to the biological—the action of extended polymer layers on biological membranes.
Donald Rau and his colleagues in the Section on Macromolecular Recognition and Assembly investigate intermolecular forces, with particular emphasis on the role of water and DNA-protein recognition reactions. During the past year, the section probed the mechanism by which osmolytes stabilize native, compact protein conformations. Several naturally occurring, small solutes protect proteins from heat shock and other denaturing environmental stresses. These osmolytes are excluded from protein surfaces by the same water-structuring forces that the section measured between many macromolecules, further emphasizing the critical role of water in molecular interactions. In demonstrating the importance of water in recognition reactions, the section measured a difference of 140 sterically sequestered water molecules between the specific complex of the restriction nuclease BamHI with its recognition DNA sequence and the nonspecific complex with noncognate DNA. The number is consistent with X-ray crystal structures, confirming the validity of the section’s approach.
The Section on Mass Spectrometry and Metabolism, led by Alfred Yergey, applies knowledge of the physical chemistry of gas phase ions to basic research in structural biology. The applied research ranges from mapping picomolar quantities of peptides extracted from proteins digested in situ from electrophoretically separated proteins, to obtaining partial peptide sequences at sub-picomolar sensitivities to facilitate the construction of nucleotide probes, to mapping epitopes of femtomolar quantities of proteins isolated by noncovalent interactions with antibodies. The section made significant progress in three areas of mass-spectrometric analysis. First, it developed a tool for generating consensus spectra from several replicates, permitting the assignment of statistically significant confidence intervals to the dot product comparison of two consensus spectra. Second, researchers applied this tool to the characterization of tubulin C-terminal peptides and to time-of-flight (TOF)/TOF peptide fragmentation spectra. Third, the section developed a means of quantifying phosphorylation stoichiometry by using a set of synthetic peptides to generate standard curves. Fourth, the section made considerable progress in developing a tool for quantifying cardiolipins in human serum for the purpose of determining the amount of cardiolipin in the blood of infants born to mothers colonized by group B streptococcal organisms.
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 parasite infection, exocytosis, and apoptosis. The section has organized an interdisciplinary attack on the mechanisms of membrane remodeling. For malaria, it is clear that a major way to stop Plasmodium, the parasite that causes the disease, is to prevent the parasites’ release from the human red blood cell, the cell in which they develop. To study the mechanisms by which cells are released in a synchronous fashion, the section developed a quantitative assay for release. The key physiological stage in enveloped viral disease is the poorly understood budding stage, which occurs when construction of the virus is complete and it buds away from the plasma membrane. This year, members of the section discovered how the protein forming the matrix of the virus interacts with membranes to cause them to bud. In a project using fluorescence photoactivation localization microscopy (FPALM), the section imaged distributions of tens of thousands of hemagglutinin (HA) molecules with subdiffraction resolution (30–40 nm) in live and fixed fibroblasts. HA molecules form irregular clusters on length scales from 30 nm up to many micrometers, consistent with results from electron microscopy. The section observed the dynamics of HA molecules within clusters and quantified them in live cells to determine an effective diffusion coefficient, interpreted the results in terms of several established models of biological membranes, and calculated the effect of an externally applied lateral tension on the line tension between two domains of different thickness in a lipid bilayer membrane.
Within the LCMB, the NASA/NIH Center for Three-Dimensional Tissue Culture, a pan–NIH facility directed by Joshua Zimmerberg, with deputy directors Leonid Margolis, Paul Blank, and Jean-Charles Grivel, provides NIH researchers with an opportunity to develop new model systems for diseases whose pathology cannot be reproduced by merely growing the appropriate 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 available, along with experienced technicians to test tissues, primary cell cultures, and cell lines under low-shear fluid conditions that seem to facilitate cell-cell interactions and promote differentiation. As a consequence of the surprise finding of immunodysfunction in human lymphoid tissue in culture and known findings of immunodysfunction in astronauts after space flight, Zimmerberg also serves as a NASA flight principal investigator on the International Space Station. The past year has seen considerable progress in understanding the culture and differentiation of normal human breast tissue. Preliminary evidence points to a proliferation of breast ducts in three-dimensional tissue culture. We have also joined forces with the Breast Cancer Stamp program for future testing of neoplastic human breast in three-dimensional culture.
