Joshua Zimmerberg, MD, PhD, Chief
Using systems whose complexity ranges from a well-defined molecular composition and structure to human tissue to investigate the physicochemical basis of molecular, physiological, and pathological processes, the Laboratory of Cellular and Molecular Biophysics (LCMB) develops novel, noninvasive technologies to probe the processes’ physical and chemical parameters. The research program focuses on the physical chemistry of gas phase ions, polymer organic chemistry, membrane biochemistry, electrophysiology, cell biology, parasitology, immunology, tissue culture, virology, and HIV pathogenesis. The past year has been unusually productive for the Laboratory. Biophysical concepts and experiments have provided new insights into key physiological processes, the life cycle, and human defenses to important pathogens.
The Section on Membrane and Cellular Biophysics, led by Joshua Zimmerberg, studies membrane mechanics, intracellular molecules, membranes, viruses, organelles, and cells involved in exocytosis, apoptosis, and viral and parasite infection. First, the Section has organized an interdisciplinary attack on the mechanisms of membrane remodeling. In the past year, the researchers discovered that the influenza virus causes its hemagluttinin (an antigen against which the flu vaccine is made) to cluster in the plane of the plasma membrane at two length scales, one for eventual budding of a new virus and one for molecular cooperativity in the fusion step of infection. These results have major implications for theories of membrane microdomain structure in that they disprove the current dominant theory. Second, the Section discovered that photo-damage had precluded a true view of the life cycle of Plasmodium, the malarial parasite; the researchers proposed a new pathway with two new intermediates and suggested that a pore initiates the release of parasites at the end of each asexual infection, ruling out membrane fusion and simple swelling and bursting as mechanisms of release. Third, members of the Section found that the insertion of glucose transporters by insulin in fat cells exploits microtubule tracks on their way to the plasma membrane and revealed constrained release of membrane cargo. The probability of tethering and fusion of these vesicles to the plasma membrane is specifically sensitive to insulin, opening a novel door to research on insulin signaling. The Section has also developed a new physical theory that quantifies conditions that allow membrane microdomains to form, further defining the parameters that control membrane merger.
The Section on Membrane Biology, led by Leonid Chernomordik, studies the mechanisms of membrane fusion. The group has expanded its working hypothesis that the hemagglutinin of influenza virus not only initiates fusion but also provides the driving force for the entire fusion reaction. The researchers found that fusion proteins of both classes (I and II) drive the entire fusion pathway rather than merely catalyze the merger of the contacting monolayers of two membranes. Their research points to the universality of this mechanism of biological fusion and supports the hypothesis that the final hairpin structure shared by diverse viral fusion proteins and by proteins involved in intracellular fusion is more important for fusion than the proteins’ initial metastable conformations. Further, the Section showed that cross-linking of surface glycoproteins by endogenous lectin-like host defense molecules apparently blocks an essential but hitherto unexplored stage of viral entry: displacement of proteins from the prospective fusion site. The researchers also discovered that entry of the unconjugated TAT peptide cells involves a clathrin-dependent endocytic pathway, a process that proceeds either via heparan sulfate receptors or without them.
The Section on Intercellular Interactions, led by Leonid Margolis, studies HIV pathogenesis in human lymphoid tissue ex vivo. This culture system, developed by the Section, 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. This year, the group discovered that R5 HIV-1 isolates from nonswitch virus patients are more cytopathic than R5 variants from switch virus patients; the difference may explain the steady decline of CD4+ T cells in patients with continuous dominance of R5 HIV-1. The level of R5 pathogenicity, as measured in ex vivo lymphoid tissue, may have a predictive value. The Section also deciphered the molecular mechanisms by which the measles virus and other pathogens alter local cytokine/chemokine networks and cause tissue microenvironments to become inhospitable to HIV-1, results that may significantly contribute to the development of effective anti–HIV therapies. Finally, the Section discovered that pertussis toxin B oligomer PTX-B and PT-9K/129G, its genetically modified variant that is used for vaccination, inhibit HIV-1 replication in ex vivo–infected human lymphoid tissue. Moreover, the proteins augmented the number of cells emigrating from the tissue blocks into the surrounding medium and stimulated the proliferation of emigrated cells, most of which were CD4+ T lymphocytes.
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 subpicomolar sensitivities to facilitate the construction of nucleotide probes, to mapping epitopes of femtotomolar quantities of proteins isolated by noncovalent interactions with antibodies. During the past year, the Section used matrix-assisted laser desorption ionization (MALDI) of peptides as a model system to study peptide ion fragmentation, ion energetics relationships between laser fluence, and peptide ion fragmentation. In addition, as host of the NICHD Mass Spectroscopy Center, the Section identified proteins isolated in the biochemical investigations of other researchers and developed new methods, such as a novel approach to providing sequence information for proteins not described in databases. The Section also characterized the C-terminal post-translational modifications of tubulins as well as the protein mass fingerprints of amniotic fluid from patients who had undergone premature labor, research that resulted in the ability to identify patients who present with evidence of a bacterial infection along with premature labor.
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 cells in monolayer culture. Several NASA-designed rotating wall vessels (RWVs), which culture cells under minimal shear forces in a well-oxygenated medium under conditions that mimic microgravity, are available to researchers, along with technicians experienced in conducting tests on tissues, primary cell cultures, and cell lines under low-shear fluid conditions that seem to facilitate cell-cell interactions and promote differentiation. Extensive consultations and a seminar determine the applicability of the Center’s resources to the aims of the interested investigator such that, together, the investigator and the staff design pilot projects. Principal investigators with successful pilot projects can apply for Center funding for salary, equipment, and consumables. A project is fully mature when its principal investigator continues work with his or her own funding and no longer requires Center funding.