Joshua Zimmerberg, MD, PhD, Chief
Using systems ranging in complexity from 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 effort is based in the physical chemistry of gas phase ions, polymer organic chemistry, membrane biochemistry, electrophysiology, cell biology, parasitology, immunology, tissue culture, virology, and HIV pathogenesis. In what has been an unusually productive year for the LCMB, biophysical concepts and experiments have shed light on significant physiological processes as well as on human defenses against important pathogens.
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 laboratory has organized an interdisciplinary attack on the mechanisms of membrane remodeling. In the past year, researchers (1) discovered that that the toxin causing E. coli disease, unlike other bacterial toxins, is highly unusual in its lipid dependence; (2) put forth a new proposal for the structure of gel-phase lipids in cell membranes; (3) developed a general formalism for how proteins can form shape on biological membranes; (4) proposed a new physical model for the exocytosis of synaptic vesicles at the synapse; and (5) performed a quantitative analysis of the nucleation and growth of membrane microdomains.
The Section on Membrane Biology, led by Leonid Chernomordik, studies the mechanistic pathway of membrane fusion. During 2006, the group both extended its biophysical analysis of fusion caused by HA, the spike protein of the influenza virus, and started to study the mechanism by which cells fuse in development. With respect to HA, researchers found that the lipid composition of the target membrane changes the relative stability of the HA molecule itself, with more fusogenic target membranes blocking its conformational change. The group found two distinct types of activation/inactivation intermediates that are thought to be attributable to differences in local HA surface density. With respect to development, the group found that the C. elegans fusion protein candidate EFF1 is a bona fide fusogen rather than a regulator of fusion. The group expressed it at the surface of insect cells and found that it initiates cell fusion, producing multinucleate syncytia. Thus, the laboratory established EFF-1 as the first known developmental fusogen. As with viral and intracellular fusion reactions, the EFF-1-mediated fusion involves hemifusion intermediates. However, the reconstituted developmental fusion differs from all fusion reactions in requiring the same fusogen (EFF-1) to be present in both fusion partners.
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. During the past year, the group showed that the concomitant effect of a series of previously identified weak barriers to the X4 variant of HIV diminishes the variant's ability to infect humans as compared with the R5 variant of HIV, countering the prevalent hypothesis that posited infection by a single mechanism. The laboratory also developed a system of intestinal explants derived from both endoscopy and surgical resection specimens. The higher frequency of R5 target cells in rectal tissue suggests that the cells may be a mechanism for preferential R5 HIV-1 transmission. Moreover, the laboratory discovered the suppression of HIV R5 (but not X4) replication by herpes virus HHV-7, a mechanism that involves the downregulation of CD4 on target cells. In addition, the group studied the effect of environment on the differentiation of embryonic stem cells (ESC). Using several extracellular matrixes, researchers compared the bioactive collagen Matrigel with a biologically inert agarose. The extent of cell-cell interaction versus cell-substrate adhesion is closely related to the pattern of ESC differentiation.
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 group's applied research ranges from mapping of 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 femtotomolar quantities of proteins isolated by noncovalent interactions with antibodies. The Section had shown earlier that rapid and extensive peptide fragmentation is associated with low laser plume densities. At higher densities, ions undergo a large number of collisions, fragmenting in a series of consecutive reactions in which the amide backbone bonds are ruptured. The laboratory is also improving protein characterization capabilities by calculating a database consisting of an exhaustive list of all amino acid compositions up to a maximum of 2 kDa, thereby improving the algorithms used for peptide sequencing and eliminating potential ambiguities arising from incomplete fragmentation. In addition, the laboratory showed that bovine and rat brain tubulins appear to have indistinguishable compositions and perhaps, more interestingly, that the tubulins associated with clathrin-coated vesicles have the same tubulin composition as a homogenate of whole brain. The group performed a quantitative determination of deamidation, mapped specific deamidation sites, and determined how to distinguish patients who deliver at term from those who have delivered prematurely under conditions wherein both groups have experienced 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 in which to study diseases whose pathology cannot be reproduced by merely growing cells in monolayer culture. The Center maintains, for example, several NASA-designed rotating wall vessels (RWVs) that culture cells under minimal shear forces in a well-oxygenated medium under conditions that mimic microgravity. In addition, the Center is staffed with experienced technicians who test 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 whether the Center's resources are compatible with the aims of an interested investigator such that, together, the investigator and staff design pilot projects. Principal investigators with successful pilot projects may then 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.
Given 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. In this capacity, Zimmerberg has, over the past year, achieved considerable progress in understanding the culture and differentiation of embryonic and progenitor stem cells and the role of extracellular matrix in cell differentiation.
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