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Laboratory of cellular and molecular biophysics

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 includes the physical chemistry of gas phase ions, polymer organic chemistry, membrane biochemistry, electrophysiology, cell biology, parasitology, immunology, tissue culture, virology, and HIV pathogenesis.

The Section on Membrane and Cellular Biophysics, led by Joshua Zimmerberg, studies membrane mechanics, intracellular molecules, membranes, viruses, organelles, and cells to understand viral and parasite infection, exocytosis, and apoptosis. The section has organized an interdisciplinary attack on the mechanisms of membrane remodeling. In the past year, the researchers discovered that a novel frame-shifted protein encoded by the influenza genome causes apoptotic pores similar to those formed by Bax and truncated Bcl and Bid; that direct intra-preterminal membrane recordings in the squid giant synapse show a dramatically different type of channel; that molecular shape determines solid amphiphile macroscopic structure from nanodiscs to punctuated icosohedra; why membrane bending is predicted to lower the energy for protein insertion into membranes; and that the N-ethylmaleimide–sensitive fusion protein NSF is not required for the fusion of purified secretory granules to each other. In addition, the section began to investigate the role of membrane microdomains in membrane fusion.

The Section on Membrane Biology, led by Leonid Chernomordik, studies the mechanistic pathway 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. Surprisingly, the researchers found that fusion proteins play a role not only in the zone of contact between membranes but also outside the zone such that “outsiders” are both necessary and sufficient for membrane fusion. In another project, the group has initiated investigations of the mechanisms of cell-cell fusion in the development of C. elegans, a model genetic system for the study of development.

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. The section’s new data may explain the nature of copathogenesis of HHV-6 and HIV-1 in coinfected patients and suggest a new way of modulating HIV infection. This past year, the group found that noninfectious virions are immunosuppressive, indicating that defective virions in vivo (the majority of viral particles) may contribute to immunodeficiency. In contrast, the group discovered that T cell depletion ex vivo requires productive infection, suggesting that triggering in vivo viral production in latently infected cells in combination with therapies may become a meaningful strategy to purge latent viral reservoirs; the group also showed that prostratin is a promising activator of latent infection. In additon, the section’s researchers found that nef, vpr, and vpu are relevant for efficient viral infection and for CD4 T cell depletion in HIV-1-infected human lymphoid tissues.

The Section on Metabolic Analysis and Mass Spectrometry, 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 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. During the past year, the section pursued new methods to estimate the fraction of laser energy delivered to gas phase ions during matrix ionization of proteins, allowing the researchers to select and optimize both matrix and laser frequency. In addition, the section developed new methods for both the complete de novo sequencing of peptides and pinpointing phosphorylation sites on proteins.

The Section on Macromolecular Analysis, led by Andreas Chrambach, pursues the improvement of analytic and preparative electrophoresis with biological and methodological aims. The biological concern is oriented toward the idea that today’s focus on an inventory of gene products will decline in the future. Correspondingly, interest is expected to increase in an inventory of the post-translational forms of macromolecules, intermolecular complexes, and subcellular particles that constitute the building blocks of biological reality. Methodologically, the section attempts to upgrade the common practice of gel electrophoresis with the following development projects: (1) theory and computer programs for deriving information from mobility regarding physical properties of analytes and polymer networks and for predicting resolution; (2) resolution of unresolved or poorly resolved analyte types such as subcellular-sized particles and the size- and charge-isomeric forms of peptides and their complexes; (3) detergent and miniaturized gel methods suitable for native membrane proteins and their complexes of a low level of occurrence; and (4) automated analytic and preparative gel electrophoresis methods. Over the past year, the section has investigated the dependence of electrophoretic retardation on membrane elasticity of secretory vesicles modulated by cholesterol content or lipid composition. By comparing the data with that obtained by dynamic light scattering of the same vesicles in different osmotic conditions, the researchers hope to test if calcium changes membrane elasticity.

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 and Paul Blank, provides NIH researchers with an opportunity to develop new model systems for diseases whose pathology cannot be reproduced by merely growing the relevant 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, 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. 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.

Given the surprise finding of immunodysfunction in human lymphoid tissue in culture and known findings of immunodysfunction in astronauts after space flight, Joshua Zimmerberg is also a NASA flight principal investigator on the International Space Station (ISS). The past year has seen considerable progress in understanding the culture and differentiation of embryonic stem cells, the culturing of the parasite that causes malaria, the role of extracellular matrix in cell differentiation, the growth of hematopoietic stem cells in the NASA RWV, and central nervous system stem and progenitor cell differentiation into functional neuronal circuits in three-dimensional collagen gels.