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 physico-chemical 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.

Joshua Zimmerberg's group, the Section on Membrane and Cellular Biophysics, studies the mechanisms of membrane remodeling in viral and parasite infection, exocytosis, and apoptosis. Using the physics of continuum bilayers and direct observations of biological fusion, analytical and numerical calculations of membrane energetics, and experiments on phospholipid bilayers, purified proteins, cell expression systems, purified organelles, and cell surface complexes, the section has elucidated the energetics of membrane tubule shape dynamics and permeability and membrane permeability during fusion. Studies of the physiological and pathogenic events of fertilization and viral infection, e.g., those involving SNAREs, calcium, and exocytosis, have led to novel uses of viral fusion proteins for gene therapy and to a new model for the cycle of exocytosis and endocytosis.

The Section on Membrane Biology, led by Leonid Chernomordik, studies the mechanistic pathway of membrane fusion. Over the past year, the section developed a technique for measuring the complete kinetics of the lipid mixing, finding evidence that distinct fusion machines generate distinct phenotypes. In another study, the researchers found that the widely used cell-penetrating peptides that readily deliver themselves, conjugated proteins, and nucleic acids into living cells enter cells by endocytosis rather than by direct translocation through the membrane, as is often hypothesized.

Leonid Margolis's group, the Section on Intercellular Interactions, found that human lymphoid tissue ex vivo, a system developed in the LCMB, 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 media, lymphocyte apoptosis, and a functional immune response, thus providing a unique way to study HIV tissue pathogenesis. In this system, both activated and nonactivated CD4+ T cells support productive infection; a combination of productive infection and activation is sufficient for T cell apoptosis; productive HIV infection is sufficient to deplete infected CD4+ T cells; and herpes virus 6, an HIV-1 copathogen, infects both memory and naïve T cells, profoundly influencing the physiology of the immune responses and the replication of different HIV-1 variants.

The Section on Metabolic Analysis and Mass Spectrometry, led by Alfred Yergey, applies the physical chemistry of gas phase ions to research in structural biology. Studies include mapping of picomolar quantities of peptides extracted from proteins digested in situ from electrophoretically separated proteins; obtaining partial peptide sequences at sub-picomolar sensitivities to facilitate the construction of nucleotide probes; and mapping epitopes of femtotomolar quantities of proteins isolated by noncovalent interactions with antibodies. 
 

Andreas Chrambach's group, the Section on Macromolecular Analysis, pursues the improvement of analytic and preparative electrophoresis with biological and methodological aims. The biological interest is oriented toward the idea that the present focus on an inventory of gene products will decline in the future. Correspondingly, interest will shift to the development of 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 through 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 currently 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. 
 

Within 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 in growing 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 conditions that seem to facilitate cell-cell interactions and promote differentiation. Extensive consultations with a group seminar determine the applicability of Center resources to the aims of the interested principal investigator (PI). A PI with successful pilot projects can apply for Center funding for salary, equipment, and consumables. Projects are fully mature when PIs continues the work with their own funding. Given the surprise finding of immunodysfunction in human lymphoid tissue in culture and the known findings of immunodysfunction in astronauts after space flight, Dr. Zimmerberg is also a NASA flight PI on the International Space Station (ISS).