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National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2015 Annual Report of the Division of Intramural Research

Program in Perinatal Research and Obstetrics

Director: Joshua Zimmerberg, MD, PhD

Human embryonic development, on which the future child’s health depends, is a complex phenomenon within the female starting with egg-spermatozoa fusion. In each individual, a plethora of molecular recognition events mediate the development of an immune system to defend against pathogens, a musculo-skeletal system to maintain the body, and flexible networks of molecular expression to manage environmental stress. Traditionally, studies on these processes have been divided among biochemistry, cell biology, virology, toxicology, etc. However Nature does not know these artificial divisions, and new understandings are emerging from the interface between mathematically minded physical scientists and biomedical researchers. The Program in Physical Biology, led by Joshua Zimmerberg, is a unique scientific body that approaches human development in normal life and pathology as an integral process and which encompass first-class cell biologists, physical chemists, biophysicists, virologists, and immunologists, who not only successfully train post-docs and students within their own fields in work on their own projects but also collaborate widely, building and uniquely promulgating multidisciplinary approaches to the most important biomedical problems in the framework of the NICHD mission. This year marks the retirement of our dear friend and colleague, Donald Rau, to whom we extend our very best wishes.

The Section on Molecular Transport, led by Sergey Bezrukov, advances biophysical methods as tools to understand molecular interactions, notably by studying, in the context of human development, disease, and pharmacological intervention, the interactions of beta-barrel membrane channels with drugs and cytosolic proteins, as regulated by upstream signaling. Using single-molecule functional approaches, one project aims to unveil the physical mechanisms regulating the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane in cell proliferation, reprogrammed cancer metabolism, kinase-regulated cell signaling, cytoprotection, and neurodegeneration. The past year's work on the VDAC focused on the effect of alpha-synuclein, an intrinsically disordered protein known to be involved in mitochondrial dysfunction in neurodegeneration. The group showed that alpha-synuclein reversibly blocks (and is able to translocated through) the VDAC, the major transport pore of the mitochondrial outer membrane, which suggests that alpha-synuclein participates in the regulation of normal mitochondrial respiration, in synuclein-induced mitochondrial dysfunction, or both. The results thus point to a plausible molecular mechanism for the protein's (patho-)physiological function. Work on the physical theory of facilitated transport concentrated on bulk-mediated surface diffusion to support experimental findings pointing to the importance of the membrane-binding step in the interaction of cytosolic proteins with beta-barrel channels.

The long-term goal of the Section on Membrane Biology, led by Leonid Chernomordik, is to understand how proteins drive membrane rearrangements in important cell biology processes. Whereas each kind of protein has its individual personality, membrane lipid bilayers have rather general properties manifested by their resistance to disruption and bending. Analysis of molecular mechanisms underlying important and diverse membrane rearrangements will clarify the generality of emerging mechanistic insights and likely bring about new strategies for treating diseases involving cell invasion by enveloped viruses, intracellular trafficking, and intercellular fusion. In recent studies, in parallel with ongoing studies on the cell-to-cell fusion stage during development and on regeneration of muscles and bones, the Section focused on the mechanisms of cell entry by cell-permeable cationic peptides.

During the last year, the Section of Intercellular Interactions, led by Leonid Margolis, pursued the following aims. The first was to study HIV-1 infectivity in human tissue by analyzing individual virions' spikes, which mediate virus-cell fusion; it was found that viruses exhibit little mosaicism—on the majority of virions either all spikes are functional or are all defective; such an all-or-nothing viral strategy is likely to aid immune evasion by subverting the focus of humoral responses to generate multiple non-neutralizing antibodies at no cost to infectious virions. The second aim was to apply the flow virometry technology developed for HIV-1 analysis to other viruses, in particular to dengue virus; analysis of individual viral particles permitted the Section to distinguish immature from mosaic virions by the presence of the prM protein on the viral surface. The third aim was to investigate pathogen-triggered changes in the cytokine network of the amniotic fluid of women in preterm labor, according to the presence or absence of intra-amniotic inflammation and microorganisms in the amniotic cavity. The study showed that the cytokine network connectivity with microbial-associated intra-amniotic inflammation is denser and more coordinated than in women with sterile inflammation or without intra-amniotic inflammation. This new network analysis provides a deeper insight into the pathophysiological mechanisms of intra-amniotic infection/inflammation in preterm labor and helps identify potentially relevant modules of cytokines that correspond to distinct disease pathways. A similar network analysis can now be applied to other pathogens. The fourth aim was to perform a broad analysis of the biomedical literature and our unpublished results to determine the possible role of immuno-activation in human disease. Immuno-activation appears to be a common denominator or general mechanism of pathogenesis and may explain similarities in pathology among otherwise unrelated human diseases. Identification of general mechanisms of immuno-activation may lead to the development of new therapeutic strategies applicable to many diseases, even before detailed knowledge of their specific etiology and pathogenesis is available.

The Section on Cell Biophysics, led by Ralph Nossal, studies physical and physical-chemical mechanisms underlying cell behavior, for which the Section develops and applies mathematical and computational methodologies and uses biochemical and cell-biological techniques. Projects currently include: (1) elaborating a mathematical model to understand the physical basis of coated vesicle biogenesis during receptor-mediated endocytosis, focusing on how the size dependence of nanoparticle uptake relies on mesoscopic cell mechanics; (2) developing quantitative measures of eukaryotic cell chemotaxis; (3) exploring how the physiological behavior of neural tissues and other cell elements is mediated by temperature-linked phase changes in the lipid bilayer of the cell membrane; and (4) understanding how certain small molecules interact with microtubules to act as antimitotic agents, and how microtubule arrays function in mitosis to produce accurate segregation of chromosomes. The Section also develops new experimental modalities to characterize these and related phenomena, with a particular interest in the ways in which cellular activities are coordinated in space and time.

The Section on Macromolecular Recognition and Assembly, headed by Donald Rau, focuses on the nature of forces, structure, and dynamics of biologically important assemblies. The group showed that measured forces differ from those predicted by current theories and interpreted the observed forces to indicate the dominant contribution of water-structuring energetics. To investigate the role of water in binding, the group measures and correlates changes in binding energies and hydration that accompany recognition reactions of biologically important macromolecules, particularly sequence-specific DNA–protein complexes. By investigating differences in water sequestered by complexes of sequence-specific DNA–binding proteins bound to different DNA sequences, members of the Section correlated binding energy and water incorporated with the energy necessary to remove hydrating water from complexes. The emphasis on water permits a different approach to recognition reactions than standard practice. The Section also continued its work on sperm nuclear DNA packing and on the effect of temperature on the packing transitions in viruses, which facilitate infection. The Section discovered a novel, temperature-mediated transition in DNA packing for both the bacteriophage lambda and human herpes simplex virus type 1 (HSV-1). Both X-ray scattering and cryoEM reconstruction indicated a transition in DNA packing that occurs at about 30°C and is characterized by a substantial increase in the amount of disordered DNA in the center of the capsid.

The Section on Membrane and Cellular Biophysics, led by Joshua Zimmerberg, studies membranes, viruses, organelles, cells, and tissues in order to understand the molecular organization of cellular membranes, the physico-chemical mechanisms of membrane remodeling, and the molecular anatomy of tissues, which will lead to deeper insights into viral, parasitic, metabolic, developmental, and neoplastic diseases. The Section aims to use the expertise and techniques it perfected over the years to address several biological problems that have in common the underlying regulation or disturbance of protein/lipid interactions. During the past year, the Section (1) performed long-term time-lapse microscopy of a novel fluorescent protein construct that reports the expression of the transcription factors OCT4, SOX2, and NANOG; (2) invented a new chamber for high-resolution, time-lapse, optical microscopy with precise control of shear forces over a time intervals, forces associated with blast shock waves known to create traumatic brain injury in the field during exposure to explosive blasts; (3) achieved direct chemical detection of the lipids around domains of influenza hemagglutinin on the plasma membrane of fibroblasts; and (4) tested the idea that the response to insulin would be gradually diminished as the metabolic health of the individual diminished; instead a biphasic response was found.

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