William A. Mohler - US grants

Cell-cell fusion is a key process in reproduction and in the morphogenesis of many multicellular organisms. However, because of the limitations of approaches used to study cell fusion, little is known about the molecular basis of the event. In the laboratory of John White at the University of Wisconsin, Madison, I propose to study the process of cell fusion in the nematode Caenorhabditis elegans. This model system offers the most tractable strategy for the study of fusion via genetic analysis and direct, detailed observation and manipulation. Information gained from this work should be readily transferred, by virtue of evolutionary conservation of both the mechanisms and genes involved, to an understanding of cell-cell fusion and the formation of various syncytial tissues of other species, including humans. Two separate directed approaches will be applied to the study of fusion in C. elegans. 1. Genes which are essential to the process of cell membrane fusion will be isolated by screening for mutants with specific deficits in cell fusion of the syncytial epidermis of C. elegans. Mapping and cloning of these genes by standard methods of C. elegans genetics will lead to identification of the molecules which direct fusion in the worm, and eventually lead to the identification of homologues in other species. 2. Individual cell-fusion events will be observed in live animals using established techniques for microscopy and the labeling of single cells in C. elegans embryos. This approach will be the basis for detailed descriptions of the structural nature and biochemical requirements of cell fusion.

The long-term goal of this proposal is to understand the mechanisms by which cell membranes become fusion competent in normal animal development. The fusion of somatic cells to form syncytial tissues is an essential part of human development, and cell fusion is an ancient process, shared in evolution by all eukaryotic phyla. Yet the molecular mechanism of cell fusion is essentially unknown in all species. Finding the molecules that drive plasma membrane fusion is therefore a major priority in understanding human health. Our studies of the nematode C. elegans have uncovered EFF-1, the first cellular membrane protein in any species that is known to be completely required for and completely specific to the process of cell membrane fusion. EFF-1 is a novel type I membrane protein, with sequence motifs that suggest modes of action via direct interaction and/or processing of membrane lipids: a phospholipase A2 active site, and a potential virus-like amphipathic fusion peptide. The absence of functional EFF-1 in mutant cells blocks cell membrane fusion at the earliest step in the process. We propose to determine the molecular function of EFF-1 in membrane fusion, and to further dissect the full mechanism of cell fusion via the following lines of inquiry: 1. Map and test the functional domains of EFF-1 through directed mutation analysis and via assays of enzymatic activity. 2. Assess the role of EFF-1 within the dynamic structural context of a cell fusion event by imaging of labeled EFF-1 protein within the fusing cells of live embryos. 3. Identify other protein components of the cell fusion machinery through isolation of molecules that interact physically and genetically with EFF-1. This proposal is submitted in response to PAS-00-067 from NICHD: "Membrane properties: Exploration of sperm/microbe susceptibility", as it focuses on topics specifically targeted by the PA. The scientific impact of the proposed research is of general import, however. Our results will increase understanding of the molecular mechanism underlying developmental cell fusion, a crucial and under-explored component of animal reproduction, development, and health.

A group of six major NIH-funded users requests funds for a spinning-microlens array laser-scanning confocal microscope. The Perkin Elmer Ultraview system requested includes a broad selection of excitation laser lines, including a special-purpose 442-nm HeCad laser for specific excitation of CFP fusion proteins and CFP/YFP ratio imaging, which is not available on an other system at UCHC. In addition, the spinning microlens array allows high-frame-rate acquisition using the full resolution of a high quantum-yield cooled CCD camera. This excitation/collection strategy has been shown to result in reduced photobleaching and photodamage in imaged samples when compared to point-scanned galvanometer confocal systems, probably because of sub-saturating instantaneous excitation and the higher signal-to-noise of the CCD detector. An additional computer-controlled dye-tunable photobleaching laser system is included for high-time/space-resolution studies of fluorescence recovery after photobleaching (FRAP), as well as dequenching measurements of fluorescence resonance energy transfer (FRET). The instrument will be housed in the Center for Biomedical Imaging Technology (CBIT) at the UConn Health Center, which offers use of high-level fluorescence microscopy instrumentation and image analysis resources to the research community both at UCHC and outside institutions, through a well-established user facility. The six major users have well-developed projects that each require some aspects that are unique the to requested system and unavailable elsewhere at UCHC. William Mohler will record high-speed 4D data and perform FRAP analysis to understand the mechanism of cell fusion in C. elegans. Vladimir Rodionov will use fluorescent speckle imaging study the cytoskeletal basis for self-centering of the centrosome. Elizabeth Eipper will study the dynamics of peptide vesicle secretion via high-speed confocal imaging and FRAP. Dianqing Wu will study the interactions of Wnt-signalling molecules in vivo via FRET. Steven Pfeiffer will study the dynamic transport of myelin proteins and their localization to lipid rafts via FRET. Bruce Mayer will visualize actin cytoskeletal changes induced by aggregation of signaling oroteins, as well as dvnamic colocalization and orotein interaction bv FRET in vivo.

DESCRIPTION (provided by applicant): The second Gordon Research Conference on Cell-Cell Fusion will take place in 2009, at Colby-Sawyer College in New Hampshire, July 19-24. This relatively new GRC follows upon a very successful first GRC meeting in 2007 and two earlier special interest subgroup meetings on cell-cell fusion that took place in 2002 and 2003 at the American Society for Cell Biology. This conference focuses on the fusion of cells with each other during development, regeneration, and disease. The Cell-Cell Fusion GRC is unique in the novelty, breadth, and depth of its coverage of new developments in the blossoming field of cell fusion. The conference will include eight topical sessions. Three of these will cover 1) structure and function of known fusogenic membrane proteins, 2) the physics of fusing membrane bilayers, and 3) novel applications of induced cell fusion in medicine. Five additional sessions will cover mechanisms of developmental control of cell fusion in important species and cell types, where the molecular details are just beginning to emerge and converge: 4) fertilization by gamete cells;5) epithelial cells;6) myoblasts and mesenchymal cells;7) macrophages and other monocytic cells;and 8) fungal and plant cells. The discussion leaders and speakers selected to date are among the most respected investigators and international leaders on cell fusion. The keynote address speaker will be one of the world's foremost experts in membrane fission, fusion, and/or trafficking. The success of Gordon Conferences as a forum for the discussion of state-of-the-art research advances is attributable to their limited size, geographical isolation, and overall structure. The Gordon Research Conference format differs from virtually all other scientific meetings in providing an extensive opportunity for open, free, and informal discussions. The number of participants is limited to about 130, and attendees include international leaders in the field from the academic and industrial sector, young investigators, postdoctoral fellows, and students. The purpose of this application is to request funds to defray the costs of registration and travel for graduate students and postdoctoral fellows. Because direct funding from the GRC organization to new meetings is limited, these funds from the NIH will aid immeasurably in allowing the Cell-Cell Fusion conference to fulfill its function, which is to stimulate creativity and interaction in this emerging field of biology. PUBLIC HEALTH RELEVANCE: Cell-cell fusion is a topic spanning many disciplines of biology and medicine, including fertility, development and repair of muscle and bone, vision, and immunity to infection. It is also an important aspect of the life cycle of many parasitic and viral pathogens. Understanding the mechanisms by which diverse cell types fuse, and the ways in which cell engineering via cell fusion can be applied in medicine, will advance the aim of the NIH to improve human health. This meeting stimulates communication among researchers in the various systems in which cell fusion is under study.

Project Summary A major challenge in neuroscience is to comprehend how differentiating embryonic cells fashion de novo the working circuits and networks that make up a nervous system. We propose WormGUIDES, a resource that would facilitate understanding the cellular origins of an entire nervous system from all angles?cell birth, migration and differentiation; neurite formation, targeted outgrowth and bundling; and ultimately, synapse formation and tuning of functional circuits?with single cell resolution, from conception until hatching. Building on the innovations we established in the first funding cycle, we propose to use ShootingStar, a novel platform for real-time cell tracking and optical manipulation to label single neurons, and isotropic high-resolution diSPIM imaging to capture nerve bundling dynamics and single cell outgrowth in the early developing embryo when the major architecture of the nervous system is constructed. We will also assign all nuclear identities and positions from the first cell division until hatching. Finally, we will share the knowledge with the community through WormGUIDES Atlas software, displaying models, supporting images and primary data. Completion of the WormGUIDES resource will ultimately complement the existing cell lineage, adult nervous system structure, and collective genomic and genetic data for the worm, creating a fourth pillar of systems-level knowledge that will enhance our understanding of neurodevelopment.