How does it all fit together?

The structure of EFF-1 is bringing insights into the mechanisms of cell fusion. We are mapping mutations and critical residues on the atomic structures of the ectodomain of EFF-1 protein. Eventually we will determine whether we can crystallize not only the wild-type forms but also some mutants. In addition to the work at atomic resolution we also explore negative staining electron microscopy (TEM) and cryoTEM to determine the structure of EFF-1 and AFF-1 at lower resolution in solution and in association with liposomes and pseudotyped viruses. We will also attempt to analyze membrane cell fusion using superresolution light microscopy at nanometric resolution. The combination of live imaging, superresolution light microscopy, EM, CryoEM and crystal structure determination will be very informative and will contribute to the understanding of the molecular mechanism of FF-mediated membrane fusion.

Identifying critical domains required for FF fusion, intermediates in membrane remodeling, and atomic structures of FF proteins will advance the fundamental understanding of developmental cell fusion mechanisms and should be relevant to research in the fields of fertility, developmental biology and cancer. The projects that we are currently pursuing are both feasible and unique since no other candidate cell-cell fusogens have been analyzed the way we propose to study EFF-1 and AFF-1. Although some of the projects going on in our lab involve certain risk, the gains that will be obtained if successful, vastly surpass the risks. Thus, we already have the structure of EFF-1 ectodomain and we expect to get the atomic structure of AFF-1 and non-nematode FF proteins within five years and to determine how cells fuse from the organism to atomic resolution.

The key gap in the knowledge of cell fusion in general is the lack of fusogens. In particular we are searching for the fertilization fusogens in C. elegans and for the muscle fusogens in mouse. We currently have an ideal interdisciplinary group to address some of the biggest questions in our field: how myoblasts and how sperm-egg fuse. We will simultaneously study how FF proteins fuse cells in vivo and in vitro and in parallel we will use all our experience, assays and reagents accumulated on cell fusion in C. elegans and in the lab dish to find and characterize the missing fusogens. In parallel we will determine how EFF-1 fuses, retracts and bends dendrites and other nanotubes.

Research vision in 2020

In the next decade we expect to have a much better understanding on the mechanism of FF-mediated cell membrane fusion and in addition we hope to find at least one new fusogen to help us understand how other fusion reactions occur. We also expect to find and study how other members of the FF family fuse cells in all kingdoms of life. In addition, we will determine how neurons develop elaborate branching patterns using proteins from the FF family that bend, fuse and twist dendrites to sculpt menorahs essential for mechanosensation. Since we are studying fundamental problems in life sciences, we think that in five years there will be many potential applications of our findings in biomedicine and biotechnology.

In summary, we are working on an ambitious multidisciplinary program of research that will determine the precise functions and mechanisms of cell fusion, organ formation, evolution of morphogenesis, neuronal arborization and fusion of neurons in a tractable genetic organism as well as in simpler in vitro systems. We anticipate that EFF-1 and AFF-1 expression in other heterologous systems may be used to fuse cells with potential applications for gene therapy and manipulation of stem cell fates.

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