Discover the mechanism of cell fusion:
We identified EFF-1 and AFF-1, two novel type I membrane proteins essential for developmental cell fusion in C. elegans. EFF-1 and AFF-1 are the founders of the first family (FF) of developmental cell fusion proteins (fusogens). EFF-1 and AFF-1 from nematodes and other species can fuse heterologous insect and mammalian cells 6. EFF-1 and AFF-1 are required in both fusing cells and the process is via hemifusion. We have purified and determined the three-dimensional structure of EFF-1 protein in collaboration with Felix Rey (Pasteur Institute, Paris) . We are testing its fusogenic activities in cells, pseudotyped viruses and in reconstituted liposomes. Our ultimate goal is to understand the molecular and physicochemical mechanisms of cell membrane fusion .
Find the sperm-egg fusion proteins (fusogens) in C. elegans:
Although gamete fusion is a fundamental process in almost all kingdoms of life, we still do not know which molecules (fusogens) fuse the membranes of gametes to form zygotes. To identify candidate proteins expressed in oocyte and sperm that can potentially act as the gamete fusogens, we will use several complementing approaches, such as bioinformatics, genetics and proteomics. Our hypothesis is that sperm-egg fusion involves heterotypic interactions of fusogens and their receptors that are essential and sufficient for cell fusion. After identifying candidate fusogens, we will test whether the proteins are (1) necessary for cell fusion (2) localize at the fusion site (3) able to mediate fusion in vivo and (4) of heterologous cells, to ascertain true fusogens have been discovered, rather than proteins involved in adhesion or other aspects of fertilization (Fig.1). If successful, our approaches can be applied to identify sperm-egg fusogens in other organisms.
Identify the myoblast fusogens in mammals:
EFF-1 and AFF-1 can fuse epithelial and myoepithelial cells in C. elegans, in heterologous Sf9 insect cells and in BHK hamster cells. This is a proof of principle that will allow us to test potential fusogens involved in mammalian myoblast fusion [5,10,11]. While candidates for muscle fusogens exist in Drosophila and vertebrates [1,12,13] none of these candidates has been shown to be both essential and sufficient for the cell membrane fusion process. Instead, the many genes involved in muscle cell fusion may be acting in earlier stages in the process that include: cell cycle arrest, recognition, alignment and adhesion (Fig. 1). We use a molecular genetic approach to identify the mammalian myoblast fusogen using expression of candidate genes in BHK cells and complementation of a C. elegans eff-1 deletion mutant with cross species expression of mouse cDNAs expressed during muscle formation. The approach and rationale is novel, risky and with extremely high potential of making a very important discovery. We use C. elegans eff-1 mutants and assays developed to study cell fusogens form nematodes and other species to fish out genes that should exist in mammals but that have never been proven to exist. This work is done in close collaboration with the lab of Leonid Chernomordik (NIH, Bethesda). Thus, we propose to find the Holy Grail of mammalian myoblast fusion. We estimate that this project, if successful, will bring a very important contribution to the muscle fusion field with potential applications in basic and applied biomedical sciences. This research is high risk, high gain with potential of bringing a major breakthrough. Positive results will probably give us the opportunity in the future to develop novel methods for gene therapy and may allow to fuse stem cells to muscles.
How cell fusion is regulated:
We have accomplished a complete description of the cellular events leading to the formation of an organ . Using genetic analyses we identify genes that function in different cell fusion events in C. elegans and in other organisms and how this process is regulated in development. In particular we are looking for posttranslational regulation of EFF-1 activities. Cellular fusion proteins require tight regulation to prevent excess fusion in developing animals. EFF-1 is a conserved fusion protein that is essential and sufficient for fusion during organ formation in C. elegans. We are studying EFF-1-mediated epidermal cell fusion using confocal live imaging and superresolution microscopy. We recently found that EFF-1 expression on the plasma membrane is transient and EFF-1 is actively removed from the plasma membrane of fusion-fated cells by RAB-5-mediated endocytosis that requires the small GTPase dynamin. In wild type embryos EFF-1 accumulates in RAB-5 positive early endosomes. We found that the cytoplasmic tail and the immunoglobulin domain of EFF-1 protein are necessary for EFF-1 trafficking. When RAB-5 activity is reduced, EFF-1 mislocalizes to apical domains of the plasma membranes and induces excess fusion thus contributing to embryonic lethality. Thus, RAB-5 controls dynamin-dependent endocytosis and sorting of EFF-1 cell fusion protein in C. elegans embryos. We expect that additional layers of regulation will control EFF-1 and AFF-1 activities.
Evolution of cell fusion and organogenesis:
We study cellular events during morphogenesis of the vulva across species [15-17]. We found that changes in the direction of cell divisions can result in differences in size and shape of the vulva. We found that evolution of most vulval characters are biased and proposed that evolution of the vulva in nematodes is governed by selection and/or selection-independent constraints and not by stochastic processes .
How neurons fuse and the mechanisms of dendritic arborization:
We discovered that EFF-1 is also required to sculpt complex neuronal trees required for sensing strong mechanical stimuli. We found that EFF-1 trims abnormal or excessive neuronal branches as a novel quality control mechanism. EFF-1 works in specific neurons by fusing excess and abnormal neuronal branches. In addition, EFF-1 retracts branches [19,20]. We have identified other genes that participate in the generation and maintenance of complex dendritic trees and we hope that our discoveries in C. elegans may help to understand and repair degenerative diseases of the nervous system and accidental breaking of neurons.