טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentAvinoam Ori
SubjectConserved Eukaryotic Fusogens Can Fuse Viral Envelopes to
Cells
DepartmentDepartment of Biology
Supervisor Professor Benjamin Podbilewicz
Full Thesis textFull thesis text - English Version


Abstract

Cell to cell fusion is a ubiquitous and evolutionary conserved process that is essential for reproduction and development in most eukaryotes. However, the majority of proteins that mediate this process (i.e. fusogens) are unknown or uncharacterized. The main objective of this work was to understand how cells fuse by characterizing fusogens (AFF-1 and EFF-1) in the nematode Caenorhabditis elegans, as paradigms of eukaryotic fusion machineries. To show that AFF-1 and EFF-1 are functional paralogs in C. elegans I studied the activity of AFF-1 in vivo and in tissue culture cells. I showed that AFF-1 is sufficient for cell fusion in vivo and in Baby Hamster Kidney cells. Furthermore, I showed that AFF-1 is required in the two fusing cells and that expression of AFF-1 on the surface of pseudotyped vesicular stomatitis virus results in AFF-dependent homotypic virus-cell fusion. Moreover, I identified functional orthologs of CeFFs in other organisms and showed that candidates from three organisms can mediate cell-cell fusion of tissue culture cells. This work suggests that CeFFs are the first members of a family of conserved fusogens and that homotypic interactions may be a common principle in the molecular mechanism of FF mediated fusion. Furthermore, it suggests that FF proteins can function as minimal fusion machineries and do not require additional co-factors to mediate membrane fusion of viruses and cells. To study the mechanism of FF mediated fusion I used computational homology modeling to predict the structure of AFF-1. Based on this analysis I hypothesized that FF proteins share a similar structure with a previously described class of viral fusogens (Class II). To test whether the fusion mechanism of CeFFs is also similar to class II viral fusogens I performed structure-function analysis. I found that despite of their similar structures the fusion mechanism diverged considerably. This work is significant first because it identifies the first family of eukaryotic proteins that form a unique minimal fusion machinery that can mediate homotypic cell fusion of viruses and cells in the absence of additional cellular co-factors. Secondly because it will help identify the missing fusogens of muscles, macrophages and gametes with possible implications in muscular dystrophies, stem cell trans-differentiation, infertility and cancer metastasis. Lastly, this work uncovers similarities between viral and cellular fusogens that suggest they descended from a common ancestor, challenging the current models of protein mediated membrane fusion.