Functional analysis of EFF-1: A novel protein necessary and sufficient for somatic cell fusion in the nematode C. elegans
Date of Completion
January 2005
Keywords
Biology, Genetics
Degree
Ph.D.
Abstract
Cell fusion is the biological process underlying the merger of cells into giant syncytia. Though cell fusion is fundamental in nature, the mechanism remains a mystery. We found that the protein EFF-1 (Epithelial Fusion Failure) is necessary and sufficient for fusion of somatic cells in C. elegans . EFF-1 is a predicted type I integral membrane glycoprotein. BLAST searches indicate that EFF-1 is not a member of any known protein family. Mutations within the extracellular domain of EFF-1 do not seem to affect prefusion events (differentiation, adhesion, etc.). However, weak and null eff-1 mutants fail to fuse cells of the hypodermis, vulva, pharynx and lateral seam tissues. Remarkably, eff-1 null mutants are fertile, demonstrating that the fusion of gametes requires alternative fusion machinery. ^ I employed two approaches to deduce the functional domains and motifs of EFF-1. (1) I changed putative structural and functional domains (phospholipase A2 active site, putative fusion peptide), via site directed mutagenesis and tested each mutant for rescue of eff-1 mutant worms. (2) I isolated several new alleles by random EMS mutagenesis, including several predicted eff-1 null mutations. ^ Observation of GFP-tagged EFF-1 localization was performed in vivo. EFF-1::GFP expression precedes embryonic cell fusion events, and the labeled protein localizes dynamically and specifically to fusion competent cell contacts. Mutant EFF-1::GFP constructs show atypical localization. Several lines of evidence, both in vivo (EFF-1::GFP localization) and in vitro (bead aggregation assay), suggest that EFF-1 has an affinity for itself. ^ I have also tested EFF-1 sufficiency in Drosophila and mammalian cell culture. Though in vitro expression of eff-1 in either case, has not generated cell fusions, I show here that in vivo expression in Drosophila is sufficient to fuse cells. ^ Based on these data, I propose a model for EFF-1 mediated cell fusion such that EFF-1 is targeted in a homotypic manner to the apical membrane moments before fusion occurs. EFF-1 molecules from each neighboring cell may undergo a series of intermediates steps before initiating a viral-like fusion pore. ^
Recommended Citation
del Campo, Jacob John, "Functional analysis of EFF-1: A novel protein necessary and sufficient for somatic cell fusion in the nematode C. elegans" (2005). Doctoral Dissertations. AAI3180196.
https://digitalcommons.lib.uconn.edu/dissertations/AAI3180196