Localization Dynamics and Cytoskeletal Dependence of the EFF-1 Fusogen in Caenorhabditis elegans Embryos

Developmental cell fusion in Caenorhabditis elegans has been studied as a model system for understanding basic processes that drive cell-cell fusion in general. In this species two distinct genes encoding bona fide fusogens have been identified; aff-1 and eff-1. These discoveries are important advancements in the field, although very little is known about the mechanisms of action involved in cell-cell fusion induced by these molecules. ^ Experimental efforts to understand the regulation of EFF-1 at the subcellular level produced an EFF-1::GFP translational fusion protein. Expression of EFF-1::GFP in C. elegans embryos resulted in a striking pattern of dynamic localization of fusogen to the junctions of fusion fated cells during epidermal morphogenesis. This observation along with supporting results from the study of EFF-1 activity in worm embryos prompted a paradigm of regulation of fusogenic activity by specific localization. Based on this system of precise localization of EFF-1::GFP to cell junctions two hypotheses have been proposed and tested in this thesis. ^ Utilizing quantitative fluorescence imaging methods analysis of the localization pathway and lateral mobility of EFF-1::GFP in developing embryos was performed. The role of the cytoskeleton in EFF-1::GFP dynamics was investigated by pharmacological manipulation of vital embryos producing results indicative of a cytoskeletal-independent pathway of fusogen localization. The lateral mobility of EFF-1::GFP localized to contacts of fusion-fated epidermal cells was quantified by a FRAP method yielding results indicative a species possessive of a high diffusion coefficient within a population exhibiting low mobile fraction. Evaluation of these microscopic studies has compelled the consideration of EFF-1 localization to sites of action by a \u27diffusion to a trap\u27 process. ^ A separate imaging-based study conducted on the EFF-1 mechanism reported in this thesis regards the actin cytoskeleton in cell fusion. Investigators intend to determine the exact role of actin in cell-cell fusions that occur in disparate model systems. Results produced by various methods have been incongruent with respect to the specific role of actin in membrane fusion; positive or negative. Fusion assays were conducted in worm embryos treated with an actin inhibitor producing results suggestive of a stage-dependent requirement for actin.