Title

Sufficiency and Regulation of the C. elegans Family of Developmental Cellular Fusogens

Date of Completion

January 2010

Keywords

Biology, Cell|Health Sciences, Human Development

Degree

Ph.D.

Abstract

Regulatory and biophysical mechanisms of cell-cell fusion are largely unknown, despite the fundamental requirement for fused cells in eukaryotic development. Only two cellular fusogens that are not of obvious viral origin have been identified to date, both in nematodes. EFF-1 and AFF-1 comprise the FF family and together they are necessary for virtually all somatic cell fusions in C. elegans. Unregulated EFF-1 expression causes lethality due to ectopic fusion between cells not developmentally programmed to fuse, highlighting the necessity of tight fusogen regulation for proper development. ^ Identifying factors that regulate EFF-1 and its paralog AFF-1 could lead to discovery of molecular mechanisms that control cell fusion upstream of the action of a membrane fusogen. Yet, FF proteins are sufficient to fuse heterologous invertebrate cell types, suggesting that they function autonomously in foreign molecular and proteomic environments. The work presented here examines whether a predicted 14-3-3 binding interaction with EFF-1A's cytoplasmic domain regulates its function and localization in hypodermal cells (hyp7). This thesis also explores the extent of FF protein functionality in an unfamiliar environment, mammalian cells that may not harbor conserved invertebrate co-factors.^ Experimental results show that membrane fusion activity appears to proceed independent of 14-3-3 binding to EFF-1. However, the EFF-1A C-terminal domain and its predicted phosphopeptide sites are required for correct morphologically significant cell fusions in vivo. Despite this requirement, the phosphorylation of these sites does not impact the localization of the fusogen. While these results suggest that FF proteins may utilize self-regulatory mechanisms rather than accessory molecules, FF protein induction in CHO-Kt mammalian cells in vitro does not produce appreciable cell fusions, suggesting that these developmental fusogens require factors not present in this mammalian cell type. Together, these results have given increased insight into FF protein domain requirements and have established the means to assess robustly the plausibility of a cell culture FF-fusion assay.^