Endocytosis regulates membrane localization and function of the fusogen EFF-1

Abstract

Cell fusion is essential for sexual reproduction and formation of muscles, bones, and placenta. Two families of cell fusion proteins (Syncytins and FFs) have been identified in eukaryotes. Syncytins have been shown to form the giant syncytial trophoblasts in the placenta. The FFs are essential to fuse cells in the skin, reproductive, excretory, digestive and nervous systems in nematodes. EFF-1 (Epithelial Fusion Failure 1), a member of the FF family, is a type I membrane glycoprotein that is essential for most cell fusions in C. elegans. The crystal structure of EFF-1 ectodomain reveals striking structural similarity to class II fusion glycoproteins from enveloped viruses (e.g. dengue and rubella) that mediate virus to cell fusion. We found EFF-1 to be present on the plasma membrane and in RAB-5-positive early endosomes, with EFF-1 recycling between these 2 cell compartments. Only when EFF-1 proteins transiently arrive to the surfaces of 2 adjacent cells do they dynamically interact in trans and mediate membrane fusion. EFF-1 is continuously internalized by receptor-mediated endocytosis via the activity of 2 small GTPases: RAB-5 and Dynamin. Here we propose a model that explains how EFF-1 endocytosis together with interactions in trans can control cell-cell fusion. Kontani et al. showed that vacuolar ATPase (vATPase) mutations result in EFF-1-dependent hyperfusion. ¹ We propose that vATPase is required for normal degradation of EFF-1. Failure to degrade EFF-1 results in delayed hyperfusion and mislocalization to organelles that appear to be recycling endosomes. EFF-1 is also required to fuse neurons as part of the repair mechanism following injury and to prune dendrites. We speculate that EFF-1 may regulate neuronal tree like structures via endocytosis. Thus, endocytosis of cell-cell fusion proteins functions to prevent merging of cells and to sculpt organs and neurons.

Keywords: AFF-1; C. elegans; Dynamin; EFF-1; RAB-5; cell-cell fusion; endocytosis; vacuolar ATPase.

Figure 1.

EFF-1 localizes to intracellular organelles in wild-type embryos. (A) Immunofluorescence using anti-EFF-1 (endogenous EFF-1, green) and anti-DLG-1 (apical junction, red) in wild-type embryo. (B) The magnification of the boxed area from (A) is showing 2 cells in the process of fusion. Endogenous EFF-1 localizes to intracellular puncta (arrowheads) and only partially on the cell membrane prior to fusion (not shown). (C) Localization of EFF-1 (green) in cells before the fusion of plasma membranes (red). (D) Anti-GFP (EFF-1*::GFP mutant, green) and anti-DLG-1 (apical junction, red) in an embryo carrying eff-1*::gfp mutant transgene. Scale bar, 20 μm. (E) The magnification of the boxed area from (D). EFF-1*::GFP shows plasma membrane mislocalization in the interface between 2 cells expressing EFF-1::GFP (arrow) and partial intracellular localization (arrowheads). (F) Localization of EFF-1*::GFP (green) in cells before fusion of plasma membranes (red).

Figure 2.

Model of epidermal cell fusion regulation in C. elegans. Cytoplasms and plasma membranes of 2 cells that are ready to fuse are colored in light and dark orange and blue. (A) EFF-1 monomers produced by an orange cell are orange, and EFF-1 monomers synthesized by a blue cell are blue. EFF-1 is delivered to the plasma membranes from both fusion-competent cells in a monomeric state. (B) Fusion is initiated by EFF-1 trans-oligomerization (hetero-dimers). (C) Formation of EFF-1 trans-oligomers (hetero-trimers). (D) Conformational change of EFF-1 trimers induces fusion of opposing membranes and mixing of the cytoplasms. (E) EFF-1 in a mixture of monomers and oligomers in a postfusion state is actively removed from the cell surface via receptor-mediated endocytosis in a dynamin- and RAB-5-dependent mechanism and EFF-1 accumulates in early endosomes. RAB-5 is represented by green, and dynamin in magenta.

Figure 3.

EFF-1 protomer showing the location of the 2 point mutations in EFF-1*. EFF-1 structural domains DI (red), DII (yellow), and DIII (blue) and the positions of T173A and N529D mutations on the protomer of the EFF-1 trimer. Stem region (magenta) connects DIII to the transmembrane domain (green). N529D is located in a region of the stem that is unstructured in the crystal. T173A is in the CD loop.