Pictures were acquired using spinning disc Revolution XD confocal system (Andor) based on Nikon Eclipse Ti microscope equipped with 100/1

Pictures were acquired using spinning disc Revolution XD confocal system (Andor) based on Nikon Eclipse Ti microscope equipped with 100/1.3 PlanFluor objective, perfect focus system; CSU-X1 spinning disc (Yokogawa) and iXon3 EMCCD camera (Andor) operated by IQ2 software (Andor). enlarged area of the fusion (Figure?2E). Arrow marks the beginning of apical junction disassembly. Note the disappearance of the bright EFF-1::GFP puncta, the localization of EFF-1::GFP on the junctions and the increase of numerous small and less bright EFF-1::GFP vesicular staining. mmc4.jpg (1.0M) GUID:?915E3B4C-404E-462F-BEF9-7405B2733C7D Movie S4. Depletion Induces EFF-1 Mislocalization to the Apical Plasma Membrane and Hyperfusion, Related to Figures 2 and S2 Animated z-stack of an embryo treated with RNAi. All cells in the dorsal hypodermis are fused to each other (hyperfusion) in contrast to three unfused syncytia (and 7) in the embryos (Figure?1A). EFF-1 is expressed on plasma membrane of hypodermis syncytia. Maximum intensity projection of the dorsal side of this embryo is shown in Supplemental Figure?S2G, S2H. mmc5.jpg (829K) GUID:?8A77DCBF-6C46-49DF-A90B-332BCDBD8C40 Movie S5. Apical Membrane EFF-1 Expression and Hyperfusion Induced by RNAi, Related to Figure?2F Animated z-stack of live embryo expressing EFF-1::GFP and DLG-1::RFP after RNAi treatment. This embryo shows EFF-1::GFP expression on the apical membrane, defects in embryogenesis, and hyperfusion. mmc6.jpg (678K) GUID:?839F9A27-4A27-4820-869E-D03F40D29BEF Movie S6. RNAi Depletion Induces EFF-1::GFP Accumulation to All Surrounding Apical Membranes, Related to Figure?6 Time lapse recording of an embryo after led to the identification of the eukaryotic fusion protein (EFF-1 fusogen), which has structural homology to class II viral fusogens. Transcriptional repression of EFF-1 ensures correct fusion fates, and overexpression of EFF-1 results in embryonic lethality. EFF-1 must be expressed on the surface of both fusing cells; however, little is known regarding how cells regulate EFF-1 surface exposure. Rabbit Polyclonal to 14-3-3 zeta Here, we report that EFF-1 is actively removed from the plasma membrane of epidermal cells by dynamin- and RAB-5-dependent endocytosis and accumulates in early endosomes. EFF-1 was transiently localized to apical domains of fusion-competent cells. Effective cell-cell fusion occurred only between pairs of cell membranes in which EFF-1 localized. Downregulation of dynamin or RAB-5 caused EFF-1 mislocalization to all apical membrane domains and excessive fusion. Thus, internalization of EFF-1 is a safety mechanism Indole-3-carbinol preventing excessive cell fusion. Graphical Abstract Open in a separate window Introduction Cell-to-cell fusion initiates the process of sexual reproduction and, following fertilization, sculpts organs such as muscle, bone, eye lens, and placenta in the developing organism (Aguilar et?al., 2013). Cell fusion is also involved in inflammation, regeneration, wound healing, and cancer (Losick et?al., 2013, Medvinsky and Smith, 2003, Oren-Suissa and Podbilewicz, 2010, Rizvi et?al., 2006). Nevertheless, little is known about mechanisms that regulate cell fusion (Chen et?al., 2007, Podbilewicz, 2014). In the nematode epithelial fusion failure 1 (EFF-1), mediates fusion of cells in the hypodermis (skin), pharynx, and vulva (Mohler et?al., 2002). Ectopic expression of EFF-1 can induce fusion of cells that normally do not fuse both in and in heterologous cells grown in culture (Avinoam et?al., 2011, Podbilewicz et?al., 2006, Shemer et?al., 2004). Fusion of these cells requires EFF-1 expression in both fusing partners (Avinoam et?al., 2011, Kim et?al., 2015, Podbilewicz et?al., 2006, Shilagardi et?al., 2013). Because EFF-1 is a potent fusogen and its Indole-3-carbinol ectopic expression induces embryonic lethality, it must be regulated in space and time. Different Indole-3-carbinol genetic pathways including Engrailed/CEH-16, GATA factors, Hox, Notch, RTK, and Wnt signaling regulate embryos. EFF-1 colocalizes with RAB-5 in early endosomes before and during fusion, whereas RAB-5 depletion results in EFF-1 mislocalization to the apical plasma membrane and induces ectopic fusion. EFF-1 localization at the apical plasma membrane is dynamic and transient due to its downregulation by dynamin- and RAB-5-dependent endocytosis. Membrane merger is initiated only when both apposing apical plasma membranes co-express EFF-1. Results EFF-1 Localizes to Intracellular Puncta To uncover the expression pattern of the EFF-1 protein during Indole-3-carbinol development, its endogenous localization was followed by immunofluorescence with specific monoclonal antibodies against the extracellular domain of EFF-1 (Fridman, 2012; K.?Fridman, C..