WASP-Arp2/3-dependent actin polymerization influences fusogen localization during cell-cell fusion in Caenorhabditiselegans embryos

Abstract

Cell-cell fusion is essential for development and physiology. Actin polymerization was implicated in the Caenorhabditiselegans fusogen EFF-1 engagement in a reconstituted Drosophila cell culture system, and the actin-binding protein spectraplakin links EFF-1 to the actin cytoskeleton and promotes cell-cell fusions in C. elegans larvae. However, it remains unclear whether and how fusogens and the actin cytoskeleton are coordinated in C. elegans embryos. Here, we used live imaging analysis of GFP knock-in and RNAi embryos to study the embryonic cell-cell fusions in C. elegans Our results show that the inhibition of WASP-Arp2/3-dependent actin polymerization delays cell-cell fusions. EFF-1 is primarily distributed in intracellular vesicles in embryonic fusing cells, and we find that the perturbation of actin polymerization reduces the number of EFF-1-postive vesicles. Thus, the actin cytoskeleton differently promotes cell-cell fusion by regulating fusogen localization to the fusing plasma membrane in larvae or to intracellular vesicles in embryos.

Keywords: Actin polymerization; Arp2/3; Caenorhabditis elegans; Cell-cell fusion; Fusogen.

Fig. 1.

The WASP-Arp2/3 promotes cell-cell fusion in C. elegans embryos. (A) Schematics of the embryonic epithelial cell fusion from the comma stage (395 min after the first cleavage, left) to twofold stage (450 min, right). Dorsal hypodermal cells 1-17 (green) and several ventral cells fuse to form the main body epithelial syncytium of hyp7. Based on Podbilewicz and White (1994). (B) Inverted fluorescence time-lapse images of the dorsal hypodermal cell fusions from comma (0 min) to twofold stage in WT, RNAi embryos or eff-1(ok1021) embryos. Cell boundaries were labeled with DLG-1::TagRFP. Arrowheads indicate the hyp7 precursor cell borders, and 15 borders exist in the comma stage. Scale bar: 10 μm. See Movie 1 for the entire series. (C) Quantification of the dorsal hypodermal cell number from comma to twofold stage; n=15-20 for each measurement. (D) Quantification of the dorsal hypodermal cell number at twofold stage. Data are mean±s.e.m.; **P<0.01, ***P<0.001 based on Student's t-test; n=15-20 for each measurement.

Fig. 2.

ARX-2 partially associates with EFF-1 during embryonic cell-cell fusions. (A) EFF-1::GFP puncta associate with ARX-2::TagRFP puncta in the two fusing hyp7 precursor cells. Areas in the rectangles are enlarged on the right. Scale bars: 5 μm. See Movie 2. (B) Fluorescence time-lapse images of EFF-1 and ARX-2 puncta (from A). Scale bar: 2 μm. Kymograph (right) of EFF-1::GFP and ARX::TagRFP motility. Horizontal bar, 2 μm; vertical bar, 10 s. n>5. (C) Ratio of EFF-1 puncta (>0.25 μm²) that colocalize with ARX-2 puncta. The number of EFF-1 puncta that colocalized with ARX-2 puncta was divided by the total number of EFF-1 puncta. Data are mean±s.e.m.; n=12. (D) Fluorescence time-lapse images of ARX-2::TagRFP knock-in and the plasma membrane (GFP::MIG-2). The right panels show the magnified region of the inset area. Arrows indicate disassembly of the plasma membrane. 0 min, fusion pore formation. Scale bars: 5 μm. See Movie 3 for the entire series.

Fig. 3.

WASP and Arp2/3 regulate vesicular localization of EFF-1. (A) Live images of EFF-1::GFP and the plasma membrane (labeled with TagRFP::MIG-2) in WT and RNAi embryos. Each bottom panel represents the magnified region of the top inset area with 90° rotation. The dorsal hypodermal hyp7 cell is outlined with a dotted line. Scale bars: 5 μm. (B) The density of EFF-1 puncta (>0.25 μm²) in hyp7 precursor cells of WT and RNAi embryos. Data are mean±s.d. **P<0.01, ***P<0.001 based on Student's t-test; n=10. (C) Quantification of the amount fluorescence intensity of EFF-1::GFP in embryos. Data are mean±s.d.; no significance based on Student's t-test; n=15. (D) Proposed models of the coordination between F-actin and EFF-1 during the C. elegans embryonic hyp7 precursor cell fusion.