1. Volume 41, Issue 1, Pages 107-120.e4 (April 2017)
Spectraplakin Induces Positive Feedback between Fusogens and the Actin Cytoskeleton to Promote Cell-Cell Fusion 
Yihong Yang, Yan Zhang, Wen-Jun Li, Yuxiang Jiang, Zhiwen Zhu, Huifang Hu, Wei Li, Jia-Wei Wu, Zhi-Xin Wang, Meng-Qiu Dong, Shanjin Huang, Guangshuo Ou 
Developmental Cell 
Volume 41, Issue 1, Pages e4 (April 2017)
DOI: /j.devcel
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2. Developmental Cell 2017 41, 107-120. e4DOI: (10. 1016/j. devcel. 2017
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3. Figure 1
Dynamics of EFF-1 and the Actin Cytoskeleton during the Epithelial seam-hyp7 Cell Fusion in C. elegans Larvae
(A) Time-lapse images of nuclear localization signal (NLS) in the hyp7 cell (upper) and the plasma membrane in seam cells during seam-hyp7 cell fusion (bottom). 0 min, fusion pore formation. The white arrowheads show the fusing cells. The yellow asterisks show the non-fusing seam cells. A schematic cartoon is shown on the left.
(B) GFP fluorescence intensity ratio of the cytoplasm between V.a and hyp7 (left) or of the plasma membrane between V.a and V.p (right).
(C) Quantification of the diffusion time of NLS-GFP in hyp7 or of GFP-tagged membrane and histone in seam cells. n = 13. SE, seam cells.
(D) Schematics of intercellular fusion between the anterior daughter cell of the seam cell (V.a) and the hyp7 cell. 0 min: the birth of V.a or the completion of V cell cytokinesis.
(E–G) Fluorescence time-lapse images of GFP-tagged EFF-1 (E), F-actin (F), and VAB-10A (G) during the seam-hyp7 cell fusion. Asterisks, V.a.
(H and I) Quantification of the EFF-1 and VAB-10A fluorescence ratios during seam-hyp7 cell fusion. Birth: the completion of V cell cytokinesis; enrichment in (H) and (I): the outline fluorescence intensity on the V.a membrane is >1.5 times higher than that of the sister non-fusing V.p cell; fusion: fusion pore formation indicated by the loss of mCherry::PH fluorescence in V.a. The fluorescence intensity ratio is quantified between V.a and V.p. n = 15. (I) Quantification of F-actin, VAB-10A, and EFF-1 enrichment times after V cell cytokinesis. n = 11–27.
(J) A cross-sectional view of seam cells (red), the hyp7 cell (blue), and F-actin (green) in a larva. D, dorsal; V, ventral; L, left; R, right. Based on the Worm Atlas ( chapter: Epithelial System Seam Cells).
(K) Left: still images of GFP-tagged moesinABD and mCherry-tagged plasma membrane in the seam cell. Upper, lateral view; bottom, cross-sectional view. Schematic cartoon is shown on the right. Arrowhead shows the enrichment of F-actin in cross-sectional view. Scale bar, 5 μm.
(L) Time-lapse images of F-actin (GFP::moesinABD) and mCherry::PH in seam cell, and the F-actin sheath (TagBFP::actin) in hyp7 cell. 0 min, fusion pore formation.
(M) Duration time of EFF-1 and F-actin in hyp7 or seam cell on the cell cortex. n = 11–30.
Scale bars, 5 μm in (A), (E) to (G), and (L). Error bars indicate the mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < based on Student's t test; n.s., not significant. See also Figure S1; Movies S2 and S3.
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4. Figure 2 WASP and Arp2/3 Promote Cell-Cell Fusion in C. elegans Larvae
(A) Gene models of wsp-1, arx-2, wve-1, and vab-10a. Exons are in blue, and red arrows indicate sgRNA sites. The green arrow indicates a mutant site of vab-10a(e698).
(B) Representative gels of the T7E1 assay for wsp-1-sg, arx-2-sg, wve-1-sg, and vab-10a-sg. Asterisk, non-specific band.
(C) Inverted fluorescence time-lapse images of F-actin (GFP::moesinABD) in a seam cell from the birth of V.a (0 min, the completion of V cell cytokinesis) to fusion pore formation (red asterisks, the loss of F-actin fluorescence in V.a) in WT and mutant animals (sg: conditional mutations generated using somatically expressed CRISPR-Cas9). Scale bar, 5 μm.
(D) Quantification of the time interval from V.a birth to V.a-hyp7 cell fusion.
(E) Quantification of the time interval from V.a birth to F-actin enrichment on the plasma membrane.
(F) Fluorescence intensity ratio of GFP-labeled moesinABD between V.a and V.p at 10 min before cell fusion.
Error bars indicate the mean ± SD. ∗p < 0.05, ∗∗∗p < based on Student's t test; n.s., not significant; n = 11–27.
See also Figures S2, S4, and S5; Movie S5.
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5. Figure 3 WASP and Arp2/3 Facilitate EFF-1 Localization
(A) Fluorescence time-lapse images of GFP::WSP-1 knockin and ARX-2::TagRFP knockin. 0 min, the completion of V cell cytokinesis. White asterisks, V.a.
(B) Quantification of the ARX-2, WSP-1, F-actin, and EFF-1 enrichment formation time. n = 10–28.
(C) Fluorescence time-lapse images of EFF-1::GFP and mCherry::PH during the seam-hyp7 cell fusion in WT and mutant animals.
(D) Outline fluorescence intensity of EFF-1::GFP and mCherry::PH on the V.a seam cell membrane. In (C) and (D), 0 min, fusion pore formation.
(E) Quantification of the fluorescence intensity ratio of EFF-1 and PH between V.a and V.p.
(F) Quantification of the ratio of EFF-1::GFP fluorescence intensity between the plasma membrane and cytoplasm.
(G) Quantification of the fluorescence intensity of EFF-1::GFP in V.a.
Scale bars, 5 μm in (A) and (C). Error bars indicate the mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < based on Student's t test; n.s., not significant. n = 15.
See also Movie S6.
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6. Figure 4 Identification of VAB-10A as a Binding Partner of EFF-1
(A) EFF-1 immunoprecipitates VAB-10A in C. elegans. HS+, heat-shock treatment induced the expression of EFF-1::GFP.
(B) Schematics of the full-length protein or the truncated domains of VAB-10A. ABD, actin-binding domain; FBD, fusogen-binding domain.
(C) SDS-PAGE analysis of recombinant His-VAB-10A domains and GST-EFF-1C (intracellular domain).
(D) Interaction between EFF-1C (intracellular domain) and VAB-10A domains in a GST fusion protein pull-down assay.
(E and F) High-speed co-sedimentation assays for the binding activities of VAB-10A domains (E) and EFF-1C (F) with actin filaments.
(G) EFF-1C was co-sedimented with F-actin in the presence of VAB-10N in a high-speed co-sedimentation assay.
See also Figure S3.
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7. Figure 5
VAB-10A Stabilizes F-Actin and Co-localizes with EFF-1C along Actin Filaments
(A and B) VAB-10N, but not EFF-1C, inhibits dilution-mediated actin depolymerization. Actin depolymerization was monitored by tracing the changes in pyrene fluorescence.
(C and D) The NBD fluorescence assay for actin polymerization shows that actin polymerization is not stimulated by VAB-10N or EFF-1C. Reactions contain 3 μM actin (20% NBD labeled) and VAB-10N and EFF-1C at the indicated concentrations.
(E) VAB-10N co-localized with EFF-1C along actin bundles. F-actin was stained with Alexa Fluor 488 Phalloidin in the absence or presence of His-VAB-10N (anti-His, blue) and GST-EFF-1C (anti-GST, magenta). Scale bars, 5 μm.
(F) Line scans of actin filaments, showing the VAB-10N and EFF-1C intensity of the indicated segments of F-actin.
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8. Figure 6 EFF-1C Enhances the F-Actin Bundling Activity of VAB-10A
(A) Low-speed co-sedimentation assays of F-actin bundles showed that EFF-1C promotes F-actin bundling activity of VAB-10A in a dose-dependent manner. Supernatant (S) and pellet (P) samples were analyzed using SDS-PAGE followed by Coomassie blue staining. Quantification is shown on the top. n = 3.
(B) Dynamics of F-actin bundle formation with VAB-10N or EFF-1C observed by time-lapse total internal reflection fluorescence microscopy (TIRFM). Two or three F-actin structures coalesced in a zipper-like fashion and attached to one another in the presence of VAB-10N (300 nM) or VAB-10N and EFF-1C (300 nM). The bundling events are highlighted with dotted lines. Arrows denote the growing ends of actin filaments. Scale bar, 2.5 μm.
(C and D) Quantifications of (B) by measuring the F-actin bundling frequency (C) and the percentage of F-actin filaments in bundles (D). ∗∗p < 0.01 based on Student's t test; n.s., not significant. n > 50.
(E) Representative TIRFM images of actin filaments in the presence or absence of VAB-10N and EFF-1C. Images were randomly captured under a 100× objective. The number indicates actin bundles in the presence of VAB-10N or VAB-10N + EFF-1C. Scale bar, 10 μm.
(F) Quantifications of the length (top) and average intensity (bottom) of F-actin filaments or actin bundles formed in the presence of indicated proteins as in (E). n ≥ 500. Error bars indicate the mean ± SD. ∗∗∗p < , based on the Mann-Whitney U test.
See also Figure S6 and Movie S7.
Developmental Cell  , e4DOI: ( /j.devcel )
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9. Figure 7
EFF-1 and VAB-10A Promote F-Actin Stability during Cell-Cell Fusion
(A) F-actin (GFP::moesinABD) in WT, vab-10a, and eff-1 mutants. Top, fluorescence images of the V.a cell; the areas indicated by yellow dotted boxes are enlarged at the bottom. Arrowheads indicate F-actin enrichment at membrane protrusions. The green dotted lines (50 s) indicate the location where the kymographs of actin dynamics in (C) were generated. Scale bars, 5 μm (top); 2.5 μm (bottom).
(B) GFP::moesinABD fluorescence intensity ratio between the plasma membrane and cytoplasm. A schematic of the measurement is shown in Figure S6B.
(C) Kymographs and corresponding lines of F-actin dynamics in WT and mutants. Horizontal bar, 1.2 μm; vertical bar, 100 s.
(D–F) Quantifications of F-actin protrusion numbers (D), protrusion length (E), and protrusion growth rate (F) in the V.a cell in WT and mutant animals. n = 30; error bars indicate the mean ± SD. ∗∗∗p < based on Student's t test.
(G) The proposed model showing the sequential events of actin assembly, EFF-1 recruitment, and plasma membrane fusion, as well as a proposed positive feedback loop in which VAB-10A, EFF-1, and F-actin promote cell-cell fusion. Question marks indicate the unknown issues of cell-cell fusions, including the signaling pathway that activates the WASP-Arp2/3 complex and the formation of the F-actin sheath in the hyp7 cell that forms after fusion pores open.
See also Movie S8.
Developmental Cell  , e4DOI: ( /j.devcel )
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