Spectraplakin Induces Positive Feedback between Fusogens and the Actin Cytoskeleton to Promote Cell-Cell Fusion

Article

Spectraplakin Induces Positive Feedback between Fusogens and the Actin Cytoskeleton to Promote Cell-Cell Fusion Graphical Abstract

Authors Yihong Yang, Yan Zhang, Wen-Jun Li, ..., Meng-Qiu Dong, Shanjin Huang, Guangshuo Ou

Correspondence [email protected]

In Brief Yang, Zhang et al. show that the C. elegans spectraplakin VAB-10A promotes cell-cell fusion by linking the actin cytoskeleton to the fusogen EFF-1. EFF-1, in turn, enhances the actin bundling activity of VAB-10A, creating a positive feedback loop that drives EFF-1 recruitment to fusion sites and promotes cell fusion.

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The C. elegans fusogen EFF-1 and F-actin are enriched at the cortex of fusing cells

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Spectraplakin directly links EFF-1 to F-actin and promotes cell-cell fusion

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EFF-1 enhances the F-actin bundling activity of Spectraplakin in vitro

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Spectraplakin and EFF-1 regulate actin dynamics in the cortex of fusing cells

Yang et al., 2017, Developmental Cell 41, 107–120 April 10, 2017 ª 2017 Elsevier Inc. http://dx.doi.org/10.1016/j.devcel.2017.03.006

Developmental Cell

Article Spectraplakin Induces Positive Feedback between Fusogens and the Actin Cytoskeleton to Promote Cell-Cell Fusion Yihong Yang,1,3 Yan Zhang,1,3 Wen-Jun Li,2 Yuxiang Jiang,1 Zhiwen Zhu,1 Huifang Hu,1 Wei Li,1 Jia-Wei Wu,1 Zhi-Xin Wang,1 Meng-Qiu Dong,2 Shanjin Huang,1 and Guangshuo Ou1,4,* 1Tsinghua-Peking Center for Life Sciences, School of Life Sciences and MOE Key Laboratory for Protein Science, Tsinghua University, Beijing 100084, China 2National Institute of Biological Science, Beijing 102206, China 3Co-first author 4Lead Contact *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2017.03.006

SUMMARY

Cell-cell fusion generally requires cellular fusogenic proteins and actin-propelled membrane protrusions. However, the molecular connections between fusogens and the actin cytoskeleton remain unclear. Here, we show that the Caenorhabditis elegans fusogen EFF-1 and F-actin are enriched at the cortex of the post-embryonic fusing cells, and conditional mutations of WASP and Arp2/3 delay cell-cell fusion by impairing EFF-1 localization. Our affinity purification and mass spectrometry analyses determined that an actin-binding protein, spectraplakin/VAB10A, binds to EFF-1. VAB-10A promotes cell-cell fusion by linking EFF-1 to the actin cytoskeleton. Conversely, EFF-1 enhanced the F-actin bundling activity of VAB-10A in vitro, and actin dynamics in the cortex were reduced in eff-1 or vab-10a mutants. Thus, cell-cell fusion is promoted by a positive feedback loop in which actin filaments that are crosslinked by spectraplakin to recruit fusogens to fusion sites are reinforced via fusogens, thereby increasing the probability of further fusogen accumulation to form fusion synapses.

INTRODUCTION Membrane fusion is a fundamental process required for viral entry, intracellular trafficking, and cell-cell fusion (Aguilar et al., 2013; Chen and Olson, 2005; Podbilewicz, 2014). The investigation of viral-host cell fusion and intracellular vesicle fusion has offered a wealth of information on the molecular components and regulatory mechanisms that mediate membrane fusion. For example, SNARE proteins and Rab GTPases have been identified as essential factors for tight apposition of vesicle and €dhof and Rothman, target membranes (Hong and Lev, 2014; Su 2009). Although cell-cell fusion plays critical roles in a diverse array of developmental and physiological events, including fertil-

ization, placenta formation, bone remodeling, myogenesis, and immune responses (Chen, 2011; Chen and Olson, 2005; Podbilewicz, 2014), little is known regarding how fusogens and other crucial factors coordinate to merge two apposed lipid bilayers belonging to separate plasma membranes. The Caenorhabditis elegans fusogenic proteins have been identified through genetic screens of fusion-defective mutants (Mohler et al., 2002; Sapir et al., 2007). Epithelial fusion failure 1 (EFF-1) encodes a type I single transmembrane protein that contains a glycosylated N-terminal ectodomain and an unstructured cytosolic tail (Mohler et al., 2002; Podbilewicz, 2006; Sapir et al., 2007). Recent structural studies have revealed that EFF-1 shares structural homology with viral class II fusion proteins (Pe´rez-Vargas et al., 2014; Zeev-Ben-Mordehai et al., 2014). Moreover, EFF-1 trans-trimerization on the surface of two fusion partners may bring transmembrane segments into close contact and then zip the two opposing membranes into one, analogous to the mechanism illustrated for the SNARE proteins during vesicle fusion (Pe´rez-Vargas et al., 2014; Zeev-Ben-Mordehai et al., 2014). EFF-1 is present only in nematodes, but its ectopic expression induced syncytium formation in cultured insect or mammalian cells (Avinoam and Podbilewicz, 2011; Podbilewicz, 2006; Sapir et al., 2007; Shilagardi et al., 2013). The actin cytoskeleton has been implicated in muscle cell fusion in Drosophila, zebrafish, and mice (Abmayr and Pavlath, 2012; Chen, 2011; Gruenbaum-Cohen et al., 2012; Shilagardi et al., 2013). During Drosophila myoblast fusion, the ‘‘attacking’’ fusion-competent cell forms an F-actin-enriched podosomelike structure to invade the ‘‘receiving’’ founder cell (Sens et al., 2010). The receiving partner mounts a myosin-mediated mechanosensory response and generates high mechanical tension, allowing the fusogenic synapse to overcome energy barriers for membrane apposition (Kim et al., 2015). To understand the interplay between the fusogen and the actin cytoskeleton, a reconstituted cell culture system was developed to induce ectopic cell-cell fusion by expressing the C. elegans EFF-1 in Drosophila S2R+ cells. The system mimics the asymmetric actin reorganization during Drosophila myoblast fusion and revealed that EFF-1 is enriched at the tip of invasive membrane protrusions and that the actin cytoskeleton promotes cell-cell fusion by facilitating EFF-1 engagement (Shilagardi et al., 2013). Using a functional

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EFF-1::GFP reporter and immunofluorescence, a recent study from C. elegans embryos demonstrated that EFF-1 may transiently localize at the fusing cell membranes and is otherwise largely distributed to vesicular puncta; the same study also revealed that endocytosis negatively regulates cell-cell fusion by removing EFF-1 from the plasma membrane (Smurova and Podbilewicz, 2016b). In addition, RNAi targeting the actin nucleation factor Arp2/3 complex and the actin nucleation-promoting WAVE complex in C. elegans did not perturb embryonic epidermal cell fusion (Patel et al., 2008; Xiong et al., 2011), which indicates that the actin cytoskeleton is dispensable for embryonic cellcell fusions in nematodes. The distinct observations from Drosophila S2R+ cells and C. elegans embryos may reflect the intrinsic differences between the two experimental systems: EFF-1 is overexpressed in Drosophila S2R+ cells but is present at an endogenous level in C. elegans embryos, and the hemocyte-like S2R+ cells may also behave differently from the worm epithelial cells. The C. elegans epithelial hyp7 syncytium affords an attractive system for studying cell-cell fusions in live animals. This syncytium contains 139 nuclei and is the largest C. elegans somatic cell. During morphogenesis in C. elegans embryos, cell-cell fusion merges 23 cells into hyp7, and the remaining 116 cells fuse during larval development (Podbilewicz and White, 1994; Sulston and Horvitz, 1977). At the beginning of each larval stage, the seam cell V lineages undergo a stem-like-cell division and the anterior daughter cells V.pna (V.a for short) fuse with hyp7 (Figures S1A and S1B) (Sulston and Horvitz, 1977). Although five times more cell-cell fusions occur in larvae than in embryos, the behavior of EFF-1 and the actin cytoskeleton in post-embryonic cell-cell fusions remains largely unexplored. Here, we have established a live cell imaging method to follow the seam and hyp7 cell-cell fusions in C. elegans larvae. We have constructed the EFF-1::GFP knockin nematode to study the dynamics of this fusogen at its endogenous level. We show that EFF-1 and F-actin are highly enriched at the cortex of fusing cells in larvae and that WASP-Arp2/3-dependent actin polymerization is involved in these cell-cell fusions by recruiting EFF-1 to fusion

sites. Furthermore, we have addressed the mechanistic connections between EFF-1 and the actin cytoskeleton by identifying VAB-10A, a homolog of the actin-binding protein spectraplakin, as a direct binding partner of EFF-1. Our biochemical and genetic analyses reveal that VAB-10A regulates cell-cell fusion by linking EFF-1 to the actin cytoskeleton. Our findings provide evidence of a positive feedback loop in which EFF-1, VAB-10A and the actin cytoskeleton function together to promote cell-cell fusion. RESULTS Dynamics of EFF-1 and Actin during Epithelial Seam-hyp7 Cell Fusion in C. elegans We developed fluorescent markers and live cell microscopy to characterize the cell-cell fusion process in C. elegans larvae. The seam and hyp7 cells can be individually identified using cell type-specific promoters (Cassata et al., 2005; Li et al., 2012), and cell-cell fusion is determined by the cytoplasmic exchange between the seam and hyp7 cells. We followed the dynamic change of nuclear localization signal-tagged GFP that is specifically expressed in hyp7 and showed that GFP enters seam cells from the seam-hyp7 contact surface and marks the cytosol and then the nucleus of the seam cell within 3.1 ± 1.2 min (mean ± SD, n = 13) (Figures 1A–1C). We also imaged a GFP-tagged plasma membrane marker in seam cells during the seam-hyp7 cell fusion. Because the cell volume of hyp7 is >100 times larger than that of a seam cell, GFP in the seam cell is too diluted to be visible after cell fusion. As a result, GFP fluorescence disappears along all the seam-hyp7 contact surface within 3.4 ± 0.8 min (n = 13) (Figures 1A–1C), which is similar to the time interval in which seam cells gain GFP fluorescence from hyp7 (Figure 1C). These observations suggest that the fusion pore may form rapidly along the contact surface between the seam and hyp7 cells. To visualize the dynamics of EFF-1 at its endogenous level, we used a CRISPR-Cas9-based genome-editing protocol to construct an EFF-1::GFP knockin animal (Dickinson et al., 2013). In embryos, EFF-1::GFP localized to intracellular puncta in the

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 (http://www.wormatlas.org, 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 mm. (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 mm in (A), (E) to (G), and (L). Error bars indicate the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 based on Student’s t test; n.s., not significant. See also Figure S1; Movies S2 and S3.

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(1) WT (2) wsp-1-sg (3) wsp-1-sg;Pceh-16::mwsp-1 (4) arx-2-sg (5) wve-1-sg (6) vab-10a (e698) (7) vab-10a-sg (8) Pceh-16::FBD(vab-10)::gfp OE (9) vab-10a;Pceh-16::vab-10N (10) syg-1 (11) syg-2 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 vab10a(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 mm. (legend continued on next page)

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cytoplasm of the epithelial hyp7 precursor cells and was not enriched at fusion sites (Figure S1C; Movie S1), which is consistent with the EFF-1 localization pattern that was previously reported (Smurova and Podbilewicz, 2016b). We expressed an mCherrytagged pleckstrin homology domain to label the plasma membrane of seam cells in C. elegans larvae (Figure 1E). After seam cell division in larvae, EFF-1::GFP was expressed in V.a but not in the non-fusing V.p (Figure 1E). In contrast to observations in embryos, EFF-1::GFP was enriched at the fusion sites 155 ± 20 min (n = 21) after V.a formation, and gradually reached its highest intensities before fusion pore formation (i.e., the exchange of cellular content between two fusing cells; 3.3-fold increase of EFF-1::GFP fluorescence in V.a compared with V.p; Figures 1E and S1F–S1G; Movie S2). Thus, EFF-1 was enriched at the fusion sites on the plasma membranes in larvae. To follow actin dynamics during the seam-hyp7 cell fusion, we expressed a GFP-tagged actin-binding domain (ABD) of moesin (GFP::moesinABD) or actin (actin::GFP) to show the actin cytoskeleton (Figures 1F and 1L). We revealed that the seam cell generated F-actin-enriched structures to make contact with the hyp7 cell (Figures 1D–1F and S1G; Movie S2). Our three-dimensional projection showed that the F-actin-enriched structures were assembled only on the lateral plane of seam cells and that EFF-1::GFP was also enriched only on the same plane (Figures 1J, 1K, and S1D; Movie S3), implying that some portions of the lateral plane that are enriched in F-actin and EFF-1 may be the potential fusion sites between seam cells and the hyp7 cell. Cortical F-actin was assembled in seam cells 103 ± 34 min (n = 27) before fusion pore formation and was immediately disassembled upon fusion pore opening. Intriguingly, a thin sheath of F-actin was formed at the boundary between the seam and hyp7 cells 10 ± 3 min (n = 10) after fusion pore formation (Figures S1E, S1H, and S1I; Movie S4). To examine these actin structures in the same animal, we expressed GFP::moesinABD and mCherry::PH in seam cells and actin::TagBFP in hyp7 (Figure 1L). Our triple-fluorescence live-imaging analysis showed that F-actin on the cortex of seam cells was disassembled upon fusion pore formation and that the F-actin sheath was formed 10 ± 2 min (n = 15) after fusion pore opening, confirming the sequential formation of these two types of actin structures (Figures 1L and 1M). These results suggest that F-actin at the cortex of seam cells may contribute to the initial fusion pore formation, whereas the F-actin sheath may be involved in the later event. WASP and Arp2/3 Promote Cell-Cell Fusions in C. elegans Larvae Next, we used the somatic CRISPR-Cas9 strategy to assess the role of the actin cytoskeleton in C. elegans larval cell-cell fusion. In this system, the expression of the Cas9 endonuclease is controlled by a heat-shock-inducible promoter (Shen et al., 2014) to generate conditional mutants of Arp2/3 (arx-2), WASP (wsp-1), and WAVE (wve-1) (Figure 2A). T7 endonuclease I-based assays demonstrated that these transgenic animals

produced molecular lesions with the expected sizes at the target loci of arx-2, wsp-1, and wve-1 after heat-shock induction of Cas9 expression (Figure 2B) (Shen et al., 2014). The arx-2, wsp-1, and wve-1 conditional mutant embryos and not the wild-type (WT) embryos exhibited embryonic lethality with penetrance of 85.1% (arx-2-sg, n = 275), 88.9% (wsp-1-sg, n = 256), and 78.0% (wve-1-sg, n = 180) (Figure S2A), consistent with the reported phenotypes of the RNAi animals (Patel et al., 2008). To identify the specific seam cells in which the arx-2 or wsp-1 locus was disrupted, we introduced the somatic CRISPR-Cas9 transgenes into the corresponding GFP knockin animals. The green fluorescence in the seam cells of 15% of ARX-2::GFP animals (n = 104) and 68% of GFP::WSP-1 animals (n = 107) was indistinguishable from the background fluorescence, suggesting that the target proteins were eliminated (Figures S2C–S2E). While we did not find any defects in seam cell cytokinesis in wsp-1-sg or arx-2-sg conditional mutant animals (Figure S2I), we detected reduced migration of the C. elegans Q neuroblasts in wve-1-sg or arx-2-sg conditional mutant animals but not in wsp-1-sg conditional mutant animals (Zhu et al., 2016). These results confirmed that WASP and Arp2/3 are not involved in cytokinesis and that cell migration is regulated by the Arp2/3 and WAVE complexes but not by WASP, which suggests that the phenotypes uncovered by using these conditional mutants may be specific. Together, we validated that these actin regulators were disrupted in the conditional mutant animals. Next, we measured the time interval from seam cell birth to fusion pore formation in WT and in conditional mutants. Seam cells take 219 ± 42 min (n = 53) to fuse with hyp7 after birth in WT animals; however, this process is significantly prolonged to 266 ± 32 min (n = 15) in arx-2 conditional mutants and to 365 ± 67 min (n = 11) in wsp-1 conditional mutants (Figures 2C, 2D, and S2B; Movie S5). To determine whether the delay of cell-cell fusion is caused by the loss of WASP, we performed rescue experiments. The wsp-1 gene carrying synonymous mutations at the sgRNA recognition site (mwsp-1) was introduced into wsp-1-sg conditional knockout animals; in this manner, the mwsp-1 gene abolishes the recognition of Cas9. The mwsp-1 transgene fully rescued the seam cell fusion phenotype caused by wsp-1-sg (Figures 2C–2E). In agreement with the prior finding that WAVE is dispensable for cell-cell fusion in C. elegans (Patel et al., 2008; Xiong et al., 2011), the seamhyp7 cell fusion appeared to be normal in wve-1 conditional mutants (217 ± 34 min, n = 16) (Figures 2D and S2B; Movie S5). The penetrance of the intercellular fusion phenotype in arx-2-sg mutants was subtler than that in wsp-1-sg mutants (Figure 2D). One possible reason for this result is that the somatic CRISPRCas9 method may not have completely removed Arp2/3 because of its stability (Figures S2C–S2E) (Wu et al., 2012). We next examined the actin cytoskeleton on the cortex of fusing cells in conditional mutants. Because the actin cytoskeleton, EFF-1 and the plasma membrane do not change in nonfusing V.p cells (Figure 1E), we used V.p as an internal control

(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 < 0.001 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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for quantifications. The enrichment of F-actin in V.a was defined as the GFP::moesinABD fluorescence intensity on the plasma membrane being 1.5-fold higher than that in V.p. In WT animals, F-actin was enriched 128 ± 34 min (n = 27) after birth. By contrast, the assembly of F-actin was markedly delayed to 159 ± 33 min (n = 11) in arx-2-sg and to 280 ± 49 min (n = 11) in wsp-1-sg, but it was not affected in wve-1-sg conditional mutants (Figure 2E). Although F-actin was eventually enriched after the delay, the fluorescence intensity of the ratio GFP::moesinABD in V.a was reduced from 2.8-fold in WT to 2.1-fold in arx-2-sg and to 1.7-fold in wsp-1-sg mutants (Figure 2F). These results indicate that F-actin assembly on the cortex depends on WASP and Arp2/3. Next, we followed the dynamics of actin regulators by using GFP knockin lines of ARX-2::GFP, GFP::WSP-1, and GFP::WVE-1 (Figures S2C–S2G). ARX-2::GFP and GFP::WSP-1 but not GFP::WVE-1 accumulated at the cortex (Figures S2C– S2G), and in an ARX-2::TagRFP and GFP::WSP-1 double knockin strain, ARX-2 and WSP-1 were associated with each other (Figure 3A). The time interval from the start of F-actin enrichment to fusion pore formation remained constant in WT, arx-2, and wsp-1 conditional mutants (Figure S2H), which suggests that cell-cell fusion is delayed in conditional mutant animals but proceeds normally after F-actin enrichment starts. Together, our results indicate that the WASP-Arp2/3-dependent actin polymerization promotes cell-cell fusion in C. elegans larvae. 112 Developmental Cell 41, 107–120, April 10, 2017

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(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 mm in (A) and (C). Error bars indicate the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 based on Student’s t test; n.s., not significant. n = 15. See also Movie S6.

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EFF-1 Recruitment Is Promoted by the Actin Cytoskeleton 1000 ARX-2, WSP-1, and F-actin are enriched on 500 the cortex approximately 30–50 min earlier than EFF-1 (Figures 1I and 3B), implying 0 WT wsp-1-sg vab-10a that F-actin is assembled first and subsequently recruits EFF-1 to the plasma membranes. To test this possibility, we introduced EFF-1::GFP to wsp-1-sg conditional mutants. The level of EFF-1::GFP on the plasma membrane of the anterior fusing seam cell, V.a, was approximately 2.9 times higher than that on the posterior non-fusing daughter seam cell, V.p, in WT animals; however, the loss of WASP (wsp-1-sg) clearly decreased the difference (n = 10) (Figures 3C–3E). Furthermore, we compared the EFF-1::GFP fluorescence on the plasma membrane and in the cytosol of V.a cells and found that the EFF-1::GFP level was approximately 1.4 times higher on the plasma membrane than in the cytoplasm of WT cells. In the wsp-1 conditional knockout V.a cells, the total fluorescence of EFF-1::GFP did not change compared with WT cells; however, EFF-1::GFP was more evenly distributed on the plasma membrane and in the cytosol (Figures 3F and 3G). These results indicate that WASP promotes the recruitment of EFF-1 from the cytoplasm to the plasma membrane and suggest that the interruption of EFF-1 enrichment in the plasma membranes may delay cell-cell fusion in wsp-1-sg animals. Thus, the WASP-Arp2/3-mediated actin polymerization regulates cell-cell fusion in C. elegans larvae, and we suggest that the actin cytoskeleton may facilitate the recruitment of EFF-1 to the fusing plasma membrane. n.s.

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Identification of VAB-10A as a Binding Partner of EFF-1 To understand the mechanistic connections between EFF-1 and the actin cytoskeleton, we used affinity purification and mass

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spectrometry to identify interacting proteins of EFF-1. Using an anti-GFP antibody, we purified GFP-tagged EFF-1 with its associated proteins from the lysate of transgenic C. elegans larvae that expressed EFF-1::GFP fusion protein or from the lysate of EFF-1::GFP knockin larvae (Figure S3A). Protein constituents were determined by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Figures S3B and S3C). One of the striking hits was VAB-10A, a giant cytoskeletal cross-linker with high similarity to the mammalian protein spectraplakin (Bosher et al., 2003; Ro¨per et al., 2002). In general, spectraplakins are evolutionarily conserved, multifunctional cytoskeletal proteins that associate with and coordinate F-actin, microtubules, and intermediate filaments. Because of their multiple promoters and many exons, spectraplakins consist of a vast array of differential splice forms with distinct functions (Jefferson et al., 2004; Ro¨per et al., 2002); however, the involvement of the spectraplakin family proteins in cell-cell fusion has not been reported. The C. elegans VAB-10A is essential for epidermis-extracellular matrix attachment and contains a calponin-type ABD, a plakin domain and plectin repeats, closely resembling plectin and BPAG1e (Bosher et al., 2003; Jefferson et al., 2004). We confirmed our LC-MS/MS analysis of EFF-1::GFP eluate by immunoblotting 3 3 FLAG- or mCherry-tagged VAB-10A in

eluates from anti-GFP immunoprecipitates of animals co-expressing EFFP 1::GFP with VAB-10A::3 3 FLAG or actin VAB-10A::mCherry (Figures 4A and S3D). We then determined whether GST-EFF-1C VAB-10A interacted directly with EFF-1. Our GST pull-down assays showed that the intracellular domain of EFF-1 (EFF1C, amino acids [aa]: 584–658) bound to a previously uncharacterized region between the ABD and Plakin domains of VAB-10A, which we named the fusogenbinding domain (FBD, aa: 275–533) (Figures 4B–4D). These results indicate that EFF-1 directly binds to VAB-10A and raise the possibility that VAB-10A may recruit EFF-1 to the actin cytoskeleton during cell-cell fusion.

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VAB-10A Promotes Cell-Cell Fusion by Recruiting EFF-1 We next investigated the physiological relevance of the interaction between VAB-10A and EFF-1. Considering the enormous size of VAB-10A protein (390 kDa), we generated a VAB10A::GFP knockin strain to examine VAB-10A dynamics during the seam-hyp7 cell fusion. After V cell cytokinesis, VAB-10A was distributed equally into both daughter cells (Figure 1G, registered time 0 min represents the completion of V.a cytokinesis; Movie S2). During intercellular fusion, VAB-10A was enriched in the plasma membranes and gradually reached its highest intensity in the anterior seam cell V.a before fusion pore formation (3.7-fold increase of VAB-10A::GFP; Figures 1G–1H and S1G). We did not notice any change in VAB-10A in V.p cells that did not fuse with other cells (Figure 1G). Using V.p as an internal control, we defined VAB-10A enrichment in a V.a cell as the fluorescence intensity on the plasma membrane being 1.5-fold higher than that in the sister V.p cell. Our quantifications indicated that F-actin and VAB-10A were enriched on the membrane at approximately the same time after V.a birth but earlier than EFF-1 (125 ± 40 min, VAB-10A; n = 27; Figure 1I). Developmental Cell 41, 107–120, April 10, 2017 113

Thus, VAB-10A and F-actin are first recruited to the cortex and then may recruit EFF-1 to the fusion sites. To examine whether VAB-10A regulates cell-cell fusion in C. elegans, we used the somatic CRISPR-Cas9 strategy to generate conditional mutant animals defective in VAB-10A. After validating that the mutant could disrupt the target gene (Figures 2A and 2B), we measured the time interval from seam cell birth to fusion pore formation. This process was significantly prolonged from 219 ± 42 min in WT (n = 38) animals to 330 ± 79 min (n = 18) in vab-10a conditional mutants or to 321 ± 61 min (n = 39) in a weak mutation of vab-10a(e698) (Figures 2C and 2D; Movie S5) (Bosher et al., 2003), which indicates that VAB-10A promotes cell-cell fusion. While we did not detect any delay of F-actin assembly on the cortex of the VAB-10Adeficient V.a cells, the GFP fluorescence intensity of actin filaments was reduced from 2.8-fold in WT to 1.5-fold in vab10a-sg, and to 1.4-fold in vab-10a(e698) mutants (Figures 2E and 2F), which indicates that VAB-10A regulates the cortical actin cytoskeleton. To further dissect the interaction between EFF-1 and VAB-10A, we introduced the EFF-1::GFP marker to vab-10a(e698) mutants and observed a marked reduction of EFF-1::GFP fluorescence on the plasma membrane from 3-fold in WT animals to 2.1-fold in vab-10a (Figures 3C–3G; Movie S6). Together, these results show that VAB-10A facilitates the recruitment of EFF-1 to the membranes during cellcell fusions. Considering that a complete removal of VAB-10A in the null alleles of vab-10a is embryonic lethal (Bosher et al., 2003), the 2.1-fold increase in EFF-1 fluorescence in the weak loss-of-function allele vab-10a(e698) may result from residual VAB-10A activity. Alternatively, an additional mechanism for EFF-1 membrane enrichment may be involved. We next studied whether the interaction between VAB-10A and EFF-1 is essential for cell-cell fusion. We reasoned that the FBD domain of VAB-10A may serve as a dominant-negative reagent to block the interaction between VAB-10A and EFF-1 in WT animals and thereby produce the cell-cell fusion phenotype. Indeed, overexpression of FBD in seam cells caused a significant delay of the seam-hyp7 cell fusion (Figures 2C, 2D, and S4). Furthermore, the VAB-10N fragment that links EFF-1 to the F-actin in vitro could partially rescue the cell-cell fusion phenotype in vab-10a(e698) mutant animals (Figures 2C–2F). Considering that VAB-10A (390 kDa) is a gigantic protein belonging to the Spectraplakin family of cytoskeletal scaffold proteins and that the VAB-10N fragment (148 kDa) only contains the ABD, FBD, and Plakin domains, the partial rescue result suggests that the full function of VAB-10A may require all of its domains (Figures 2C–2F). We further explored whether VAB-10A is involved in vulval cell-cell fusions that are mediated by EFF-1 in C. elegans larvae (Mohler et al., 2002). We used a TagRFP-tagged DLG-1 to mark the boundary of the vulC and vulD precursor cells in the developing vulva. In WT animals, all of the boundaries between the precursor cells of vulC and vulD completely dissolved at the L4 larval stage (Figure S5A) (Podbilewicz, 2006; Sulston and Horvitz, 1977), whereas the boundary was maintained in 38% (n = 24) of vab-10a(e698) or 100% (n = 15) of eff-1(ok1021) larvae at the same stage (Figures S5A and S5B), which indicates that the loss of VAB-10A impairs vulval cell fusions. Consistent with this model, VAB-10A and EFF-1 localize at 114 Developmental Cell 41, 107–120, April 10, 2017

the fusion sites of vulC and vulD precursor cells during fusion (Figures S5C and S5D). These results suggest that VAB-10A contributes to different types of EFF-1-dependent cell-cell fusions. EFF-1 Augments the F-Actin Bundling Activity of VAB-10A In Vitro To directly characterize the effects of VAB-10A and EFF-1 on the actin cytoskeleton in vitro, we prepared the recombinant N-terminal region of the VAB-10A protein (VAB-10N, aa: 1–1353) or other VAB-10A domains and EFF-1C (Figure 4C). Our F-actin pelleting assay showed that VAB-10A co-sedimented with actin filaments through its ABD domain and that other regions of VAB-10A or EFF-1C did not co-sediment with F-actin (Figures 4E and 4F). Importantly, EFF-1C could be copelleted by actin filaments in the presence of VAB-10N (Figure 4G), which provides direct biochemical evidence that VAB10A connects EFF-1 to the actin cytoskeleton. To further validate the co-sedimentation of EFF-1C, VAB-10N, and actin filaments in vivo, we constructed a triple-fluorescence knockin animal expressing VAB-10A::TagBFP, EFF-1::GFP, and ARX-2::TagRFP. As shown in Figure S6A, the three fluorescence makers are enriched in the cortex of the fusing V.a cells. We next determined whether VAB-10A has the capacity to stabilize actin filaments using a standard dilution-mediated actin depolymerization assay (Huang et al., 2005). Without any protection, actin filaments depolymerized into G-actin after dilution, causing a reduction in fluorescence (Figure 5A). The addition of the VAB-10N domain (aa: 1–1353), but not of EFF1C, inhibited the disassociation of the G-actin monomer from the existing F-actin in a dose-dependent manner (Figures 5A and 5B). Neither VAB-10N nor the combination of VAB-10N and EFF-1C promoted actin polymerization in vitro (Figures 5C and 5D). The biochemical results support our live-imaging observations that VAB-10A was not involved in the initial assembly of F-actin but was required for the stability of the assembled actin filaments (Figures 2C–2E). We further investigated the interaction of VAB-10A, EFF-1, and F-actin. We showed that the purified VAB-10N and EFF1C proteins co-localized along actin filaments under confocal microscopy (Figures 5E and 5F). Unexpectedly, actin filaments in vitro appeared to be more crosslinked in the presence of both EFF-1C and VAB-10N than those with VAB10N alone, which suggests that EFF-1 and VAB-10N together crosslinked actin filaments into higher-order structures (Figure 5E). We further explored the potential enhancement of EFF-1 on the ability of VAB-10A to bundle actin filaments. Under a low centrifugation force, the presence of EFF-1C increased the amount of VAB-10N-bundled actin filaments in the pellet in a dosage-dependent manner (Figure 6A; see the STAR Methods). We then used total internal reflection fluorescence microscopy to monitor the formation of F-actin bundles. In the standard F-actin bundling assay using 200 nM G-actin (Amann and Pollard, 2001), actin alone or actin in the presence of EFF-1C did not generate actin bundles, whereas VAB-10N bundled F-actin (Figures 6B–6D). Importantly, the combination of VAB-10N and EFF-1C markedly increased the F-actin bundling frequency and the number of actin filaments per bundle compared with VAB-10N alone

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(A and B) VAB-10N, but not EFF-1C, inhibits dilutionmediated 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 mM 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-VAB10N (anti-His, blue) and GST-EFF-1C (anti-GST, magenta). Scale bars, 5 mm. (F) Line scans of actin filaments, showing the VAB10N and EFF-1C intensity of the indicated segments of F-actin.

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(Figures 6B–6D; Movie S7). By measuring the length and fluorescence intensity of actin filaments at the end of the F-actin bundling assay, we showed that both parameters were significantly higher in the presence of both EFF-1C and VAB-10N than with VAB-10N alone. Together, our results indicated that EFF-1C could enhance the F-actin bundling activity of VAB-10N (Figures 6E and 6F). We next sought to understand how EFF-1 augments the F-actin bundling activity of VAB-10A. The enrichment of VAB10A at the fusion site could increase its local concentration and contributes to the augmentation by itself. Moreover, EFF1-driven fusion of cells entails trans-trimerization, such that transmembrane domains anchored in the two opposing membranes are brought into contact at the tip of the EFF-1 trimer (Pe´rez-Vargas et al., 2014; Zeev-Ben-Mordehai et al., 2014), which raises the possibility that EFF-1 may induce the dimerization of VAB-10A at fusion sites. The calponin-type ABDs were previously reported to form dimers that bundle actin filaments (Fontao et al., 2001), and dimerization of ABD dimers might further augment their F-actin bundling activity. In agreement

with this model, the GST-tag-mediated EFF-1C dimer, but not the MBP-tagged EFF-1C monomer, enhanced the F-actin bundling activity of VAB-10A (Figures 6 and S6C–S6E). Hence, EFF-1 localization at the cortex of a fusing cell involved actin polymerization and its interaction with VAB-10A. The stability of actin filaments is then dependent on VAB-10A, the F-actin bundling activity of which can be enhanced by EFF-1 that is present on the cortex. Newly synthesized EFF-1 is originally deposited randomly in the plasma membrane; if, at a certain position, WASP-Arp2/3-dependent actin nucleation happens to occur, leading to the formation of actin filaments toward the fusion site, EFF-1 is selectively recruited to this site by VAB-10A. Deposition of EFF-1 further stimulates F-actin bundling and stability through VAB-10A, leading to the formation of a fusogenic synapse. Interplay among VAB-10A, EFF-1, and F-Actin In Vivo To assess the above positive feedback circuit in live animals, we used a 1503 objective to examine actin dynamics on the cortex of eff-1(ok1021) or vab-10a(e698) mutant animals (Figures 7A, 7B, and S6B). Our kymograph analysis revealed that the number of F-actin puncta on the cortex was reduced from 10.2 ± 1.9 per cell (n = 30) in WT to 4.4 ± 1.6 (n = 30) in eff-1(ok1021) and to 4.7 ± 1.7 (n = 30) in vab-10a(e698) mutants (Figures 7C and 7D; Movie S8). Similarly, the length and growth rates of these F-actin puncta were also significantly decreased from 1.1 ± 0.2 mm and 2.0 ± 0.4 mm/min in WT (n = 30) to 0.7 ± 0.1 mm and 0.8 ± 0.3 mm/min in eff-1 (n = 30) and to 0.6 ± 0.1 mm and 0.9 ± 0.4 mm/min in vab-10a mutant cells (n = 30) (Figures 7E and 7F). These results indicate that EFF-1 and VAB-10A contribute to the length and growth of the cortical actin cytoskeleton during cell-cell fusion. Furthermore, the Developmental Cell 41, 107–120, April 10, 2017 115

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observed kinetics of the enrichment of EFF-1 and VAB-10A on the plasma membrane strongly resembled each other, and all measures of GFP fluorescence reached their highest levels before fusion pore formation (Figure 1H), suggesting that the positive feedback loop may serve as a continuous amplification mechanism that amplifies a small initial enrichment of fusogens to high dosages until the amount of fusogens is sufficient to open the fusion pore. DISCUSSION This study has identified VAB-10A as the molecular linker between a fusogen and the actin cytoskeleton. Our findings support the existence of a spectraplakin-mediated positive feedback loop in promoting cell-cell fusion. The fusogen is first recruited to the cortex by the actin cytoskeleton and spectraplakin, and then augments the local F-actin stability, increasing the probability of further fusogen accumulation to the fusion sites (Figure 7G). Two fusing cells mixed their cellular content within minutes, and the positive feedback may contribute to the quick formation of the fusion pore, which is reminiscent of the positive feedback loop at the rapid metaphase-anaphase transition (Holt et al., 2008). We have demonstrated that EFF-1 has distinct localization patterns at the embryonic and larval stages in the formation of a single epithelial syncytium. We showed that knocked in EFF-1::GFP does not accumulate at fusion sites in embryos, 116 Developmental Cell 41, 107–120, April 10, 2017

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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 VAB10N 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 mm. (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 EFF1C. Images were randomly captured under a 1003 objective. The number indicates actin bundles in the presence of VAB-10N or VAB-10N + EFF-1C. Scale bar, 10 mm. (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 R 500. Error bars indicate the mean ± SD. ***p < 0.0001, based on the Mann-Whitney U test. See also Figure S6 and Movie S7.

supporting the recent result on EFF-1 localization (Smurova and Podbilewicz, 2016b). Importantly, we revealed the previously undetected enrichment of EFF-1 and the actin cytoskeleton at fusion sites in larvae, providing evidence for EFF-1 accumulation at the fusing plasma membranes in a real physiological setting (Figure 1E). The discrepancies between different cell-cell fusion events may result from the ways in which two fusion partners make contact with each other. The larval seam and hyp7 cells establish contact only at the lateral plane, whereas the embryonic hyp7 precursor cells interact across the entire border. As a result, embryonic cell-cell fusion may require a low dosage of EFF-1 that is transiently localized at fusion sites. Moreover, tight juxtaposition of the fusing membranes may be achieved more easily in embryos because hundreds of embryonic cells are closely packed within the eggshell. By contrast, no exogenous force from the neighboring tissues is known to push the larval seam and hyp7 cells into close proximity for fusogens engagement and fusion pore formation. Consequently, seam cells may produce the cortical F-actin puncta that locally concentrate EFF-1 and push two plasma membranes together to engage the fusogens. The upstream signaling pathway that activates the WASPArp2/3 complex during the C. elegans cell-cell fusion remains unclear. A pair of immunoglobulin-domain-containing adhesion molecules, Sns and Duf, are involved in the assembly of actin filaments at the fusogenic synapse during Drosophila myoblast fusion (Sens et al., 2010; Shilagardi et al., 2013). The

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C. elegans homologs of Duf and Sns, SYG-1/Duf and SYG-2/ Sns, respectively, determine the location of specific neuronal synapses (Shen et al., 2004). We did not detect any defects of F-actin organization or cell-cell fusion during the seam-hyp7 cell fusion in syg-1(ky652) and syg-2(ky673) null mutants (Figures 2D and S2B), suggesting that different types of cell-cell fusion in different species may utilize distinct regulatory components to initiate actin polymerization. The actin cytoskeleton may play a more vital role in cell-cell fusion than is demonstrated by this study. The inhibition of WASP and Arp2/3 delayed cell-cell fusion without completely preventing it (Figures 2C and 2D), which may be the result of an incomplete depletion of WASP or Arp2/3 using conditional mutants. Because Arp2/3 is essential for cell viability (Wu et al., 2012), our phenotype quantifications were based on embryos or larvae that escaped the early lethality. Conditional mutations led to the death of 80% of larvae, in which cell-cell fusion was perhaps more severely prolonged or even blocked, as in eff-1 mutants (Figure 2C). In support of this view, the larval cell-cell fusion was further prolonged to 478 ± 116 min (n = 12) in wsp-1 and arx-2 double conditional knockouts, likely resulting

from the additive disruption of WASP and Arp2/3. The recruitment of EFF-1 to the fusion sites may be one of the functions of the actin cytoskeleton in C. elegans cell-cell fusion. In Drosophila myoblasts, F-actin-enriched podosome-like structure facilitates tight juxtaposition of the fusion plasma membrane (Kim et al., 2015; Sens et al., 2010); similarly, the cortical F-actin puncta in seam cells may help push two plasma membranes into close contact. In addition, the localization of EFF-1 in RAB-5positive early endosomes suggests that actin-dependent endocytosis may be involved in EFF-1 recycling from the plasma membrane after cell-cell fusion (Smurova and Podbilewicz, 2016a, 2016b). A truncated version of EFF-1 lacking its cytoplasmic tail was still able to mediate hypodermal cell fusion, but the course of the fusion was prolonged (Shinn-Thomas et al., 2015). This result resembles the delay of cell-cell fusion when WASP, Arp2/3, and VAB-10A were disrupted (Figures 2C–2E). Because of cell lethality in vab10a null mutant animals, this study examined the function of VAB-10A in cell-cell fusion using vab-10a weak allele or conditional mutants, which may not completely deplete the activity of VAB-10A. Overexpression of VAB-10A’s FBD delayed cell-cell fusion, suggesting that the VAB-10A-EFF-1 interaction is involved in this process. However, considering that EFF-1 can be recruited to the fusion sites in the absence of its cytoplasmic domain, alternative pathway(s) can recruit EFF-1 to the fusion sites. Developmental Cell 41, 107–120, April 10, 2017 117

This work implies that distinct cell types recruit fusogens differently. During larval cell fusion, hyp7 may use an actin-independent mechanism to recruit EFF-1. This assessment is based on the observation that F-actin in hyp7 was not assembled around fusion sites until the fusogen disappeared from the plasma membrane (Figures 1L and S1E). Consistently, ARX2::GFP was restricted in the plasma membrane of seam cells (Figure S2G). The F-actin sheath at the seam-hyp7 boundary was formed after WASP and Arp2/3 delocalized from fusion sites. Importantly, F-actin was still assembled when the Arp2/ 3 level was reduced. Other actin nucleation factors, such as formin or VASP-family proteins (Skau and Waterman, 2015), may compensate for the absence of Arp2/3 during cell-cell fusion. The F-actin sheath forms in the hyp7 cell when the intercellular junction dissolves (Mohler et al., 1998), and it remains unclear whether the actin sheath contributes to the dissolution of the junction or whether they are independent events. We cannot exclude the possibility that WASP-Arp2/3 and VAB10A may also contribute to the seam-hyp7 cell fusion from the hyp7 syncytium, because the somatic CRISPR-Cas9 technique does not enable the conditional mutation of all the copies of the target gene in this cell that contains 139 nuclei. Nevertheless, our cell biological data show that the F-actin sheath assembles after fusion pore formation in the hyp7 cell, whereas WASP-Arp2/3 and VAB-10A are enriched on the cortex of seam cells before fusion pore formation, suggesting that WASP-Arp2/3-based actin polymerization and VAB-10A may function in seam cells. The activity of fusogens needs to be precisely regulated in space and time. Various transcription factors and signaling pathways direct EFF-1 expression and ensure the fidelity of cell-cell fusion (Aguilar et al., 2013; Cassata et al., 2005; Podbilewicz, 2006). For example, transcriptional repression of eff-1 expression in V.p cells by a GATA-type transcription factor was demonstrated to restrict the production of EFF-1 in V.a but not V.p; the elt-1(RNAi) animals ectopically expressed eff-1 in V.p and caused this cell to inappropriately fuse with the hyp7 syncytium (Brabin et al., 2011). At the protein level, EFF-1 is actively removed from the plasma membrane by endocytosis (Smurova and Podbilewicz, 2016b). Although the fusogen is necessary and sufficient to mediate cell-cell fusion, our results show that spectraplakin and the actin cytoskeleton significantly accelerate this process. The modulation of spectraplakin localization and actin organization could provide additional programs to ensure the fidelity and efficiency of cell-cell fusion. Similarly, SNARE complexes can drive membrane fusion in vitro, and numerous Rab GTPases and tethering factors contribute to the high degrees of specificity, speed, and fidelity of the intracellular membrane fusion (Hong and Lev, 2014; Parlati et al., 2000; Shi et al., 2012). Based on this idea, we speculate that additional components must be involved in cell-cell fusion. The signaling pathway that stimulates the WASP-Arp2/3 complex to polymerize actin at fusion sites remains unknown. Our study has established an experimental system and techniques to isolate the missing components and to decipher their roles in cell-cell fusion protein machinery. Future studies will provide greater insight into the regulation of intercellular fusion and other biological processes, such as axon fusion, which re-establishes the connection between injured neurons via the fusogen EFF-1 (Neumann et al., 2015). 118 Developmental Cell 41, 107–120, April 10, 2017

STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d d

d

KEY RESOURCES TABLE CONTACT FOR REAGENT AND RESOURCE SHARING EXPERIMENTAL MODEL AND SUBJECT DETAILS METHOD DETAILS B Genetics and DNA Manipulations B Live Cell Imaging in C. elegans Embryo and Larvae B Immunoprecipitation in C. elegans B Mass Spectrometry Analysis B GST Protein Pull-Down Assay B Protein Preparation B F-actin Co-sedimentation Assay B Actin Polymerization and De-polymerization Assays B F-actin Binding Assays B TIRFM Assays QUANTIFICATION AND STATISTICAL ANALYSIS

SUPPLEMENTAL INFORMATION Supplemental Information includes six figures, two tables, and eight movies and can be found with this article online at http://dx.doi.org/10.1016/j. devcel.2017.03.006. AUTHOR CONTRIBUTIONS G.O., Y.Z., Y.Y., M.D., H.S., W.L., J.W., and Z.W. conceived the experiments; Y.Y., Y.Z., W.-J.L., Y.J., H.H., and Z.Z. performed the experiments; and G.O., Y.Z., Y.Y., and W.-J.L. wrote the manuscript. ACKNOWLEDGMENTS We thank Drs. J. James, E. Chen, K. Shen, R. Li, A. Chisholm, and Y. Jin for discussion or comments. This study was supported by the National Natural Science Foundation of China to Y.Y., Y.Z., G.O., and W.L. (31100972, 31501131, 31525015, 31201048, 31222035, 31101002, 31171295, and 31190063), the National Basic Research Program of China to W.L. and G.O. (973 Program, 2013CB945600, 2012CB966800, and 2012CB945002). Received: October 14, 2016 Revised: January 18, 2017 Accepted: March 10, 2017 Published: April 10, 2017 REFERENCES Abmayr, S.M., and Pavlath, G.K. (2012). Myoblast fusion: lessons from flies and mice. Development 139, 641–656. Aguilar, P.S., Baylies, M.K., Fleissner, A., Helming, L., Inoue, N., Podbilewicz, B., Wang, H., and Wong, M. (2013). Genetic basis of cell–cell fusion mechanisms. Trends Genet. 29, 427–437. Amann, K.J., and Pollard, T.D. (2001). Direct real-time observation of actin filament branching mediated by Arp2/3 complex using total internal reflection fluorescence microscopy. Proc. Natl. Acad. Sci. USA 98, 15009–15013. Avinoam, O., and Podbilewicz, B. (2011). Eukaryotic cell-cell fusion families. Curr. Top. Membr. 68, 209–234. Bosher, J.M., Hahn, B.-S., Legouis, R., Sookhareea, S., Weimer, R.M., Gansmuller, A., Chisholm, A.D., Rose, A.M., Bessereau, J.-L., and Labouesse, M. (2003). The Caenorhabditis elegans vab-10 spectraplakin isoforms protect the epidermis against internal and external forces. J. Cell Biol. 161, 757–768.

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with

enhanced

sensitivity

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STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Mouse anti-GFP

Roche

Cat# 11814460001; RRID: AB_390913

Mouse monoclonal anti-FLAG(M2)

Sigma-Aldrich

Cat# F1804; RRID: AB_262044

Mouse monoclonal anti-mCherry

EarthOx

Cat# E022110

Antibodies

Chemicals, Peptides, and Recombinant Proteins GFP-Trap_A beads

Chromoteck

GTA20

Glutathione Sepharose 4B beads

GE HealthCare

170756

Nickel-affinity chromatography

GE HealthCare

175268

amylose-affinity chromatography

New England BioLabs

E8021

cOmplete protease inhibitor

Roche

04693159001

Trypsin Gold, Mass Spectrometry Grade

Promega

V5280

T7 endonuclease I

New England BioLabs

M0302

Alexa 488-phalloidin

Invitrogen

A12379

NEM-myosin

(Zhang et al., 2010)

N/A

In-Fusion Advantage PCR Cloning Kit

Clontech

639619

Phusion High-Fidelity PCR master Mix

New England BioLabs

M0531S

Critical Commercial Assays

Experimental Models: Organisms/Strains C. elegans strains, see Table S1

This paper

Oligonucleotides Targets of CRISPR and Primers for Molecular Analysis, see Table S2.

This paper

Primers for cloning, see Table S2

This paper

Primers for PCR Products for C. elegans Transgenesis, see Table S2.

This paper

Recombinant DNA pDD162

(Dickinson et al., 2013)

Addgene, #47549

pDD162-Phsp-16.2:Cas9+PU6::Empty sgRNA

(Shen et al., 2014)

N/A

Plasmids for C. elegans Transgenesis, see Table S2.

This paper

Software and Algorithms mManager

(Chai et al., 2012)

https://www.micro-manager.org/

ImageJ

(Chai et al., 2012)

http://rsbweb.nih.gov/ij/

CONTACT FOR REAGENT AND RESOURCE SHARING Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Guangshuo Ou ([email protected]). EXPERIMENTAL MODEL AND SUBJECT DETAILS Wild-type or transgenic Caenorhabditis elegans (C. elegans) of the N2 strains were used for all experiments. Strains were maintained on the nematode growth medium (NGM) plates seeded with E. coli OP50 following standard protocols at 20 C. METHOD DETAILS Genetics and DNA Manipulations CRISPR-Cas9-assisted conditional knockout and knock-in animals were generated and analyzed as described previously (Dickinson et al., 2013; Shen et al., 2014). To construct knock-in repair template plasmids, we amplified the 1-1.5kb upstream and downstream Developmental Cell 41, 107–120.e1–e4, April 10, 2017 e1

homologous arms from the N2 genomic DNA and inserted them into pPD95.77 via In-Fusion Advantage PCR Cloning Kit (Clontech, 639619). To avoid the cleavage of the homologous repair template by Cas9, synonymous mutations were introduced to Cas9 target site of the template. The sgRNA plasmid and the knock-in repair template plasmid were co-injected into N2 animals. The knock-in worms were selected and examined by PCR and Sanger sequencing. Transgenic worms were generated by microinjection of DNA plasmids or PCR products to the germline at 10 - 50 ng/ml with co-injection markers pRF4 [rol-6 (su1006)] and Podr-1::dsRed into N2 or co-injection the unc-76-rescuing plasmid p76-16B into unc-76 (e911) animals. Our knock-in animals of EFF-1, ARX-2, WSP-1, WVE-1 and VAB-10A do not show any obvious abnormality in cell-cell fusion or the overall development of C. elegans. All the primers, plasmids, PCR products and strains were listed in Tables S1 and S2. For T7 endonuclease I (T7EI)-based assays of conditional knock-out mutants, the molecular lesions were detected according to previously described methods (Shen et al., 2014). In brief, the worms with or without heat-shock treatment were lysed and used as templates for amplifying DNA fragments containing CRISPR-Cas9 targets. PCR products were purified and digested by T7 endonuclease I (New England BioLabs, M0302) at 37 C for 30 min. The indel rate was determined by the formula 100 3 {1 – [1 – (b + c)/(a + b + c)]1/2}, where a is the intensity of intact band, and b and c are the intensities of digested small bands. For the survival rate assay in conditional knockout animals, approximately 100 eggs that were laid after 4 hours were subjected to heat-shock treatment at 33  C for 1 hour and then recovered at 20  C overnight. We counted the hatched worms at the L3 larval stage. The survival rate was calculated as the number of the hatched worms divided by the number of the original eggs. For live cell imaging of cell-cell fusions in conditional knockout larvae, eggs were subjected to heat-shock treatment at 33  C for 1 hour after laid 10 hours. Live Cell Imaging in C. elegans Embryo and Larvae C. elegans L2 larvae were anesthetized using 0.1 mmol/L levamisole in M9 buffer, and eggs or larvae were mounted on 3% agarose pads at 20  C (Chai et al., 2012). Live cell images were collected by an Axio Observer Z1 microscope (Carl Zeiss MicroImaging, Inc.) equipped with a 1003, 1.45 N.A. objective or an Olympus IX83 microscope equipped with a 1503, 1.45 N.A. oil objective, an EM CCD camera (Andor iXon+ DU-897D-C00-#BV-500), and the 405 nm, 488 nm and 568 nm lines of a Sapphire CW CDRH USB Laser System with a spinning disk confocal scan head (Yokogawa CSU-X1 Spinning Disk Unit). Time-lapse images were acquired with an exposure time of 200 msec at every 2 min for imaging the entire cell-cell fusion process. Images were acquired by mManager software (https://www.micro-manager.org/) and processed and quantified by ImageJ software (http://rsbweb.nih.gov/ij/). Immunoprecipitation in C. elegans Mix-staged C. elegans larvae were cultured at 20  C on one hundred 90-mm NGM plates seeded with OP50 bacteria. Before harvest, EFF-1::GFP expression was induced by a heat-shock treatment at 33  C for 10 min. After a 30-minute recovery at 20  C, the animals were harvested and washed with M9 buffer to yield 1–1.5 ml packed worms. 1 ml of packed worms was mixed with 1 ml of 2x lysis buffer (50 mM Tris-HCl, pH 7.4, 300 mM NaCl, 1% NP-40, 20% glycerol) and 4 ml of 0.5-mm diameter glass beads. The mixture was lysed using FastPrep-24 (MP Biomedicals) at 6.5 m/sec, 15 sec/pulse 3 5 pulses with 5-min intervals on ice. Worm lysates were then cleared by centrifugation at 14,000g for 30 min at 4  C. For anti-GFP immunoprecipitation (IP), the supernatant was incubated with GFP-Trap A beads (Chromoteck, GTA20) for 1 hr. The beads were then washed 3 times with the lysis buffer and boiled in 2x SDS loading buffer for Western blot analysis with anti-GFP antibody (Roche, 11814406001), or anti-Flag (Sigma-Aldrich, F1804) or anti-mCherry (Earthox, E022111). Alternatively, the beads were treated with Urea to elute immunoprecipitates for mass spectrometry analysis (see below) (Tao et al., 2013). Mass Spectrometry Analysis EFF-1::GFP immunoprecipitates were dissolved from the GFP-IP beads in 100 mM Tris, pH 8.5 and 8 mol/L Urea. After reduction with 5 mM TCEP and alkylation with 10 mM Iodoacetamide, the proteins were digested with trypsin at 37  C overnight. The resulting peptides were analyzed twice on a Q Exactive mass spectrometer (ThermoFisher Scientific) interfaced with an Easy-nLC1000 liquid chromatography system (ThermoFisher Scientific). Peptides were loaded on a 75 mm x 4 cm trap column packed with 10 mm, 120 A˚ ODSAQ C18 resin (YMC Co., Ltd. Kyoto, Japan) and connected to a 75 mm x 10 cm analytical column packed with 1.8 mm, 100 A˚ Luna C18 resin (Welch Materials, Shanghai, China). After desalting, peptides were separated at a flow rate of 200 nL/min with a 79-min linear gradient from 5% buffer B (100% ACN, 0.1% FA), 95% buffer A (0.1% FA) to 28% buffer B followed by a 10-min gradient to 80% buffer B, 3-min gradient to 100% buffer B and maintaining at 100% buffer B for 10 min. The top 15 most abundant precursor ions from the survey scan were selected for Higher-energy Collisional Dissociation (HCD); R = 70,000 in full scan, R = 17,500 in HCD scan; AGC targets were 3e6 for FTMS full scan, 1e5 for MS2; minimal signal threshold for MS2 = 5e4; +1 and unassigned precursors are excluded; normalized collision energy is 27 for HCD; dynamic exclusion is 30 s; ion 445.12003 is used for internal calibration. Raw data were extracted using the RawXtract software. The MS/MS spectra were searched against the C. elegans protein database using with Prolucid (Xu et al., 2015) and the search results of the two technical replicates were filtered separately using DTASelect 2 (Tabb et al., 2002) by requiring 1% FDR at the peptide level, precursor mass accuracy % 5 ppm, Z score R 4, and a minimum of one peptide for a protein ID. The FDR rates at the protein level were 0.61% and 1.76% respectively.

e2 Developmental Cell 41, 107–120.e1–e4, April 10, 2017

GST Protein Pull-Down Assay To examine the interaction between EFF-1 and VAB-10A, E. coli strain (DE3) was used to express GST, GST-EFF-1C (EFF-1 intracellular domain) and His-tagged truncations of VAB-10A. Bacterial cultures were disrupted in the lysis buffer [25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Nonidet P40, 10% glycerol]. The cleared supernatants were mixed and incubated with Glutathione Sepharose 4B beads (GE HealthCare, 170756) at 4  C for 2 hr. After three times wash with the lysis buffer, the bounded proteins were eluted with 13 SDS loading buffer. Protein Preparation Actin was purified from rabbit skeletal-muscle acetone powder by Sephacryl S-300 chromatography and stored on ice in buffer G (5 mM Tris-HCl, pH 8.0, 0.2 mM CaCl2, 0.2 mM ATP,0.5 mM DTT, and 0.01% NaN3) (Pollard, 1984; Spudich and Watt, 1971). Actin was labeled with pyrene iodoacetamide or 7-chloro-4-nitrobenzeno-2-oxa-1,3-diazole (NBD) for fluorometry assay and oregongreen-488 iodoacetamide for direct visualization of actin filaments by total internal reflection fluorescence microscopy (TIRFM) as described(Amann and Pollard, 2001). 6xHis-VAB-10A domains [Figure 4B; ABD (actin binding domain): 1-300 aa; FBD (fusogen binding domain): 275-533 aa; PLAKIN (plakin domain): 533-1353 aa; VAB-10N (containing the N-terminal ABD, FBD and plakin domains): 1-1353 aa], GST-EFF-1C and MBP2-EFF-1C were expressed in E. coli strain BL21 (DE3) by the induction with 0.3 mM IPTG at 16  C overnight. VAB-10A domains were purified with Nickel-affinity chromatography (GE Healthcare, 175268). VAB-10N was further purified by ion exchange and gel filtration chromatography (Source-15Q/15S and Superdex-200, GE Healthcare) in the presence of 5 mM CaCl2. Purification of GSTEFF-1C or MBP2-EFF-1C was performed with glutathione-affinity chromatography (GE Healthcare) or with amylose-affinity chromatography (New England Biolabs, NEB, E8021). Purified proteins were dialyzed in 5 mM Tris-HCl, pH 8.0 and flash-frozen in liquid nitrogen and stored at -80  C with 10% glycerol. F-actin Co-sedimentation Assay F-actin co-sedimentation assay were performed as previously described(Kovar et al., 2000). All the proteins, including G-actin were pre-cleared by centrifugation at 150,000g for 30 min at 4  C. F-actin (3 mM) were incubated with 1 mM His-VAB-10N or 1 mM His-ABDFBD or 1 mM His-FBD or 5 mM His-PLAKIN or 3 mM GST-EFF-1C or both 1 mM VAB-10N and different concentrations GST-EFF-1C for 20 min at 20  C. Reactions were centrifuged at 100,000g for 30 min at 4  C in the high speed co-sedimentation assay or 25,000 g for 20 min at 4  C in the low speed co-sedimentation assay. Supernatant and pellet fractions were analyzed on 12% SDS–PAGE gels by Coomassie Brilliant Blue R 250 (Sigma-Aldrich) staining and quantified by scanning densitometry using ImageJ (http://rsbweb.nih. gov/ij/; version1.38). Actin Polymerization and De-polymerization Assays NBD-actin polymerization assays were adapted from previously described protocols (Huang et al., 2005; Ojala et al., 2002). 3 mM G-actin (20% NBD-labeled) were mixed with increasing concentrations (133 nM to 2 mM) of His-VAB-10N or 532 nM His-VAB10N plus (133 nM to 1.2 mM) of GST-EFF-1C in G buffer by the addition of one-tenth volume of 103KMEI (13contains 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, and 10 mM imidazole-HCl, pH 7.0). Reactions were measured by changes in NBD fluorescence (lex = 547 nm and lem = 530 nm) using a fluorescence spectrophotometer (model Safas Xenius, Safas SA, Monaco). To test the stabilizing effect of VAB-10N or EFF-1C on F-actin, the dilution-mediated actin de-polymerization was performed according to published methods (Zhang et al., 2010). Briefly, 8 mM F-actin (100% pyrene labeled) was diluted 15-fold in G buffer with different concentrations of His-VAB-10N or GST-EFF-1C. Pyrene fluorescence was monitored at lex = 365 nm and lem = 407 nm using a fluorescence spectrophotometer. F-actin Binding Assays To visualize the binding of VAB-10N and EFF-1C to actin filaments, in vitro immunofluorescence labeling was performed as previously described (Zhu et al., 2013). Preassembled actin filaments were incubated with His-VAB-10N or GST-EFF-1C or both at 20  C for 30 min. F-actin was labelled with Alexa 488-phalloidin (Invitrogen, A12379) (1:1). The samples were mixed with rabbit anti-GST and mouse anti-His antibody (1:1000). After incubating the mixture for 15 min, Alexa 549 goat anti-rabbit IgG and Alexa 405 goat anti-mouse IgG (1:400) were added. 1-2 mL of the reaction were placed onto poly-L-Lysine-treated coverslip for visualization by an Axio Observer Z1 confocal microscope. TIRFM Assays To directly visualize of the F-actin bundling activity of VAB-10A, TIRFM (Total internal reflection fluorescence microscopy) assays were adapted from previously described (Amann and Pollard, 2001) . Briefly, 200 nM G-actin (50% Oregon-green labeled) were injected into a NEM-myosin-treated flow chamber and incubated for 10 min in dark. The flow chamber was washed by 13TIRFM buffer (10 mM imidazole, pH 7.0, 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 50 mM DTT, 0.2 mM ATP, 50 mM CaCl2, 15 mM glucose, 20 mg/mL catalase, 100 mg/mL glucose oxidase, and 0.5% methylcellulose) with or without His-VAB-10N or GST-EFF-1C or MBP2-EFF-1C. Actin filaments were observed for 5 min with an interval of 3 sec using a Photometrics cascade II 512 CCD camera (Major Instruments) with mManager software by TIRF illumination with an Olympus IX81 microscope equipped with a 1003 oil objective (1.49 N.A.). The bundling frequency of actin filaments was quantified as the number of bundle per unit area per unit time (n/mm2 min). The bundling Developmental Cell 41, 107–120.e1–e4, April 10, 2017 e3

percentage of actin filaments was quantified as the bundled actin filaments over the total number of actin filaments from the timelapse recording. The bundling events of two or three or more actin filaments were separately counted in Figure 6D. By considering a direct correlation between the fluorescence intensity of actin bundles and the number of filaments they include, F-actin length and intensity were measured from TIRFM micrographs after 40 min incubation (Hoffmann et al., 2014; Jiang et al., 2015). QUANTIFICATION AND STATISTICAL ANALYSIS The birth of seam cell V.a was defined as the completion of cytokinesis of the V cell. The fusion pore formation was determined by the loss of the fluorescence from seam cells or the entry of the fluorescence from the hyp7 cell. Because a seam cell is approximately 1/100 smaller than the hyp7 syncytium, its fluorescence was too diluted to be visible after cell-cell fusion. Fluorescence intensity of GFP-tagged EFF-1, F-actin and VAB-10A were measured on the plasma membrane of V cells (V.a and V.p). F-actin or EFF-1 or VAB10A enrichment on the plasma membrane of V.a was defined as the fluorescence intensity of V.a was 1.5-folds higher than that of V.p. All the quantifications are from the V2-V4 daughter cells, and we did not notice any significant variations in cell-cell fusions of these cells: V.2a, V.3a and V.4a respectively took 246 ± 29 min (n =10), 236 ± 47 min (n =10) and 228 ± 29 min (n =10) to fuse with the hyp7 cell after birth, respectively. Fluorescence intensity ratios of the markers were quantified as fluorescence intensity on V.a membrane divided by the one on its non-fusion sister V.p of the same animals (as shown in Figure S1F). F-actin sheath assembled time was defined as from the fusion pore formation to the fluorescence of GFP::MoesinABD or ACT::TagBFP ring is highest intensities in hyp7 cell. The duration time of the enrichment of F-actin or EFF-1 on the plasma membrane was defined as the time interval from the fluorescence enrichment to the complete loss of the fluorescence. We used the Student’s t-test to determine significant differences in cell-cell fusion between WT and mutants as indicated in the figure legends. All data are represented as mean ± SD. For quantifications of the length and average intensity of F-actin filaments or actin bundles formed in the presence of indicated proteins in vitro, we used the Mann–Whitney U-test.

e4 Developmental Cell 41, 107–120.e1–e4, April 10, 2017