RSU-1 Maintains Integrity of Caenorhabditis elegans Vulval Muscles by Regulating α-Actinin

Egg-laying behavior in Caenorhabditis elegans is a well-known model for investigating fundamental cellular processes. In egg-laying, muscle contraction is the relaxation of the vulval muscle to extrude eggs from the vulva. Unlike skeletal muscle, vulval muscle lacks visible striations of the sarcomere. Therefore, vulval muscle must counteract the mechanical stress, caused by egg extrusion and body movement, from inducing cell-shape distortion by maintaining its cytoskeletal integrity. However, the underlying mechanisms that regulate the cellular integrity in vulval muscles remain unclear. Here, we demonstrate that C. elegans egg-laying requires proper vulval muscle 1 (vm1), in which the actin bundle organization of vm1 muscles is regulated by Ras suppressor protein 1 (RSU-1). In the loss of RSU-1, as well as RasLET-60 overactivation, blister-like membrane protrusions and disorganized actin bundles were observed in the vm1 muscles. Moreover, RasLET-60 depletion diminished the defected actin-bundles in rsu-1 mutant. These results reveal the genetic interaction of RSU-1 and RasLET-60 in vivo. In addition, our results further demonstrated that the fifth to seventh leucine-rich region of RSU-1 is required to promote actin-bundling protein, α-actinin, for actin bundle stabilization in the vm1 muscles. This expands our understanding of the molecular mechanisms of actin bundle organization in a specialized smooth muscle.


a-actinin
Egg-laying Ras RSU-1 Vulval muscle cells Hermaphrodite Caenorhabditis elegans fertilize their oocytes with their own sperms and stores 10 to 15 fertilized eggs in the uterus, after which the eggs are expelled into the environment through the vulva in every 20 min (Waggoner et al. 1998). Although this egg-laying behavior has been studied intensively to understand the function and development of the nervous system (Schafer 2006), studying the development of vulval muscle cells also helps in elucidating diverse fundamental cellular mechanisms, including cell migration, adhesion, and signal transduction (Bastiani et al. 2003). Animals harbor two types of musclesstriated somatic muscle and nonstriated smooth muscle. Whereas somatic muscle consists of multiple sarcomeres, which are functional contractile units featuring a specialized organization of the M-line, dense body (Z-disk), actin filaments, and myosin filaments (Moerman and Williams 2006), smooth muscle lacks these highly organized sarcomeres. In C. elegans, body-wall muscle is striated muscle (Gieseler et al. 2018), while pharyngeal, vulval, uterine, anal, and intestinal muscle are nonstriated smooth muscle. Proper egg-laying in C. elegans requires smooth muscle contraction and relaxation. Vulval muscle cells function in regulating the opening of the vulva. Two types of vulval muscle cells have been identified in C. elegans, vm1 and vm2. The four vm2 1986). Conversely, the four vm1s display a similar pattern as vm2s and are electrically coupled to vm2s, but the vm1s do not receive any synaptic input (White et al. 1986). Therefore, vm1s might facilitate the vulval opening process in a neuronal circuit-independent manner.
The proteins that localize at the dense body of sarcomeres are equivalent to the proteins in the integrin-mediated cell-adhesion complex. Integrins, which are transmembrane heterodimeric receptors highly conserved from nematodes to mammals, facilitate the mechanical linkage of the extracellular matrix to the cytoskeleton (Brown 2000;Hynes 2002;Huveneers et al. 2008). Integrin-mediated signaling pathways regulate several cellular processes, including cell adhesion, migration, proliferation, and differentiation (Schwartz and Ginsberg 2002;Huveneers et al. 2007;Humphries et al. 2019). Integrins (e.g., PAT-2 and PAT-3 in C. elegans) consist of a and b subunits, each containing a large extracellular domain, a single transmembrane domain, and a short cytoplasmic tail. Once activated to the extended-open conformation, Integrin PAT-3 recruits integrin-linked kinase (ILK  ), particularly interesting Cys-His-rich protein (PINCH UNC-97 ), and parvin  to form the IPP (Integrin-PINCH-Parvin) complex (Tu et al. 1999;Zhang et al. 2002;Wu 2004;Legate et al. 2006;Wickström et al. 2010). The IPP complex regulates cell adhesion, F-actin polarization, vascular smooth muscle contractility, and microtubule organization and dynamics (Wu 2004;Legate et al. 2006). Loss of Integrin PAT-3 , ILK PAT-4 , and PINCH UNC-97 in C. elegans causes Pat (Paralyzed and Arrested elongation at Twofold) and Unc (uncoordinated) phenotypes (Williams and Waterston 1994;Gettner et al. 1995;Mackinnon et al. 2002), which are related to the dysfunction of muscular tissues.
Besides the key components of the IPP complex, RSU-1, a Ras suppressor, localizes at focal adhesion sites. RSU-1 coimmunoprecipitates with the PINCH-ILK complex through its interaction with the LIM5 domain of PINCH UNC-97 (Kadrmas et al. 2004). RSU-1 is a 33-kD protein with 7 leucine-rich repeat (LRR)-containing domains. Human cell lines lacking RSU-1 are viable, but the proliferation and migration of the cells are markedly compromised (Gonzalez-Nieves et al. 2013); conversely, the overexpression of RSU-1 disrupts actin-filament networks (Masuelli and Cutler 1996). Moreover, RSU-1 might function in the absence of PINCH-ILK functions. In Drosophila exhibiting PINCH-ILK interaction deficiency, RSU-1 is essential for viability (Elias et al. 2012); RSU-1 participates in regulating the p38 MAP kinase signaling pathway (Gonzalez-Nieves et al. 2013); and RSU-1 promotes the localization of extrasynaptic acetylcholine receptors (AChRs) at the neuromuscular junctions (NMJs) of dorsal and ventral SAB motor neurons (Pierron et al. 2016). These findings indicate that RSU-1 potentially participates in multiple cellular processes, and the underlying molecular mechanisms on how RSU-1 suppress Ras is largely unclear.
Here, we report that RSU-1 functions in concert with Ras LET-60 to regulate actin bundling in the vulval muscle cells of C. elegans. We demonstrated that egg-laying ability was substantially reduced in a rsu-1 null mutant and a Ras LET-60 overactivated mutant. By performing genetic investigations and live imaging of C. elegans vulval muscle cells, we further demonstrated that RSU-1 and proper levels of Ras LET-60 are required for maintaining normal cellular structure and actin bundling in vm1 vulval muscles; the data from our genetic analyses also indicated that RSU-1 inhibits the activity of Ras LET-60 . In addition, by rescue experiments, we demonstrated that the fifth to seventh leucine-rich domain of RSU-1 is required to regulate Ras  . Lastly, we showed that a-actinin (ATN-1) acts as a downstream effector in RSU-1-mediated signaling to promote F-actin bundling. Based on our findings, we propose that RSU-1 inhibits Ras LET-60 activity to regulate proper actin bundling in C. elegans vulval muscle.

MATERIALS AND METHODS
C. elegans strains All nematode strains were cultured at 22°on NGM (nematode growth medium) plates seeded with Escherichia coli OP50. All strains used in this study are listed in Table S1.

RNAi
RNAi experiments were performed using the feeding method (Timmons and Fire 1998). We obtained let-60(RNAi) clones from Julie Ahringer's library (Kamath and Ahringer 2003), and we constructed rsu-1(RNAi) and atn-1(RNAi) by inserting the corresponding DNA fragments into L4440 vectors. RNAi plasmids were transformed into bacteria HT115. Primers used for DNA amplification are listed in Table S2. RNAi feeding plates were prepared using NGM containing 100 mg/ml ampicillin and 1 mM isopropylb-thiogalactoside. HT115 carrying RNAi plasmids were grown in 5 ml of LB containing 100 mg/ml ampicillin at 37°overnight and then seeded on the RNAi plates at room temperature for 10 h. Embryos were hatched on the RNAi plates, and then at the L4 stage, were transferred to new RNAi plates for 12 h and used for confocal imaging.

Egg-laying assays
The average numbers of laid eggs were quantified as described (Trent et al. 1983): N2 worms in L4 stage-30 each from control and rsu-1(tm6690) strains-were treated with 100 ml of serotonin (3 mg/ml) for 60 min in each condition, and then the eggs expelled from each worm were counted. The developmental stages of the eggs were examined using confocal imaging. Whole-worm images were acquired using a Nikon A1R confocal microscope equipped with a 40·/1.4 numerical aperture (NA) objective. Images were processed using ImageJ.

Fluorescence imaging
Young adult C. elegans hermaphrodites were selected for fluorescence imaging; the worms were anesthetized with 0.5% tetramisole on a 5%agarose pad placed on a glass slide and then were turned over to the ventrolateral position. GFP was visualized using a 488-nm laser, whereas mKate and mCherry were visualized using a 561-nm laser. To obtain images of vulval muscle and the actin filaments in vulval muscle, Z-stack images were acquired using either a Nikon A1R confocal microscope equipped with a 100·/1.4 NA oil-immersion lens and photomultiplier tube (PMT) detector, or an Olympus IX83 spinning-disk confocal microscope equipped with a 60·/1.4 NA objective and a charge-coupled device (CCD) camera. Images were processed using ImageJ.

Phalloidin staining
To visualize actin filaments in vulval muscle, young adult worms were stained with iFlour 555-conjugated phalloidin (Shanghai YEASEN Biotechnology; CAT: 40737ES75). Worms were fixed with 3% formaldehyde for 3 h at room temperature, washed thrice with phosphatebuffered saline (PBS), and treated with 100% acetone at -20°for 5 min. Next, the worms were washed with PBS and stained with iFlour 555phalloidin (0.1 mg/ml) overnight at 4°in the dark, and after rinsing with PBS, the stained worms were examined using a Nikon A1R confocal microscope equipped with a 100·/1.4 NA objective.

TEM analysis
Worms were washed with M9 buffer, fixed overnight with the primary fixative solution (0.5% glutaraldehyde and 1.5% paraformaldehyde), and then incubated with the secondary solution (2% osmium tetroxide) for 4 h at room temperature. After washing with distilled water, the samples were dehydrated using 25%, 50%, 75%, 90%, and 100% acetone, infiltrated with spur resin at 25%, 50%, 75%, and 100%, and then embedded in a plastic capsule at 60°for 2 days. Samples were sectioned at 60-nm thickness, and the thin sections were treated with uranyl acetate and lead citrate before examination by using a Hitachi HT-7700 TEM instrument.

Image analysis and quantification
To examine actin filaments in vulval muscle cells ( Figure 4A, Figure  5B & E), Z-stack images (0.6-mm intervals) were acquired on a Nikon A1R confocal microscope equipped with a 100·/1.4 NA objective. The images in different focal planes were projected (maximum projection) for further analysis in ImageJ. The midline of actin bundles was determined using the "freehand section tool" in ImageJ.
For fluorescence-intensity analysis, images of mCherry::RSU-1, GFP::PINCH UNC-97 , and Integrin PAT-3 ::GFP were obtained using a spinning-disk confocal microscope equipped with a 60·/1.4 NA objective ( Figure 2D). The intensities of the cytoplasm, focal adhesion patches, and adhesion sites were analyzed using ImageJ, and the C/F ratios were calculated using this formula: C/F = (Intensity cytoplasmic -Intensity background )/(Intensity focal adhesion -Intensity background ). The intensities measured in different groups were compared using two-tailed t-tests in GraphPad Prism.
To analyze a-actinin ATN-1 ::GFP patches in vulval muscle ( Figure  5D & E), Z-stack images of a-actinin ATN-1 ::GFP were again acquired using the spinning-disk confocal microscope equipped with the 60·/ 1.4 NA objective. The images in different focal planes were projected (maximum projection) for further analysis in ImageJ. The areas of a-actinin ATN-1 ::GFP patches were measured using the "analyze particles tool" in ImageJ. Data from different groups were compared using two-tailed t-tests in GraphPad Prism.

Data availability
All strains used in this work are available upon request or at the Caenorhabditis Genetics Center (CGC). All data to support the conclusions of this article are present in figures and tables. Supplemental material available at figshare: https://doi.org/10.25387/g3.12319226.
Next, we wonder whether the egg-laying circuits is compromised in rsu-1(RNAi) and rsu-1 mutant worms. Therefore, we stimulated adult worms to lay eggs by applying the monoamine neurotransmitter serotonin. If RSU-1 acts in the egg-laying circuits, similar amount of laid-eggs will be obtained in the control, rsu-1(RNAi), and rsu-1(tm6690) adult worms after treating with serotonin. Otherwise, less embryos will be expelled in rsu-1(RNAi) or rsu-1(tm6690). In control adult worms treated with serotonin, 15 6 2 eggs were expelled in 1 h. However, this rate decreased to 11 6 1 egg in rsu-1(RNAi) and it was almost halved in rsu-1(tm6690), only obtained 8 6 2 eggs per hour ( Figure 1D). These results demonstrated that even the egg-laying circuit was stimulated by serotonin, the number of eggs laid in rsu-1(RNAi) and rsu-1(tm6690) adult worms was less than control worms. Collectively, these data suggest that the egg-laying is impaired in rsu-1 null mutant worms.

RSU-1 expresses in vm1 muscles
Considering our finding that RSU-1 contributes to the egg-laying behavior, we further suspected that RSU-1 acts in the vulval muscles, and thereby regulates the vulval opening. First, the subcellular localization of RSU-1 in vulval muscles was examined. We generated a transgenic fluorescent C. elegans strain by using the CRISPR/Cas9 genome-editing technique to insert the mCherry-coding DNA sequence into the 5ʹ-end of endogenous rsu-1 on Chromosome III. Whole-worm imaging revealed that mCherry::RSU-1 was primarily expressed in body-wall muscles and vulval muscles (Figure 2A and B). Then, the subcellular localization of RSU-1 in vulval muscles was further examined by high resolution confocal imaging. We observed that mCherry::RSU-1 were expressed in vm1 muscles. They were primarily accumulated around the vulva, forming large patch in each vm1 muscles ( Figure 2C, asterisks), and localized as small puncta throughout the vm1 muscles ( Figure 2C, arrowheads). These mCherry::RSU-1-labeled patches and puncta were colocalized with Integrin PAT-3 ::GFP, and GFP::PINCH UNC-97 ( Figure 2C).
We also noted that mCherry::RSU-1 appeared to have a higher cytosolic fluorescence signal in vm1 muscles as compared to Integrin PAT-3 ::GFP and GFP::PINCH UNC-97 ( Figure 2D). Therefore, we speculated that an alternative pool of RSU-1 might be diffusely distributed throughout the cytosol in vm1 muscles. To assess this, we measured the average fluorescence intensity in the cytosol and at adhesion sites in vm1 muscles, and then calculated the cytosol-to-adhesion-site intensity ratio (Intensity cytoplasmic -Intensity background )/(Intensity focal adhesion -Intensity background ; C/F ratio) ( Figure 2E). The C/F ratios in vm1 muscles expressing mCherry::RSU-1 was significantly higher than that in Integrin PAT-3 ::GFP and GFP::PINCH UNC-97 ( Figure 2F). Altogether, these results suggest that majority of RSU-1 localizes to the focal adhesion sites, while some of them are present in the cytosol of vm1 muscles.
RSU-1 regulates Ras LET-60 to maintain the cellular structure of vm1 muscles Given that RSU-1 expresses in vm1 muscles, we further investigate the function of RSU-1 by examining the morphology of vm1 muscles in control, rsu-1(RNAi), and rsu-1(tm6690) worms. When cytosolic GFP expression is driven by the vm1 muscle-specific egl-15 promoter (Pegl-15::GFP), the morphology of vm1 muscles can be visualized by using confocal imaging. In controls, the 4 vm1 muscles were rod-like in shape and displayed straight and smooth cell boundaries, and cells were oriented toward each other around the vulva and formed a cross shape (Figure 2A, Figure 3A). By contrast, $50% of rsu-1(RNAi) and rsu-1(tm6690) vm1 muscles were irregular in shape and harbored blister-like protrusions ( Figure 3A and B). These results indicate that RSU-1 is required for cell morphology control.

RSU-1 promotes actin-bundle organization
Blebs are generally generated in cells with abnormal cortical cytoskeleton (Gagliardi et al. 2015;Caswell and Zech 2018), therefore we further speculate that RSU-1 stabilizes the cytoskeleton in vm1 muscles to prevent deformation. To test this, we fixed adult worms and stained actin filaments with iFlour 555-phalloidin for actin bundle analysis. In control worms, most of the actin bundles in vm1 muscles were straight and parallel ( Figure 4A). By contrast, curved actin bundles were observed in rsu-1(tm6690) vm1 muscles, let-60(n1700) vm1 muscles, and let-60(n1046) vm1 muscles ( Figure 4A). Notably, in agreement with the results of vm1 muscle-cell morphological analysis, the disorganization of actin bundles was suppressed in let-60(RNAi);rsu-1(tm6690) vm1 muscles. We also examined the actin bundles in fixed rsu-1(tm6690) by using transmission electron microscopy (TEM): whereas straight and parallel actin filaments were detected in control vm1 muscles, curved actin filaments with irregular empty spaces between actin filaments were observed in rsu-1(tm6690) vm1 muscles ( Figure 4B). More importantly, in live-worm confocal imaging, the disorganization of actin bundles and membrane blebbing were also observed in the rsu-1(RNAi) vm1 muscles expressing LifeAct::mKate and Pegl-15::GFP, which were used for (respectively) labeling actin filaments and visualizing the morphology of vm1 muscles ( Figure 4C). These live-imaging data verified the reliability of the data from the analysis of fixed samples stained with phalloidin. Collectively, our findings demonstrate that RSU-1 regulates actin-bundle organization.
RSU-1 regulates the subcellular localization of a-actinin ATN-1 Actin bundles are stabilized by the interaction between many crosslinking proteins with actin filaments, forming a higher-order actin filaments structures in cells (Bartles 2000;Pollard 2016). To investigate how RSU-1 regulates actin bundling, we examined the cellular morphology of vm1 muscles in the depletion of actinbundling proteins. After screening of several candidates by RNAi (Table S3), we observed that loss of a-actinin ATN-1 leads to membrane blebbing ( Figure 5A). Furthermore, the blister-like protrusions were detected in both rsu-1(tm6690);atn-1(RNAi) and let-60(n1046);atn-1(RNAi) vm1 muscles ( Figure 5A). These results indicate that a-actinin ATN-1 is required to maintain proper cellular shape in vm1 muscle, and that the protein might function downstream of RSU-1 and Ras  .
Lastly, we analyzed the structure of actin bundles in vm1 muscles. Curved actin bundles were observed in atn-1(RNAi) and atn-1(ok84) cells ( Figure 5B), which resembled the phenotype in RSU-1 mutant vm1 muscles ( Figure 4A). We also examined the subcellular localization of a-actinin ATN-1 by expressing vm1 tissue-specific a-actinin ATN-1 ::GFP, which revealed that a-actinin ATN-1 ::GFP patches of different sizes were formed and were localized at the interspace between the actin bundles in vm1 muscles ( Figure 5C). Notably, these patches were fewer and smaller in rsu-1(RNAi) or let-60(n1046) vm1 muscle cells than control cells, while the patches in let-60(n1046);let-60(RNAi) were similar to control cells ( Figure 5D and E). These results demonstrated that RSU-1 regulates a-actinin ATN-1 subcellular localization through Ras LET-60 .

RSU-1 functions independently of the focal adhesion pathway
Body-wall muscles are striated muscles that consist of multiple sarcomeres (Gieseler et al. 2018), whereas nonstriated smooth muscles, like vulval muscles in C. elegans, lack highly organized sarcomeres. The two major components of sarcomeres are M-lines and dense bodies, both of which are composed of several proteins associated with integrin, a transmembrane receptor that facilitates cell-extracellular matrix adhesion (Mackinnon et al. 2002;Lin et al. 2003). Previous work and our study have shown that RSU-1 is mainly expressed in body-wall muscles and colocalizes with Integrin PAT-3 -localized structures, but RSU-1 is not required for the integrity of M-lines and dense bodies in body wall muscle (Fig. S4A & B) (Pierron et al. 2016). Concurrently, in vm1 muscles, our results also demonstrated that although the majority of RSU-1 is localized at focal adhesion sites, these sites remain intact in the loss of RSU-1 (Fig. S5A & B). Altogether, these findings suggest that RSU-1 may not function in the focal adhesion or it acts in parallel with an unknown protein in the downstream signaling of the focal adhesion pathway.
Intriguingly, in vm1 muscles, we identified blister-like protrusions containing defected actin bundles in rsu-1(RNAi) and rsu-1(tm6690) mutant worms (discussed below) but they were eliminated in the loss of Ras LET-60 . Furthermore, we observed that RSU-1 was diffusely distributed in the cytoplasm of vm1 muscles. Therefore, based on the results of our genetic studies and subcellular-localization analysis, we propose that the cytosolic RSU-1 performs a cellular function that is independent of the integrin-mediated adhesion pathway in vm1 muscles. In human cells, RSU-1 was reported to regulate p38 activity in a PINCH UNC-97 -independent manner (Gonzalez-Nieves et al. 2013), and was further reported to promote the viability of the Drosophila larva in a PINCH UNC-97 -ILK PAT-4 interaction-defective mutant (Elias et al. 2012). Thus, it would be of interest to comprehensively investigate the molecular functions of RSU-1 in cellular processes.
RSU-1 regulates the cellular structure of vm1 muscles Cytoplasmic protrusions that perform specialized cellular functions, such as lamellipodia, filopodia (Mogilner and Rubinstein 2005), cytonemes and tunneling nanotubes (Buszczak et al. 2016), display specific cytoskeletal arrangements. However, dysfunctional cytoplasmic protrusions, named blebs or blister-like protrusions, have been observed in apoptotic cells (Sgonc and Gruber 1998), injured cells (Malorni et al. 1991), and tumor cells (Sahai and Marshall 2003). These blebs are generally generated by the actomyosin contraction that squeezes the cytosol outward at sites exhibiting local delamination of the cortical cytoskeleton (Caswell and Zech 2018). In this study, blister-like protrusions were observed in vm1 muscles lacking RSU-1 ( Figure 3A) or a-actinin ATN-1 ( Figure 5A). Unexpectedly, highly curved actin bundles, visualized by means of phalloidin staining or LifeAct::mKate labeling, were concomitantly detected in the cells under these conditions. Therefore, although we did not examine the dynamics of the cortical cytoskeleton, we suggest that these disorganized actin bundles could explain the bleb formation observed in vm1 muscles. Moreover, the blister-like protrusions were mainly detected in adult worms and not in L4-stage worms (Fig. S6). This further suggests that RSU-1 might not regulate the early development of vulval muscle but might contribute to the stabilization of actin bundles and thus enable the cells to withstand the mechanical stresses generated by muscle contraction during body locomotion and egg-laying.

RSU-1 promotes actin-bundle stabilization
Actin filaments assemble into higher-order crosslinked bundles and thereby regulate cellular shape and cell adhesion (Bartles 2000). The actin-bundling process is mediated by actin-bundling proteins, including vinculin, fascin (Winkelman et al. 2016), and a-actinin (Bartles 2000). In human cells, actin-bundling proteins are closely colocalized with actin bundles (Hoffmann et al. 2014), although these proteins might form aggregates in certain specific cell types, such as in transformed rat kidney cells (Stickel and Wang 1987). Here, we also showed that in vm1 muscles, a-actinin colocalizes with rod-shaped patches located between well-defined actin bundles ( Figure 5C), and further that a-actinin is required for maintaining the cellular structure and actin-bundle organization in vm1 muscles ( Figure 5A and B). These results suggest that the a-actinin-containing patches hold actin bundles tightly together to prevent actin-bundle distortion.
In summary, our results demonstrated that RSU-1 depletion, Ras LET-60 overactivation, and a-actinin ATN-1 depletion lead to the formation of blister-like protrusions in vm1 muscles. Furthermore, the results of confocal imaging and TEM analyses showed that the actin-bundle integrity in vm1 muscles was compromised under these three conditions. Consequently, egg-laying ability was diminished in the worms in which vm1 muscles harbored the protrusions. Our genetic studies further revealed that RSU-1 regulates actin-bundle stabilization by inhibiting a-actinin ATN-1 subcellular localization through Ras LET-60 ( Figure 6D). Although the mechanism by which RSU-1 inhibits Ras LET-60 remains unclear, our data in the rescue experiments indicate that the fifth to seventh LRR-containing domains (LRR5-7) are critical for the inhibition. Therefore, the binding of RSU-1 and Ras LET-60 need to investigate further. Nevertheless, the interaction of a LRR protein, FLI-1, with Ras was reported in C. elegans during embryonic cytokinesis and germline development (Deng et al. 2007;Lu et al. 2008). In addition, another LRR protein, SOC-2 also directly interact with Ras and Raf to positively modulate Ras pathway in C. elegans (Sieburth et al. 1998;Selfors et al. 1998). Besides, previous studies have shown that a-actinin ATN-1 is activated by PIP2 binding and forms an antiparallel dimer (Ribeiro et al. 2014), and that Ras LET-60 activates PI3K to phosphorylate PIP2 into PIP3 (Kölsch et al. 2008). Therefore, we cannot rule out the possibility that the inhibition might not be simply due to a direct protein-protein interaction: RSU-1 might inhibit Ras LET-60 -dependent activation of PIP2 phosphorylation, and this, in turn, might promote the activation of a-actinin ATN-1 for actin-bundle stabilization. This poorly understood mechanism underlying actin organization warrants further investigation.