The RhoGAP SPV-1 regulates calcium signaling to control the contractility of theC. elegansspermatheca during embryo transits

Abstract

Running head: The RhoGAP SPV---1 regulates calcium signaling.Abbreviations used: RhoGAP, GTPase activating protein toward Rho family small GTPases; ROCK, Rho---associated protein kinase; sp---ut, spermatheca uterine valve.Highlight Summary: Through in vivo imaging of the calcium sensor GCaMP, we show that the RhoGAP SPV---1 is a key regulator of calcium signaling in the C. elegans spermatheca. Our data suggests SPV---1 acts at least partially through the small GTPase CDC---42 to modulate calcium signaling, while also acting on RHO---1 to modulate Rho---ROCK signaling. This places SPV---1 as a central regulator of cellular contractility. 1 AbstractContractility of the non---muscle and smooth muscle cells that comprise biological tubing is regulated by the Rho---ROCK and calcium signaling pathways. Although many molecular details about these signaling pathways are known, less is known about how they are coordinated spatiotemporally in biological tubes. The spermatheca of the C. elegans reproductive system enables study of the signaling pathways regulating actomyosin contractility in live adult animals. The RhoGAP SPV---1 was previously identified as a negative regulator of RHO---1/Rho and spermathecal contractility. Here, we uncover a role for SPV---1 as a key regulator of calcium signaling. spv---1 mutants expressing the calcium indicator GCaMP in the spermatheca exhibit premature calcium release, elevated calcium levels, and disrupted spatial regulation of calcium signaling during spermathecal contraction. Although RHO---1 is required for spermathecal contractility, RHO---1 does not play a significant role in regulating calcium. In contrast, activation of CDC---42 recapitulates many aspects of spv---1 mutant calcium signaling. Depletion of cdc---42 by RNAi does not suppress the premature or elevated calcium signal seen in spv---1 mutants, suggesting other targets remain to be identified. Our results suggest SPV---1 works through both the Rho---ROCK and calcium signaling pathways to coordinate cellular contractility.

Redox signaling modulates Rho activity and tissue contractility in theC. elegansspermatheca

“…After a regulated period of time, the distal spermatheca contracts while the sp-ut valve relaxes, resulting in expulsion of the embryo into the uterus (McCarter et al, 1999). When this process is misregulated, embryos can become trapped in the spermatheca (Bui and Sternberg, 2002;Kariya et al, 2004;Kelley et al, 2018;Kovacevic and Cram, 2010;Kovacevic et al, 2013;Wirshing and Cram, 2017;Wirshing and Cram, 2018), or embryos can be pushed prematurely into the uterus and become misshapen or damaged (Bouffard et al, 2019;Kelley et al, 2018;Kovacevic and Cram, 2010;Norman et al, 2005;Tan and Zaidel-Bar, 2015).…”

Activation and organization of the actomyosin cytoskeleton in the C. elegans spermatheca

“…Finally, the sp-ut valve must relax, while the distal neck cells and bag cells squeeze in coordination to push the embryo in the right direction and at the right time into the uterus (Kelley et al, 2018;McCarter et al, 1999). Miscoordination of the timing or amplitude of contraction can result in embryos becoming trapped in the spermatheca, pinched or misshapen oocytes, embryo reflux back into the gonad arm, failure of eggshell formation and subsequent decreased fertility (Bouffard et al, 2019;Bui and Sternberg, 2002;Kariya et al, 2004;Kelley et al, 2018;Kovacevic and Cram, 2010;Kovacevic et al, 2013;Norman et al, 2005;Tan and Zaidel-Bar, 2015;Wirshing and Cram, 2017;Wirshing and Cram, 2018). Other biological tubes also require similar spatiotemporal control of myosin activation and actin organization.…”

“…MLCK-1 is activated by calcium indirectly through calmodulin (Hong et al, 2011). Our lab has studied how calcium waves and oscillations are regulated in amplitude and frequency, and how these oscillations are coordinated across the spermathecal tissue (Bouffard et al, 2019;Kovacevic et al, 2013). We were curious if there was any feedback of calcium-activated MLCK-1 on the calcium signal itself.…”

“…Oocyte entry into the spermatheca stretches the membrane and displaces SPV-1, allowing activation of RHO-1 and CDC-42. GTP-bound RHO-1 activates LET-502/ROCK, which activates contractility, and GTP-bound CDC-42 elevates calcium signaling through an undetermined signaling pathway (Bouffard et al, 2019;Tan and Zaidel-Bar, 2015).…”

“…After a regulated period of time, the distal spermatheca contracts while the sp-ut valve relaxes, resulting in expulsion of the embryo into the uterus (McCarter et al, 1999). When this process is misregulated, embryos can become trapped in the spermatheca (Bui and Sternberg, 2002;Kariya et al, 2004;Kelley et al, 2018;Kovacevic and Cram, 2010;Kovacevic et al, 2013;Wirshing and Cram, 2017;Wirshing and Cram, 2018), or embryos can be pushed prematurely into the uterus and become misshapen or damaged (Bouffard et al, 2019;Kelley et al, 2018;Kovacevic and Cram, 2010;Norman et al, 2005;Tan and Zaidel-Bar, 2015).…”