Gα/GSA-1 works upstream of PKA/KIN-1 to regulate calcium signaling and contractility in the Caenorhabditis elegans spermatheca.

Correct regulation of cell contractility is critical for the function of many biological systems. The reproductive system of the hermaphroditic nematode C. elegans contains a contractile tube of myoepithelial cells known as the spermatheca, which stores sperm and is the site of oocyte fertilization. Regulated contraction of the spermatheca pushes the embryo into the uterus. Cell contractility in the spermatheca is dependent on actin and myosin and is regulated, in part, by Ca2+ signaling through the phospholipase PLC-1, which mediates Ca2+ release from the endoplasmic reticulum. Here, we describe a novel role for GSA-1/Gαs, and protein kinase A, composed of the catalytic subunit KIN-1/PKA-C and the regulatory subunit KIN-2/PKA-R, in the regulation of Ca2+ release and contractility in the C. elegans spermatheca. Without GSA-1/Gαs or KIN-1/PKA-C, Ca2+ is not released, and oocytes become trapped in the spermatheca. Conversely, when PKA is activated through either a gain of function allele in GSA-1 (GSA-1(GF)) or by depletion of KIN-2/PKA-R, the transit times and total numbers, although not frequencies, of Ca2+ pulses are increased, and Ca2+ propagates across the spermatheca even in the absence of oocyte entry. In the spermathecal-uterine valve, loss of GSA-1/Gαs or KIN-1/PKA-C results in sustained, high levels of Ca2+ and a loss of coordination between the spermathecal bag and sp-ut valve. Additionally, we show that depleting phosphodiesterase PDE-6 levels alters contractility and Ca2+ dynamics in the spermatheca, and that the GPB-1 and GPB-2 Gß subunits play a central role in regulating spermathecal contractility and Ca2+ signaling. This work identifies a signaling network in which Ca2+ and cAMP pathways work together to coordinate spermathecal contractions for successful ovulations.

The RhoGAP SPV-1 regulates calcium signaling to control the contractility of the Caenorhabditis elegans spermatheca during embryo transits.

Contractility of the nonmuscle and smooth muscle cells that comprise biological tubing is regulated by the Rho-ROCK (Rho-associated protein kinase) 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 Caenorhabditis elegans reproductive system enables study of the signaling pathways regulating actomyosin contractility in live adult animals. The RhoGAP (GTPase--activating protein toward Rho family small GTPases) 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 RNA interference does not suppress the premature or elevated calcium signal seen in spv-1 mutants, suggesting other targets remain to be identified. Our results suggest that SPV-1 works through both the Rho-ROCK and calcium signaling pathways to coordinate cellular contractility.

Regulating distal tip cell migration in space and time.

Gonad morphogenesis in the nematode C. elegans is guided by two leader cells, the distal tip cells (DTC). The DTCs migrate along a stereotyped path, executing two 90° turns before stopping at the midpoint of the animal. This migratory path determines the double-U shape of the adult gonad, therefore, the path taken by the DTCs can be inferred from the final shape of the organ. In this review, we focus on the mechanism by which the DTC executes the first 90° turn from the ventral to dorsal side of the animal, and how it finds its correct stopping place at the midpoint of the animal. We discuss the role of heterochronic genes in coordinating DTC migration with larval development, the role of feedback loops and miRNA regulation in phenotypic robustness, and the role of RNA binding proteins in the cessation of DTC migration.

CACN-1 is required in the Caenorhabditis elegans somatic gonad for proper oocyte development.

CACN-1/Cactin is a conserved protein identified in a genome-wide screen for genes that regulate distal tip cell migration in the nematode Caenorhabditis elegans. In addition to possessing distal tip cells that migrate past their correct stopping point, animals depleted of cacn-1 are sterile. In this study, we show that CACN-1 is needed in the soma for proper germ line development and maturation. When CACN-1 is depleted, sheath cells are absent and/or abnormal. When sheath cells are absent, hermaphrodites produce sperm, but do not switch appropriately to oocyte production. When sheath cells are abnormal, some oocytes develop but are not successfully ovulated and undergo endomitotic reduplication (Emo). Our previous proteomic studies show that CACN-1 interacts with a network of splicing factors. Here, these interactors were screened using RNAi. Depletion of many of these factors led to missing or abnormal sheath cells and germ line defects, particularly absent and/or Emo oocytes. These results suggest CACN-1 is part of a protein network that influences somatic gonad development and function through alternative splicing or post-transcriptional gene regulation.

Proteomic analysis reveals CACN-1 is a component of the spliceosome in Caenorhabditis elegans.

Cell migration is essential for embryonic development and tissue formation in all animals. cacn-1 is a conserved gene of unknown molecular function identified in a genome-wide screen for genes that regulate distal tip cell migration in the nematode worm Caenorhabditis elegans. In this study we take a proteomics approach to understand CACN-1 function. To isolate CACN-1-interacting proteins, we used an in vivo tandem-affinity purification strategy. Tandem-affinity purification-tagged CACN-1 complexes were isolated from C. elegans lysate, analyzed by mass spectrometry, and characterized bioinformatically. Results suggest significant interaction of CACN-1 with the C. elegans spliceosome. All of the identified interactors were screened for distal tip cell migration phenotypes using RNAi. Depletion of many of these factors led to distal tip cell migration defects, particularly a failure to stop migrating, a phenotype commonly seen in cacn-1 deficient animals. The results of this screen identify eight novel regulators of cell migration and suggest CACN-1 may participate in a protein network dedicated to high-fidelity gonad development. The composition of proteins comprising the CACN-1 network suggests that this critical developmental module may exert its influence through alternative splicing or other post-transcriptional gene regulation.