Castaneda, Perla G. et al. (2021) MicroPubl Biol

Cram, Erin J., Lee, Myeongwoo, Castaneda, Perla G., Qiu, Zhongqiang, Wu, Nan

[MicroPubl Biol, 2021]

In C. elegans, oocytes are ovulated into the spermatheca, where they are fertilized before being pushed into the uterus. Contraction in the C. elegans spermatheca is driven by circumferential acto-myosin fibers. The C. elegans zyxin homolog, zyx-1, is expressed in the body wall muscle, pharynx and spermatheca. To our surprise, a CRISPR-generated zyx-1 deletion allele results in no overt developmental phenotypes, and the spermathecal actin cytoskeleton appears wild type, however, oocyte transit through the spermatheca is slower than in wild type animals. This suggests ZYX-1/Zyxin may regulate spermathecal contraction magnitude or timing of spermathecal bag contraction and/or spermathecal-uterine valve dilation.

Castaneda, Perla et al. (2021) International Worm Meeting "Galpha/GSA-1 works upstream of PKA/KIN-1 to regulate calcium signaling and contractility in the Caenorhabditis elegans spermatheca"

Pettit, Hannah, Castaneda, Perla, Cram, Erin, Cecchetelli, Alyssa

[International Worm Meeting, 2021]

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/Galphas, 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/Galphas 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/Galphas 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 Gbeta 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.

Pettit, Hannah N et al. (2019) International Worm Meeting "G-protein alpha subunit, GOA-1, is a regulator of contraction and relaxation in the C. elegans spermatheca."

Cram, Erin J, Pettit, Hannah N, Cecchetelli, Alyssa D, Castaneda, Perla

[International Worm Meeting, 2019]

Caenorbabditis elegans is a transparent roundworm used to study a multitude of cellular processes in an in vivo system. The C. elegans reproductive system is an ideal model to study mechanotransduction and contractility. Fertilization occurs in hermaphroditic worms in the spermatheca, when oocytes get pumped in to the spermatheca, are fertilized, and then pumped out as mature embryos. This process is highly regulated and requires cellular communication and coordination between the cells of the reproductive system. In a genetic screen for regulators of this process, we identified the small G protein GOA-1 as an important regulator of calcium signaling in the spermatheca. GOA-1 is a heterotrimeric G-protein a-subunit that can inhibit the production of second messengers such as cAMP. Normally, oocyte entry into the spermatheca triggers a characteristic set of calcium transients that increase in intensity, peaking concomitantly with the strong contractions that push the fertilized embryo into the uterus. GOA-1 deficient animals have a delayed and uncoordinated calcium response, which results in defects in tissue contractility and delayed embryo exit. In order to better understand the role of GOA-1 in the proposed pathway, we created a gain of function GOA-1 strain to determine how hyperactivation of GOA-1 would affect calcium signaling in the spermatheca. Additionally, in order to understand the role of GOA-1 in cellular communication, GOA-1 was locally rescued in specific cells of the spermatheca in a GOA-1 deficient animal. Data collected from these strains will help elucidate the role of GOA-1 in modulating Ca2+ signaling in vivo, allowing us to better understand the signaling networks that drive actomyosin contractility in a conserved, intact biological system.