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.

Chen X et al. (2015) Mol Neurodegener "Erratum to: Ethosuximide ameliorates neurodegenerative disease phenotypes by modulating DAF-16/FOXO target gene expression."

McCue HV, Chen X, Morgan A, Kashyap SS, Barclay JW, Kraemer BC, Burgoyne RD, Wong SQ

[Mol Neurodegener, 2015]

The original version of this article [1] unfortunately contained a mistake. The author list contained a spelling error for the author Hannah V. McCue. The original article has been corrected for this error. The corrected author list is given below:Xi Chen, Hannah V. McCue, Shi Quan Wong, Sudhanva S. Kashyap, Brian C. Kraemer, Jeff W. Barclay, Robert D. Burgoyne and Alan Morgan

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.

Mintzer EA et al. (2002) FEBS Lett "Behavior of a photoactivatable analog of cholesterol, 6-photocholesterol, in model membranes."

Mintzer EA, Wilschut J, Waarts BL, Bittman R

[FEBS Lett, 2002]

6-Photocholesterol, a new photoactivatable analog of cholesterol in which a diazirine functionality replaces the 5,6-double bond in the steroid nucleus, was used recently to identify cholesterol-binding proteins in neuroendocrine cells [Thiele, C., Hannah, M.J., Farenholz, F. and Huttner, W.B. (2000) Nat. Cell Biol. 2, 42-49], to track the distribution and transport of cholesterol in Caenorhabditis elegans [Matyash, V., Geier, C., Henske, A., Mukherjee, S., Hirsh, D., Thiele, C., Grant, B., Maxfield, F.R. and Kurzchalia, T.V. (2001) Mol. Biol. Cell 12, 1725-1736], and to probe lipid-protein interactions in oligodendrocytes [Simons, M., Kramer, E.M., Thiele, C., Stoffel, W. and Trotter, J. (2000) J. Cell Biol. 151, 143-154]. To determine whether 6-photocholesterol is a faithful mimetic of cholesterol we analyzed the ability of this probe, under conditions in which it is not photoactivated to a carbene, to substitute for cholesterol in two unrelated assays: (1) to condense 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine monomolecular films and (2) to mediate the fusion of two alphaviruses (Semliki Forest and Sindbis) with liposomes. The results suggest that this analog is a suitable photoprobe of cholesterol.

Hannah L. Craig et al. (2006) European Worm Meeting "Understanding the Role of a Non-Peptidase Member of the ACE Family in Nematode Moulting"

R.Elwyn Isaac, Hannah L. Craig, Clare Hunton, Darren R. Brooks

[European Worm Meeting, 2006]

Hannah L. Craig1, Clare Hunton1, Darren R. Brooks2, R.Elwyn Isaac1 Analysis of the large number of protease-like genes present in animal genomes has shown that a considerable proportion of these genes code for proteins that are structurally related to peptidases, but lack key catalytic residues. These proteins are classified as non-peptidase family members. One such protein is the single member of the angiotensin-converting enzyme (ACE) family of zinc metallopeptidases found in C. elegans. ACN-1 (ACE-like non-metallopeptidase) shares considerable identity with ACE from other organisms, but lacks the conserved active site residues required for proteolytic activity. ACN-1 also differs from other ACEs in having a proline/glutamic acid-rich domain, and, towards the C-terminus, a cysteine-rich region, a proline and threonine/serine rich region and a potential GPI anchor attachment site. It has recently become apparent that an alternatively-spliced form of acn-1 may be expressed at low levels. A single EST with 5 and 3 sequence identical to acn-1, but missing a major portion of the central ACE-like domain, has been identified. Sequences showing considerable similarity to acn-1 are also present in EST and genomic datasets from several parasitic nematodes.. ACN-1 has an essential role in C. elegans development and morphogenesis. GFP-tagged ACN-1 expression was observed in the hypodermal cells and developing vulva of hermaphrodites and also the ray papillae of the male tail. Deletion of a 692 bp fragment (strain tm844, provided by Shohei Mitani, Tokyo Womens Medical College) comprising part of exon 7 and all of exons 8 and 9 resulted in arrested embryonic development and acn-1 RNAi resulted in larval arrest due to a moulting defect. Further examination showed that acn-1 RNAi-treated adults had disrupted alae, an incomplete seam syncytium and a protruding vulva. SEM images of arrested larvae showed a failure to shed the old cuticle after RNAi. Adult males displayed similar hypodermal defects, including a severely disrupted tail. The nuclear hormone receptors nhr-23 and nhr-25 were shown by RNAi to regulate expression of acn-1::gfp, hence positioning acn-1 downstream of these nuclear hormone receptors in the genetic cascade controlling moulting.

Martin L Hudson et al. (2003) International Worm Meeting "Functions of ephrins in embryonic and post-embryonic development: exploring connections with Kallman syndrome protein and cadherins"

Andrew D Chisholm, Martin L Hudson

[International Worm Meeting, 2003]

During C. elegans gastrulation, embryonic cells rearrange to form tissue layers. Endodermal and mesodermal cells move into the interior of the embryo, leaving a transient ventral cleft that is sealed up by the short-range movements of ventral neuroblasts. These ventral neuroblasts and their progeny provide the substrate for the subsequent enclosure movements of the epidermis. Mutants with abnormal ventral neuroblast movements display enlarged ventral clefts that later result in epidermal enclosure defects. Previous work in our lab showed that signaling via the C. elegans Eph receptor VAB-1 and its ephrin ligand EFN-1/VAB-2 regulate neuroblast movements at the end of gastrulation (George et al. 1998; Chin-Sang et al. 1999). The divergent ephrin EFN-4 is also required for neuroblast movements but appears to function independently of the VAB-1 receptor (Chin-Sang et al. 2002). A third pathway is defined by the LAR-like receptor phosphotyrosine phosphatase PTP-3 (Harrington et al. 2002). These pathways appear to play related and partly redundant roles in ventral neuroblast movements.We have examined whether other mutants with epidermal morphogenesis phenotypes affect neuroblast movements and have used double mutant analysis to determine if these genes might define new pathways or can be placed in the VAB-1/EFN-4/PTP-3 pathways. KAL-1 is the C. elegans homolog of human Kallman syndrome protein, anosmin-1 (Rugarli et al. 2002). A kal-1 mutation displays low penetrance defects in epidermal morphogenesis. Using 4-D analysis, we find that kal-1 mutants display delayed closure of the ventral cleft. We also find that a kal-1 mutation enhances the morphogenetic phenotypes of all mutants tested (vab-1(null), efn null mutations and ptp-3), suggesting that KAL-1 may define a separate signaling pathway. We are also studying the post-embryonic functions of ephrins. EFN-4 is expressed in the primary cells of the developing vulva (Hudson and Chisholm, 2002 WCWM, abstract 144). efn-4 mutants display very low penetrance defects in vulval morphogenesis and induction. The cadherin CDH-3 is also expressed in vulval cells (Pettit et al. 1996). A cdh-3 mutation causes epidermal morphogenesis defects (6) reminiscent of those in weak efn-4 mutants. We find that the cdh-3 mutation does not enhance efn-4(null) mutant phenotypes but significantly enhances efn-1(null) mutant phenotypes, suggestive of a role for cdh-3 in efn-4 signaling. Further analysis of the phenotypes will be presented.

Stephen T Sewell et al. (2002) Worm Breeder's Gazette "Analysis of cdh-3 upstream regulatory region to find sequences necessary for expression"

Stephen T Sewell, Helen M Chamberlin, Guojuan Zhang

[Worm Breeder's Gazette, 2002]

EGL-38 is a Pax transcription factor important for the development of several organs in C. elegans, including the hindgut. Two genes are known to act downstream of egl-38 : lin-48 and cdh-3 . Genetic studies indicate that lin-48 and cdh-3 are part of two separate pathways. The two genes also function differently, as LIN-48 is responsible for the specification of cell fates, while CDH-3 coordinates development and epithelial morphogenesis 1,2 . Previous studies have shown that lin-48 is a direct target for EGL-38 3 . To investigate whether cdh-3 might also be a target of EGL-38, we have characterized its upstream regulatory sequence. In comparison studies between C. elegans and C. briggsae , we found that there are five domains of consensus sequence within a 1000bp region upstream of the start ATG of cdh-3 . We chose to focus on these areas, reasoning that these were the most likely spots for protein binding. We constructed clones including different amounts of upstream sequence driving expression of the green fluorescent protein (gfp) and injected them into animals to study their expression. From this data (Table 1), we found that two regions are important for expression of cdh-3 in the seam cells and hindgut cells. Deletion of the region between 677 and 555 eliminated expression in the seam cells and deletion of the region between 555 and 526 eliminated expression in both seam cells and the cells of the hindgut. To confirm the function of these two regions, we generated point mutations in the reporter gene with the 824bp upstream sequence (Table 2). A clone with a mutation in the more distal site (pSS1) was not expressed in seam cells but maintained hindgut expression. In contrast, clones with a mutation in the more proximal site (pSS20) or in both sites (pSS21) were not expressed in either cell type. This indicates that the proximal element is important for expression in both cell types. GATA-binding factors have been shown to play a role in seam cell development 4,5 , and the distal region contains a block of sequence conserved between C. elegans and C. briggsae that includes a GATA sequence motif. In contrast, although EGL-38 plays a role in cdh-3 hindgut expression, the conserved sequence in the more proximal 555-526 region does not resemble the binding site for Pax proteins. Thus we suspect the relationship between egl-38 and cdh-3 is indirect. 1 Pettit et al., 1996 Development 2 Chamberlin et al., 1999 Genetics 3 Johnson et al., 2001 Development 4 Page et al., 1997 Genes and Development 5 Koh and Rothman, 2001 Development Table 1 Upstream sequence in clone (bp) Expression in hindgut (F+U cells) Expression in seam cells n 824 66.5% 96% 82 677 48.5% 65% 95 555 64% 0% 58 526 0% 0% 33 467 0% 0% 31 Table 2 clone Expression in hindgut Expression in seam cells n pSS1 84% 3% 37 pSS20 0% 0% 38 pSS21 0% 0% 38