Kislyak Y et al. (2000) East Coast Worm Meeting "Biochemical Characterization of C. elegans RNA Helicase A and Malate Dehydrogenase"

Kislyak Y, Walstrom KM, Do IP

[East Coast Worm Meeting, 2000]

In my laboratory, we are overexpressing and biochemically characterizing two different proteins from C. elegans. RNA helicase A (RHA-1) is essential for proper development in many organisms, including C. elegans. Human RHA is involved in RNA metabolism. For example, it is capable of mediating an association between CREB binding protein (CBP) and RNA polymerase II and is suggested to be required for the activation of transcription by CBP (Nakajima et al. (1997) Cell 90, 1107-1112). Human RHA is also required for RNA export from the nucleus (Tang et al. (1999) MCB 19, 3540-3550). We are attempting to determine the RNA binding targets of RHA-1 in the cell in order to elucidate its function in C. elegans. In another project, malate dehydrogenase (MDH), the last enzyme in the citric acid cycle, has been overexpressed and purified. This enzyme is active, and the enzyme kinetics of C. elegans MDH are being studied. Recent results from both projects will be presented. We are grateful to Yuji Kohara for providing the cDNA clones of these enzymes.

Peter Swoboda et al. (2002) European Worm Meeting "A multifaceted approach to elucidate the regulation of ciliogenesis in C. elegans"

Evgeni Efimenko, Peter Swoboda

[European Worm Meeting, 2002]

Cilia are evolutionarily conserved subcellular organelles functioning in cell motility, movement of extracellular fluids, sensory perception (e.g. smell) and determination of left-right asymmetry. While a great deal is known about the structure, function and motility of cilia, very little is known about the molecular mechanisms that regulate ciliogenesis in a cell-type specific and developmental manner. How do cilia become functional? How can they do what they do?

Ashrafi K (2022) Cell Host Microbe "Better living through communal eating."

Ashrafi K

[Cell Host Microbe, 2022]

Caenorhabditis elegans do not grow on either Staphylococcus saprophyticus or heat-killed Escherichia coli, but do so when exposed to both. In this issue of Cell Host & Microbe, Geng and colleagues have identified E.coli-derived signals as well as the host's neural and innate immunity pathways that promote digestion of S.saprophyticus.

Mikoshiba K (2011) Cell Calcium "Role of IP receptor in development."

Mikoshiba K

[Cell Calcium, 2011]

IP receptor is a Ca(2+) release channel localized on the endoplasmic reticulum. IP(3) receptor is composed of three isoforms, which are expressed in various cells and tissues, and play variety of roles throughout development. I here describe the role of IP receptor from oogenesis, meiotic maturation and fertilization. I also describe the Ca(2+) signaling at meiosis and mitosis, and especially the role in early embryogenesis to determine dorso-ventral axis formation. Loss of function mutation of type 1 IP receptor in mouse, both by gene targeting and spontaneous mutations shows severe ataxia and other phenotypes. Interestingly, double knockouts of type 1 and type 2 exhibit cardiogenesis arrest and that of type 2 and type 3 results in exocrine secretion deficit. IPR of Drosophila or Caenorhabditis elegans is single gene and mutation results severe phenotype of behavior. All the data described here show that IPRs are essential for life and abnormality of IP(3)Rs results in severe abnormality in its structure and function of organism.

Xu X et al. (2007) Exp Cell Res "Role of phosphatidylinositol-4-phosphate 5' kinase (ppk-1) in ovulation of Caenorhabditis elegans."

Lee M, Wycuff DL, Guo H, Xu X

[Exp Cell Res, 2007]

During Caenorhabditis elegans ovulation, the somatic gonad integrates signals from germ cells and propels a mature oocyte into the spermatheca for fertilization. Previous work suggests that phosphoinositide signaling plays important roles in C. elegans fertility. To fully understand inositol-1,4,5-trisphosphate (IP(3)) signaling in ovulation, we have examined the function of phosphatidylinositol-4-phosphate 5'' kinase (PIP5K) in C. elegans. Our results show that the C. elegans PIP5K homolog, ppk-1, is essential for ovulation in C. elegans; ppk-1 is mainly expressed in somatic gonad, and depletion of ppk-1 expression causes defective ovulation, reduced gonad sheath contractility, and sterility. Increased IP(3) signaling compensates for ppk-1 (RNAi)-induced sterility, suggesting that ppk-1 is linked to IP(3) signaling. These results demonstrate that ppk-1 plays an essential role in IP(3) signaling and cytoskeleton organization in somatic gonad.

Xing J et al. (2010) Am J Physiol Cell Physiol "Phosphatidylinositol 4,5-bisphosphate and loss of PLCgamma activity inhibit TRPM channels required for oscillatory Ca2+ signaling."

Xing J, Strange K

[Am J Physiol Cell Physiol, 2010]

The Caenorhabditis elegans intestinal epithelium generates rhythmic inositol 1,4,5-trisphosphate (IP(3))-dependent Ca(2+) oscillations that control muscle contractions required for defecation. Two highly Ca(2+)-selective transient receptor potential (TRP) melastatin (TRPM) channels, GON-2 and GTL-1, function with PLCgamma in a common signaling pathway that regulates IP(3)-dependent intracellular Ca(2+) release. A second PLC, PLCbeta, is also required for IP(3)-dependent Ca(2+) oscillations, but functions in an independent signaling mechanism. PLCgamma generates IP(3) that regulates IP(3) receptor activity. We demonstrate here that PLCgamma via hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP(2)) also regulates GON-2/GTL-1 function. Knockdown of PLCgamma but not PLCbeta activity by RNA interference (RNAi) inhibits channel activity approximately 80%. Inhibition is fully reversed by agents that deplete PIP(2) levels. PIP(2) added to the patch pipette has no effect on channel activity in PLCgamma RNAi cells. However, in control cells, 10 microM PIP(2) inhibits whole cell current approximately 80%. Channel inhibition by phospholipids is selective for PIP(2) with an IC(50) value of 2.6 microM. Elevated PIP(2) levels have no effect on channel voltage and Ca(2+) sensitivity and likely inhibit by reducing channel open probability, single-channel conductance, and/or trafficking. We conclude that hydrolysis of PIP(2) by PLCgamma functions in the activation of both the IP(3) receptor and GON-2/GTL-1 channels. GON-2/GTL-1 functions as the major intestinal cell Ca(2+) influx pathway. Calcium influx through the channel feedback regulates its activity and likely functions to modulate IP(3) receptor function. PIP(2)-dependent regulation of GON-2/GTL-1 may provide a mechanism to coordinate plasma membrane Ca(2+) influx with PLCgamma and IP(3) receptor activity as well as intracellular Ca(2+) store depletion.

Rafael P Vazquez-Manrique et al. (2007) International Worm Meeting "PLC-1/Phospholipase C epsilon regulates embryonic morphogenesis through the IP3 receptor in Caenorhabditis elegans."

Sung Ly, Howard A Baylis, Olivia Bales, Rafael P Vazquez-Manrique, James C Legg

[International Worm Meeting, 2007]

Signalling through the second messenger IP₃ is required at several stages of embryonic development in C. elegans. Previous work has demonstrated a role for IP₃ signalling in the rearrangement of epidermal cells during morphogenesis. However little is known about the molecules acting in this pathway. IP₃ is produced by phospholipase C enzymes (PLC). In order to identify signalling pathways upstream of IP₃ production, in epidermal morphogenesis, we sought to identify which of the five active PLCs in C. elegans is important for this process. We tested the function of PLC genes using RNAi and mutant strains, and we found that depletion of PLC-1/PLC-<font face=symbol>e</font> produced substantial embryonic lethality. 96% of these arrested embryos show morphogenetic defects, which include aberrant dorsal intercalation and defective ventral migration. As a consequence plc-1 loss of function results in ruptured embryos with Gex phenotype (gut in the exterior) and lumpy larvae. Thus PLC-1 is involved in the regulation of morphogenesis. However null or severe loss of function plc-1 mutants do not show the same level of embryonic lethality as some itr-1 mutants. We therefore tested if other PLCs may act redundantly with PLC-1. Using double mutants of PLCs together with plc-1, we show that PLC-3/PLC-<font face=symbol>g</font> and EGL-8/PLC-<font face=symbol>b</font> can compensate for the lack of PLC-1 activity. Finally, we demonstrate that PLC-1 acts through the IP₃ receptor of C. elegans (ITR-1), a molecule known to be involved in the regulation of C. elegans morphogenesis.

Doitsidou M et al. (2008) Nat Methods "Automated screening for mutants affecting dopaminergic-neuron specification in C. elegans."

Flames N, Lee AC, Boyanov A, Doitsidou M, Hobert O

[Nat Methods, 2008]

We describe an automated method to isolate mutant Caenorhabditis elegans that do not appropriately execute cellular differentiation programs. We used a fluorescence-activated sorting mechanism implemented in the COPAS Biosort machine to isolate mutants with subtle alterations in the cellular specificity of GFP expression. This methodology is considerably more efficient than comparable manual screens and enabled us to isolate mutants in which dopamine neurons do not differentiate appropriately.

Lee M-H et al. (1997) International C. elegans Meeting "TOWARD THE IDENTIFICATION OF IN VIVO RNA TARGETS OF GLD-1."

Lee M-H, Schedl TB

[International C. elegans Meeting, 1997]

gld-1 is a female germ cell specific tumor suppressor gene that is essential for normal oocyte differentiation and meiotic prophase progression (Francis et al., 1995a,b). GLD-1 is a member of a small family of proteins, including the mouse/human Sam68 and Quaking proteins, that are highly related over an ~ 200 amino acid region which contains a 70 to 100 amino acid RNA binding domain called the KH motif (Jones and Schedl, 1995). Extensive mutational analysis of gld-1 has demonstrated the importance of conserved sequences for its in vivo function. GLD-1 is a cytoplasmic protein and therefore is likely to control mRNA translation or RNA stability in the cytoplasm (Jones et al., 1996). Currently, no obvious RNA targets have been identified from genetic analysis. We have employed a biochemical approach to identify in vivo RNA targets of GLD-1. The strategy is based on the ability of anti GLD-1 antibodies to immunoprecipitate (IP) GLD-1 from a worm lysate and the strong likelihood that GLD-1 present in the lysate is functional and bound to RNAs. GLD-1 was IPed with affinity purified polyclonal antibodies from a cytosol extract of adult hermaphrodites or with rabbit IgG as a control. RNAs co-IP with GLD-1, as well as with rabbit IgG, were converted into cDNAs. Non-specifically trapped RNAs from the GLD-1 IP were eliminated by subtracting cDNAs from the GLD-1 IP with cDNAs from the control IP. The difference product after 4 rounds of subtraction (DP4) was used to screen a Lambda ZAP II cDNA library constructed with the cDNAs from the GLD-1 IP. Duplicate filters were also screened with a probe made from control IP cDNA. Clones that are positive only with the DP4 probe are currently being characterized. Francis et al., 1995a Genetics 139, 579-606; Francis et al., 1995b Genetics 139, 607-630; Jones and Schedl, 1995 Genes Dev. 9, 1491-1504; Jones et al., 1996 Dev. Biol. 180, 165-183. This work was supported by NIH Grant HD25614.

P Dal Santo et al. (1999) International C. elegans Meeting "The IP3 Receptor is a Timekeeper for the Defecation Cycle Rhythm"

EM Jorgensen, P Dal Santo, MA Logan, AD Chisholm

[International C. elegans Meeting, 1999]

The C. elegans defecation cycle is characterized by the activation of three distinct muscle contractions every 45-50 seconds. How is cycle time maintained? To address this question we identified and characterized mutants with abnormal cycle times. Specifically, we cloned the dec-4 gene and demonstrated that it encodes the inositol trisphosphate receptor (IP 3 receptor). Mutations in the IP 3 receptor alter the timing of the defecation cycle. Null mutations eliminate the cycle, hypomorphic mutants have a slow cycle, and overexpression of the protein results in a fast cycle time. The IP 3 receptor is expressed in several different tissues including the pharynx, the somatic gonad, and the intestine. Using mosaic analysis we demonstrated that IP 3 receptor expression in the intestine is necessary and sufficient for normal timing of the cycle. This indicates that the periodic muscle contractions can be controlled non-autonomously from the intestine. IP 3 receptors are localized to the endoplasmic reticulum and are known to function as calcium release channels. Activation of the channel by a phospholipid signaling pathway leads to the production of temporally and spatially regulated calcium oscillations. Our current data predicts a model in which calcium oscillations in the intestine trigger the defecation motor program. To test this model we injected calcium-sensitive dyes into the posterior intestinal cells of adult animals. Our results demonstrate that calcium oscillations occur in the intestine. The frequency of calcium oscillations are consistent with the timing of the defecation cycle and peak calcium levels occur just before the onset of the muscle program. We conclude that periodic calcium release in the intestinal cells initiates the muscle contractions of the defecation cycle and that the IP 3 receptor is a central component of the timekeeping mechanism that regulates this behavioral rhythm.