Gin P et al. (2003) J Biol Chem "The Saccharomyces cerevisiae COQ6 gene encodes a mitochondrial flavin-dependent monooxygenase required for coenzyme Q biosynthesis."

Lee PT, Gin P, Clarke CF, Rothman SC, Jonassen T, Tzagoloff A, Hsu AY

[J Biol Chem, 2003]

Coenzyme Q (Q) is a lipid that functions as an electron carrier in the mitochondrial respiratory chain in eukaryotes. There are eight complementation groups of Q-deficient Saccharomyces cerevisiae mutants, designated coq1-coq8. Here we have isolated the COQ6 gene by functional complementation and, in contrast to a previous report, find it is not an essential gene. coq6 mutants are unable to grow on nonfermentable carbon sources and do not synthesize Q but instead accumulate the Q biosynthetic intermediate 3-hexaprenyl-4-hydroxybenzoic acid. The Coq6 polypeptide is imported into the mitochondria in a membrane potential-dependent manner. Coq6p is a peripheral membrane protein that localizes to the matrix side of the inner mitochondrial membrane. Based on sequence homology to known proteins, we suggest that COQ6 encodes a flavin-dependent monooxygenase required for one or more steps in Q biosynthesis.

Simske, Jeffrey et al. (2015) International Worm Meeting "Glucose intolerance during C. elegans embryogenesis."

Dong, Yi, Simske, Jeffrey

[International Worm Meeting, 2015]

Glycogen debranching enzyme AGL-1 and AMP kinase signaling are required for normal C. elegans embryogenesis. One remarkable phenotype of both agl-1 and AMPK alpha subunit (aak-2) mutants is the dramatic enhancement of embryonic arrest phenotypes following glucose feeding. For agl-1, lethality is 18% when grown on NGM/OP50 and 99% when supplemented with 0.2% glucose. For aak-2 mutants, there essentially is no lethality on NGM/OP50 and as high as 77% lethality on glucose, depending on the regimen of starvation recovery and dietary restriction. agl-1 sensitivity to glucose requires daf-2, since mutations in daf-2 suppress the agl-1 glucose-dependent embryonic lethality. The role of insulin signaling pathway genes in embryonic development is further supported by the observations that certain daf-2 alleles alone or in daf-16 double mutants, display embryonic defects [1]. Interestingly, over-expression of a daf-16::gfp reporter increases agl-1 embryonic lethality (but only a modest shift of daf-16::gfp to the nucleus is observed in agl-1 mutants). To determine whether glucose intolerance is exclusive to agl-1 and aak-2, other mutants in carbohydrate metabolism were tested for embryonic arrest phenotypes. In one case, mutations in glycogen phosphorylase (tm5211) increased the penetrance of embryonic arrest when grown on glucose or under dietary restriction conditions, reinforcing the importance of maternal diet in embryonic development. To better understand glucose tolerance during embryogenesis, a genetic screen was carried out to identify mutants that display increased embryonic lethality in the presence of elevated glucose. So far, two gin (for Glucose INtolerance) alleles, gin-1(cv10) and gin-2(cv11) have been isolated and both display increased penetrance and severity of embryonic arrest phenotypes in the presence of 0.2% glucose. gin-1(cv10) is 73% viable on NGM/OP50 and 30% viable with glucose supplement. gin-2(cv11) is 21% viable on NGM/OP50 and 0.17% viable with glucose. Complementation tests indicate gin-1 and gin-2 are not agl-1 alleles. gin-1 and gin-2 hermaphrodites grown with glucose recover and lay a higher fraction of hatching eggs when transferred to NGM/OP50, similar to agl-1 mutants. The gin-2 mutant appears to have both a maternal and zygotic requirement. gin-2 embryonic arrest on normal OP50 occurs more frequently during morphogenesis, whereas in embryos grown with glucose, cell divisions cease prior to morphogenesis and intracellular inclusion bodies increase in size and number. Further study of these gin alleles and additional screens are in progress in an effort to characterize the pathways required for glucose tolerance during embryogenesis.1. Gems, D., et al., Genetics, 1998. 150(1): p. 129-55.

Nadeem Moghal et al. (2001) International C. elegans Meeting "Modulation of inductive signaling during C. elegans vulval development"

Nadeem Moghal, Paul W Sternberg, L Rene Garcia, Chieh Chang

[International C. elegans Meeting, 2001]

In C. elegans hermaphrodites, the vulval precursor cells (VPCs) P3.p-P8.p can adopt vulval or hypodermal fates. In wild-type animals, the anchor cell secretes LIN-3, which activates its receptor, LET-23, and causes P5.p-P7.p to adopt vulval fates. Although P3.p, P4.p, and P8.p also express LET-23, it is thought that their distance from the anchor cell prevents levels of activated LET-23 from accumulating necessary to overcome default hypodermal fates. We have used two approaches to study modulation of responses to this inductive signal. In one approach, we tested candidate loci for interaction with this pathway. We find that a loss of function mutation in the egl-15 negative regulator, clr-1 , a receptor tyrosine phosphatase, increases responsiveness of all 6 VPCs to the inductive signal. In contrast, an activated allele of egl-30 Gq alpha facilitates responsiveness of P5.p-P7.p, but not P3.p, P4.p, and P8.p. In a second approach, we identified EMS-induced mutations that enhance the choice of vulval fates by P3.p, P4.p, and P8.p in the presence of a gain-of-function allele of let-23 . Some of these mutations, such as sy622 , strongly enhance the responsiveness of P3.p, P4.p, and P8.p, while only weakly affecting P5.p-P7.p. These results indicate that there are both pan-Pn.p, as well as Pn.p-specific mechanisms operating to modulate vulval induction. Pan-Pn.p mechanisms might function as buffers to lower the overall noise inappropriately contributing to vulval induction. In conjunction with pathways defined by egl-30(gf) and sy622 which increase responsiveness of P5.p-P7.p and decrease responsiveness of P3.p, P4.p, and P8.p, respectively, a robust response by only a subset of cells might be guaranteed during inductive signaling.

Min Zheng et al. (2001) International C. elegans Meeting "Cell fate specification during vulva formation in Pristionchus pacificus"

Ralf J Sommer, Min Zheng

[International C. elegans Meeting, 2001]

We compare vulva development between Caenorhabditis elegans and Pristionchus pacificus to study the evolution of developmental processes. Cell fate specification during vulva development differs in these two nematodes. In C.elegans , P8.p is part of the vulva equivalence group and adopts a 3deg fate under wild-type conditions. In contrast, several experiments indicate that P8.p in P.pacificus represents a new cell type. First, P8.p is unable to respond to an inductive signal from the gonad and fuses with the epidermis, but can respond to lateral signal from P6.p and form vulval tissue. Second, P8.p provides a lateral inhibitory signal that prevents P5.p and P7.p to adopt a 1deg fate. Third, P8.p provides a negative signal that inhibits gonad-independent vulva formation of P(5-7).p. To study P8.p specification we screened for mutants with P8.p specification defects and identified three candidate mutants in which P8.p behaves like a normal VPC. In one of the mutants, named tu151 , the nucleus of P8.p has an abnormal size. Cell ablation experiments in tu151 revealed that P8.p is able to respond to the inductive signal from the gonad to form vulval tissue. Also, P8.p in this mutant cannot provide the lateral inhibitory signal so that after P6.p ablation, P5.p and P7.p can adopt the 1deg fate. In addtion, P8.p loses the function of providing negative signal in tu151 mutant animals since gonad-independent vulva formation occurs. Positional cloning of tu151 is currently in progress.

Min Zheng et al. (2002) European Worm Meeting "Genetic and molecular analysis of the vulval patterning mutants ped-7 and ped-17 in Pristionchus pacificus"

Min Zheng, Ralf J Sommer

[European Worm Meeting, 2002]

We compare vulva development between Caenorhaditis elegans and Pristionchus pacificus to study the evolution of developmental processes. Cell fate specification during vulva development differs in these two nematodes. In C. elegans, P8.p is a member of the vulva equivalence group and adopts a 3 fate under wild-type conditions. In contrast, several experiments indicate that P8.p in P. pacificus represents a novel cell type. First, P8.p is unable to respond to an inductive signal to form vulval tissue, but it can respond to lateral signal from the neighboring 1 cell P6.p. Second, P8.p provides a lateral inhibitory signal that prevents P5.p and P7.p, but not P6.p, from adopting a 1 fate. Third, P8.p provides a negative signal that inhibits gonad-independent vulva formation of P(5-7).p. To study P8.p specification, we screened for mutants with P8.p specification defects and identified three mutants in which P8.p behaves like a normal VPC. In one of these mutants, namely ped-17, the nucleus of P8.p has an abnormal size. Cell ablation experiments in ped-17 revealed that P8.p is able to respond to inductive signal from the gonad to form vulval tissue. Also, P8.p in this mutant cannot provide the lateral inhibitory signal. After P6.p ablation in these animals, either P5.p or P7.p can adopt a 1 fate. In addition, P8.p loses the function of providing negative signal in ped-17 mutant animals since gonad- independent vulva formation occurs. In another mutant, ped-7, P7.p migrates towards P8.p and forms an ectopic invagination. Genetic analysis revealed that ped-7 represents a multivulva mutation. We have mapped both mutants to the genetic linkage map of P. pacificus. ped-7 maps to chromosome V and is located between the markers S130 and S13. ped-17 is on chromosome II and is located between S111 and S24. Positional cloning of ped-7 and ped-17 is currently ongoing.

Sternberg PW et al. (1982) Worm Breeder's Gazette "cell-cell interactions in P redivivus ventral hypodermis development."

Sternberg PW, Horvitz HR

[Worm Breeder's Gazette, 1982]

In C. elegans the gonad, specifically the anchor cell, induces the ventral hypodermal precursors P(5-7).p to generate the vulva (Kimble, Develop. Biol. 87, 286, 1981). Ablation of any of these vulval precursor cells leads to regulation (Sulston and White, Develop. Biol. 78, 577, 1980). Similar regulation is seen in C. elegans males. We have performed a series of gonadal, P, and Pn.p cell laser ablation experiments in P. redivivus females and males. In P. redivivus, P(5- 8).p generate the vulva; the other Pn.p cells join the hypodermal syncytium. [In C. elegans, P(3,4,8).p divide and their progeny join the syncytium; P(1,2,9-11).p join the syncytium directly.] Our results can be summarized as follows. (1) The P. redivivus gonad is required for female Pn.p divisions and vulval development. The anchor cell (Z4. aaa) is required for the third round of Pn.p divisions. Z4 is required for vulval induction, although a few Pn.p cells can divide in its absence. In males of both species the gonad is not required for Pn.p divisions. (2) Regulation occurs as in C. elegans: the ablation of certain Pn.p cells can result in the replacement of the ablated cell by another Pn.p cell. (3) As in C. elegans, there is a hierarchy of replacements. A higher ranked cell can be replaced by a lower ranked cell but will not replace a lower ranked cell. In C. elegans hermaphrodites, the fate of P6.p is primary (ranks highest in the hierarchy), the fates of P(5,7).p are secondary, and the fates of P(3,4,8).p are tertiary. In P. redivivus females, the fates of P(6,7) .p are primary, the fates of P(5,8).p are secondary, and the fates of P(4,9).p are tertiary. (4) In C. elegans hermaphrodites, P(3-8).p can participate in vulval development and thus define the vulval equivalence group. In P. redivivus females, at least P(4-9).p can participate in vulval development. Thus, the vulval equivalence group differs between the species. (5) In males of both species, P10.p and P11.p generate cells of a pre-anal sensillum (called the hook sensillum in C. elegans); P9.p joins the ventral hypodermal syncytium or, in some C. elegans males generates two daughters that join the syncytium. As in C. elegans, P9.p can replace P10.p. Thus, in P. redivivus, P9.p belongs to both the male and female equivalence groups, indicating that equivalence groups in the two sexes need not be distinct. The differences in vulval regulation between the two species suggest that there are at least four independent levels of control specifying Pn.p fate in vulval development. Different sets of instructions seem to specify: (a) which Pn.p cells can participate in vulval development; (b) which Pn.p cells do participate in vulval development; (c) which Pn.p cells follow primary or secondary fates; and (d) what lineage is generated by cells with the primary, secondary and tertiary fates.

Alper SD et al. (1997) International C. elegans Meeting "Regulation of Pn.p Cell fusion in C. elegans"

Kenyon CJ, Alper SD

[International C. elegans Meeting, 1997]

Cell fusion is a ubiquitous process in C. elegans, with approximately one third of all somatic nuclei in adults found in variably sized multinucleate syncytia. In hermaphrodites, the hypodermal cells P1.p, P2.p, and P(9-11).p all fuse with hyp7 during late L1 while P(3-8).p remain unfused and become members of the vulval equivalence group. In males, P(3-6).p and P(9-11).p remain unfused; P(9-11).p subsequently become the male equivalence group. The Pn.p fusion decision is regulated by the Hox genes lin-39 and mab-5. lin-39 is expressed in P(3-8).p and in hermaphrodites prevents fusion of these cells. mab-5 is expressed in P(7-11).p. In males, lin-39 and mab-5 each individually prevent Pn.p cell fusion in P(3-6).P and P(9-11).p, respectively. However, in P7.p and P8.p, where both Hox genes are expressed in the same cell, they somehow neutralize one another's effects, so that P7.p and P8.p fuse with hyp7. We are carrying out a screen to identify additional genes that affect Pn.p cell fusion. In a lin-12(d) background, those Pn.p cells that remain unfused all generate ectopic pseudovulvae or hooks that protrude from the ventral surface of the worm. We are screening for mutations that change the pattern of ventral protrusion in lin-12(n137); him-5(e1467); thus far, we have identified five mutants with extra ventral protrusions in the hermaphrodite and one mutant with extra protrusions in the male. MH27, a monoclonal antibody that stains adherins junctions, has been used to verify that extra Pn.p cells fail to fuse with hyp7 in some of these mutants. Preliminary experiments suggest that some of these mutations may affect upstream regulators of the Hox genes. We also hope to find genes that integrate the Hox signals as well as genes required for the actual process of cell fusion.

Gino Poulin et al. (2006) European Worm Meeting "A Candidate-Based Approach for Genetic Interaction with gap-1 identifies a WD40 Encoding Gene"

Julie Ahringer, Gino Poulin

[European Worm Meeting, 2006]

Gino Poulin and Julie Ahringer. Vulval fate induction is an established readout to identify regulators of multiple signalling pathways including the canonical RTK/RAS/RAF/MPK signalling pathway. Vulval fate adoption requires an inductive signal (LIN-3 EGF) that emanates from the anchor cell (AC) and activates Ras signalling in three of the six vulval precursor cells (VPCs). The VPC closest to the source of LIN-3 EGF adopts the primary fate and produces the lateral signal that activates LIN-12 NOTCH signalling in the neighbouring secondary VPCs. In these cells, negative regulators of Ras ensure down regulation of Ras signalling in a Notch dependant manner, an important process to maintain vulval cell patterning. The last three VPCs adopt a non-vulval fate, because of the Ras inhibiting activity of the genetically redundant synMuv genes. GAP-1 is a GTPase activating protein that directly regulates LET-60 RAS by stimulating its GTPase activity and therefore increases the rate of LET-60 RAS bound to GDP, which is the inactive form. In absence of gap-1 animals develop a normal vulva, however vulval fate adoption becomes more susceptible to elevated Ras signalling. Hence, additional loss of a negative regulator of Ras causes extra vulval precursor cells to adopt the vulval fate and produces the Muv phenotype. We have previously performed genome-wide RNAi screens for new synMuv genes. RNAi of about half of our initial candidates caused a synMuv independent low percentage Muv phenotype. We reassessed these and the synMuv interacting genes (100 genes) in a gap-1 background and found seven where RNAi caused a high percentage Muv phenotype. We identified the members of the sumoylation pathway, two components of the NuRD complex, sys-1, and a novel WD40 encoding gene, that we named gin-1, for gap interactor-1. We focused our analysis on gin-1 and found that it inhibits Ras signalling upstream of sem-5 (Grb2). We also found that RNAi of gin-1 suppresses a let-23 EGFR loss-of-function mutant, suggesting that the inhibition occurs at or downstream of the receptor. In addition, using egl-17::cfp as a marker for primary cells, we found that gin-1(RNAi) animals show persistent expression in the normally non-expressing secondary cells, showing that gin-1 plays a role in Ras signalling inhibition during vulval fate adoption. To rule out the possibility that anchor cell duplication is responsible for our observations, we used zmp-1::yfp as an anchor cell marker and found no defect at this level. Taken together, our candidate-based approach has identify seven novel gap-1 interactors; one of these, gin-1, appears to regulate Ras signaling between the receptor and sem-5.

Simske JS et al. (1995) International C. elegans Meeting "SEQUENTIAL SIGNALING during vulval development."

Kim SK, Simske JS

[International C. elegans Meeting, 1995]

During the induction of the C. elegans vulva, cell signaling causes initially equipotent cells to express a reproducible pattern of cell fates. The position of the anchor cell determines the pattern of vulval precursor cell fates, such that the closest precursor cell (P6.p) expresses the primary cell fate, the next closest cells (P5.p and P7.p) both express the secondary cell fate, and each of the precursor cells located at a distance (P3.p, P4.p, and P8.p) express the uninduced, tertiary cell fate. We present data indicating that this stereotypical pattern of cell fates can be generated by sequential signals. We identified genetic mosaic animals in which P5.p and P7.p were defective in the anchor cell signal transduction pathway and observed that these cells adopted the secondary cell fate, indicating that anchor cell signal transduction in P5.p and P7.p is not required for the expression of the secondary cell fate. These results suggest that the anchor cell induces P6.p to express the primary cell fate, and that P6.p subsequently induces P5.p and P7.p to express the secondary cell fate.

Loo TW et al. (2013) Biochemistry "A salt bridge in intracellular loop 2 is essential for folding of human p-glycoprotein."

Clarke DM, Loo TW

[Biochemistry, 2013]

There is no high-resolution structure of the human P-glycoprotein (P-gp, ABCB1) drug pump. Homology models based on the crystal structures of mouse and Caenorhabditis elegans P-gps show extensive contacts between intracellular loop 2 (ICL2, in the first transmembrane domain) and the second nucleotide-binding domain. Human P-gp modeled on these P-gp structures yields different ICL2 structures. Only the model based on the C. elegans P-gp structure predicts the presence of a salt bridge. We show that the Glu256-Arg276 salt bridge was critical for P-gp folding.