The promotion of gonadal cell divisions by the Caenorhabditis elegans TRPM cation channel GON-2 is antagonized by GEM-4 copine

The initiation of postembryonic cell divisions by the gonadal precursors of C. elegans requires the activity of gon-2. gon-2 encodes a predicted cation channel (GON-2) of the TRPM subfamily of TRP proteins and is likely to mediate the influx of Ca(2+) and/or Mg(2+). We report here that mutations in gem-4 (gon-2 extragenic modifier) are capable of suppressing loss-of-function alleles of gon-2. gem-4 encodes a member of the copine family of Ca(2+)-dependent phosphatidylserine binding proteins. Overall, our data indicate that GEM-4 antagonizes GON-2. This antagonism could be mediated by a direct inhibition of GON-2 by GEM-4, since both proteins are predicted to be localized to the plasma membrane. Alternatively, GEM-4 could affect GON-2 activity levels by either promoting endocytosis or inhibiting exocytosis of vesicles that carry GON-2. It is also possible that GEM-4 and GON-2 act in parallel to each other. Mutation of gem-4 does not suppress the gonadal defects produced by inactivation of gon-4, suggesting that gon-4 either acts downstream of gem-4 and gon-2 or acts in a parallel regulatory pathway.     

CATP-6, a C. elegans Ortholog of ATP13A2 PARK9, Positively Regulates GEM-1, an SLC16A Transporter

In previous work, we found that gain-of-function mutations that hyperactivate GEM-1 (an SLC16A transporter protein) can bypass the requirement for GON-2 (a TRPM channel protein) during the initiation of gonadogenesis in C. elegans. Consequently, we proposed that GEM-1 might function as part of a Mg²⁺ uptake pathway that functions in parallel to GON-2. In this study, we report that CATP-6, a C. elegans ortholog of the P5B ATPase, ATP13A2 (PARK9), is necessary for gem-1 gain-of-function mutations to suppress the effects of gon-2 inactivation. One possible explanation for this observation is that GEM-1 serves to activate CATP-6, which then functions as a Mg²⁺ transporter. However, we found that overexpression of GEM-1 can alleviate the requirement for CATP-6 activity, suggesting that CATP-6 probably acts as a non-essential upstream positive regulator of GEM-1. Our results are consistent with the notion that P5B ATPases govern intracellular levels of Mg²⁺ and/or Mn²⁺ by regulating the trafficking of transporters and other proteins associated with the plasma membrane.     

Gon-2, a Gene Required for Gonadogenesis in Caenorhabditis Elegans

The gonad of the Caenorhabditis elegans hermaphrodite is generated by the postembryonic divisions of two somatic precursors, Z1 and Z4, and two germline precursors, Z2 and Z3. These cells begin division midway through the first larval stage. By the end of the fourth larval stage, Z1 and Z4 produce 143 descendants, while Z2 and Z3 give rise to ~1000 descendants. The divisions of Z2 and Z3 are dependent on signals produced by Z1 and Z4, but not vice versa. We have characterized the properties of five loss-of-function alleles of a newly described gene, which we call gon-2. In gon-2 mutants, gonadogenesis is severely impaired; in some animals, none of the gonad progenitors undergo any postembryonic divisions. Mutations in gon-2 have a partial maternal effect: either maternal or zygotic expression is sufficient to prevent the severe gonadogenesis defects. By cell lineage analysis, we found that the primary defect in gon-2 mutants is a delay (sometimes a complete block) in the onset and continuation of gonadal divisions. The results of upshift experiments using a temperature-sensitive allele suggest that zygotic expression of gon-2 begins early in embryogenesis, before the birth of Z1 and Z4. The results of downshift experiments suggest that Z1 and Z4 can generate the full complement of gonadal tissues even when gon-2 function is inhibited until the end of the second larval stage. Thus, gon-2 activity is probably not required for the specification of gonadal cell fates, but appears to be generally required for gonadal cell divisions.     

gon-4, a cell lineage regulator required for gonadogenesis in Caenorhabditis elegans

The gon-4 gene is required for gonadogenesis in the nematode Caenorhabditis elegans. Normally, two precursor cells, Z1 and Z4, follow a reproducible pattern of cell divisions to generate the mature somatic gonadal structures (e.g., uterus in hermaphrodites, vas deferens in males). In contrast, in gon-4 mutants, the Z1/Z4 cell lineages are variably aborted in both hermaphrodites and males: Z1 and Z4 divide much later than normal and subsequent divisions are either absent or severely delayed. In gon-4 adults, normal somatic gonadal structures are never observed, and germ-line and vulval tissues, which depend on somatic gonadal cues for their development, are also aberrant. In contrast, nongonadal tissues and the timing of other developmental events (e.g., molts) appear to be normal in gon-4 mutants. The gon-4 alleles are predicted to be strong loss-of-function or null alleles by both genetic and molecular criteria. We have cloned gon-4 in an attempt to learn how it regulates gonadogenesis. The gon-4 gene encodes a novel, acidic protein. A GON-4::GFP fusion protein, which rescues a gon-4 mutant to fertility, is expressed in somatic gonadal cells during early gonadal development. Furthermore, this fusion protein is nuclear. We conclude that gon-4 is a regulator of the early lineage of Z1 and Z4 and suggest that it is a part of a genetic program common to the regulation of both hermaphrodite and male gonadogenesis.     

gon-14 functions with class B and class C synthetic multivulva genes to control larval growth in Caenorhabditis elegans

Previous work showed that C. elegans gon-14 is required for gonadogenesis. Here we report that gon-14 encodes a protein with similarity to LIN-15B, a class B synMuv protein. An extensive region of GON-14 contains blocks of sequence similarity to transposases of the hAT superfamily, but key residues are not conserved, suggesting a distant relationship. GON-14 also contains a putative THAP DNA-binding domain. A rescuing gon-14::GON-14::VENUS reporter is broadly expressed during development and localizes to the nucleus. Strong loss-of-function and predicted null gon-14 alleles have pleiotropic defects, including multivulval (Muv) defects and temperature-sensitive larval arrest. Although the gon-14 Muv defect is not enhanced by synMuv mutations, gon-14 interacts genetically with class B and class C synMuv genes, including lin-35/Rb, let-418/Mi-2beta, and trr-1/TRRAP. The gon-14; synMuv double mutants arrest as larvae when grown under conditions supporting development to adulthood for the respective single mutants. The gon-14 larval arrest is suppressed by loss of mes-2/E(Z), mes-6/ESC, or mes-4, which encodes a SET domain protein. Additionally, gon-14 affects expression of pgl-1 and lag-2, two genes regulated by the synMuv genes. We suggest that gon-14 functions with class B and class C synMuv genes to promote larval growth, in part by antagonizing MES-2,3,6/ESC-E(z) and MES-4.     

Novel Alleles of gon-2, a C. elegans Ortholog of Mammalian TRPM6 and TRPM7, Obtained by Genetic Reversion Screens

TRP (Transient Receptor Potential) cation channels of the TRPM subfamily have been found to be critically important for the regulation of Mg²⁺ homeostasis in both protostomes (e.g., the nematode, C. elegans, and the insect, D. melanogaster) and deuterostomes (e.g., humans). Although significant progress has been made toward understanding how the activities of these channels are regulated, there are still major gaps in our understanding of the potential regulatory roles of extensive, evolutionarily conserved, regions of these proteins. The C. elegans genes, gon-2, gtl-1 and gtl-2, encode paralogous TRP cation channel proteins that are similar in sequence and function to human TRPM6 and TRPM7. We isolated fourteen revertants of the missense mutant, gon-2(q338), and these mutations affect nine different residues within GON-2. Since eight of the nine affected residues are situated within regions that have high similarity to human TRPM1,3,6 and 7, these mutations identify sections of these channels that are potentially critical for channel regulation. We also isolated a single mutant allele of gon-2 during a screen for revertants of the Mg²⁺-hypersensitive phenotype of gtl-2(-) mutants. This allele of gon-2 converts a serine to phenylalanine within the highly conserved TRP domain, and is antimorphic against both gon-2(+) and gtl-1(+). Interestingly, others have reported that mutation of the corresponding residue in TRPM7 to glutamate results in deregulated channel activity.     

The gon-1 gene is required for gonadal morphogenesis in Caenorhabditis elegans

In wild-type Caenorhabditis elegans, the gonad is a complex epithelial tube that consists of long arms composed predominantly of germline tissue as well as somatic structures specialized for particular reproductive functions. In gon-1 mutants, the adult gonad is severely disorganized with essentially no arm extension and no recognizable somatic structure. The developmental defects in gon-1 mutants are limited to the gonad; other cells, tissues, and organs appear to develop normally. Previous work defined the regulatory "leader" cells as crucial for extension of the gonadal arms (J. E. Kimble and J. G. White, 1981, Dev. Biol. 81, 208-219). In gon-1 mutants, the leader cells are specified correctly, but they fail to migrate and gonadal arms are not generated. In addition, gon-1 is required for morphogenesis of the gonadal somatic structures. This second role appears to be independent of that required for leader migration. Parallel studies have shown that gon-1 encodes a secreted metalloprotease (R. Blelloch and J. Kimble, 1999, Nature 399, 586-590). We discuss how a metalloprotease may control two aspects of gonadal morphogenesis.     

Gonadal cell lineages of the nematode Panagrellus redivivus and implications for evolution by the modification of cell lineage

To explore the nature of cell lineage modifications that have occurred during evolution, the gonadal cell lineages of the nematode Panagrellus redivivus have been determined and compared to the known gonadal lineages of Caenorhabditis elegans (J. Kimble and D. Hirsh, 1979, Develop. Biol.70, 396–417). Essentially invariant lineages generate the 143 somatic cells of the male gonad and at least 326 somatic cells of the female gonad of P. redivivus. The basic program of gonadogenesis is strikingly similar among both sexes of both species. For example, the early division patterns of the somatic gonad precursors Z1 and Z4 are almost identical. Later division patterns are more divergent and, in a few cases, generate structures that are species specific. In general, similar cell types are produced after similar patterns of cell divisions. Differences among the Z1 and Z4 cell lineages appear to reflect phylogenetic modifications of a common developmental program. The nature of these differences suggests that the evolution of cell lineages involves four distinct classes of alterations: switches in the fate of a cell to that normally associated with another cell; reversals in the polarity of the lineage generated by a blast cell; alterations in the number of rounds of cell division; and an “altered segregation” of developmental potential, so that a potential normally associated with one cell instead becomes associated with its sister. A number of cell deaths occur during gonadogenesis in P. redivivus. The death of Z4.pp, a cell that controls the development of the posterior ovary in C. elegans, probably prevents the development of a posterior ovary in P. redivivus and hence is responsible for the gross difference in the morphologies of the gonads of the P. redivivus female and the C. elegans hermaphrodite. As exemplified by the death of Z4.pp, an alteration in the fate of a “regulatory cell” could facilitate rapid and/or discontinuous evolutionary change.     

Quantitative Proteomics Reveals the Essential Roles of Stromal Interaction Molecule 1 (STIM1) in the Testicular Cord Formation in Mouse Testis

Testicular cord formation in male gonadogenesis involves assembly of several cell types, the precise molecular mechanism is still not well known. With the high-throughput quantitative proteomics technology, a comparative proteomic profile of mouse embryonic male gonads were analyzed at three time points (11.5, 12.5, and 13.5 days post coitum), corresponding to critical stages of testicular cord formation in gonadal development. 4070 proteins were identified, and 338 were differentially expressed, of which the Sertoli cell specific genes were significant enrichment, with mainly increased expression across testis cord development. Additionally, we found overrepresentation of proteins related to oxidative stress in these Sertoli cell specific genes. Of these differentially expressed oxidative stress-associated Sertoli cell specific protein, stromal interaction molecule 1, was found to have discrepant mRNA and protein regulations, with increased protein expression but decreased mRNA levels during testis cord development. Knockdown of Stim1 in Sertoli cells caused extensive defects in gonadal development, including testicular cord disruption, loss of interstitium, and failed angiogenesis, together with increased levels of reactive oxygen species. And suppressing the aberrant elevation of reactive oxygen species could partly rescue the defects of testicular cord development. Taken together, our results suggest that reactive oxygen species regulation in Sertoli cells is important for gonadogenesis, and the quantitative proteomic data could be a rich resource to the elucidation of regulation of testicular cord development.Male gonadogenesis is a complex process that requires the formation and assembly of several cell types that come together to form a functional organ. These cell lineages coordinate to maintain testicular cord development but do not differentiate independently (1, 2). Shortly after the activation of Sox9, when the genital ridges are still long and very thin, pre-Sertoli cells start to aggregate around germ cell clusters and form cords; they are then referred to as Sertoli cells. Partitioning of this mass of cells into cord-forming units coincides with endothelial cell invasion and expansion of interstitial space (3, 4). In mice, organization of the testicular cords begins with aggregate of germ cell and Sertoli progenitors in the gonad. Previous studies using confocal analysis and three-dimensional modeling have reported that testicular cord formation involves three basic steps (5, 6): pre-Sertoli cells and germ cells coalesce between 10.5 and 12.5 days post coitum (dpc)¹; cords partition at 12.5 dpc with a clear basal lamina surrounding the cords, and all cords are characterized as “external” cords, defined as a single transverse loop located just under the celomic epithelium that surrounds the gonad at this stage; and refinement of cords continues at 13.5 dpc. Although Sertoli cells acting as a organizing center in testicular cord formation has been well known (3) and studies in knockout mouse models have revealed several genes associated with testicular cord formation (7–10), how these cell types assemble into a functional organ remains to be explored (2, 11).Proteomics technology has been widely used in postnatal testis development and function research in mice (12–16). Two proteomics studies have been carried out in the fetal gonads in mice, and identified more than 1000 proteins expressed in gonads (17, 18), however, the temporal proteome changes have not been elucidated during gonadogenesis. Additionally, mRNA abundance may not always predict the quantity of the corresponding functional protein, and proteomic approach can provide a systemic view of protein level regulation in a large scale (18). Therefore, this study aimed to obtain a better understanding of male gonadogenesis by establishing a first temporal proteomic profile during the initiation of gonad development in male mice. After confirming the specific time point by immunofluorescence (IF) staining, we performed a comparative proteomic analysis of samples of male mouse gonads obtained at 11.5, 12.5, and 13.5 dpc. Bioinformatics analysis and functional studies demonstrate that reactive oxygen species (ROS) regulation in Sertoli cells may be important for testicular cord formation, and functional characterizing the of stromal interaction molecule 1 (stim1), a Sertoli cell specific protein, supported this hypothesis. Our categorized protein lists can serve as a useful resource for further exploring the molecular mechanisms involved in gonadal development.     

Notch signaling regulates bile duct morphogenesis in mice

Background

Alagille syndrome is a developmental disorder caused predominantly by mutations in the Jagged1 (JAG1) gene, which encodes a ligand for Notch family receptors. A characteristic feature of Alagille syndrome is intrahepatic bile duct paucity. We described previously that mice doubly heterozygous for Jag1 and Notch2 mutations are an excellent model for Alagille syndrome. However, our previous study did not establish whether bile duct paucity in Jag1/Notch2 double heterozygous mice resulted from impaired differentiation of bile duct precursor cells, or from defects in bile duct morphogenesis.

Methodology/Principal Findings

Here we characterize embryonic biliary tract formation in our previously described Jag1/Notch2 double heterozygous Alagille syndrome model, and describe another mouse model of bile duct paucity resulting from liver-specific deletion of the Notch2 gene.

Conclusions/Significance

Our data support a model in which bile duct paucity in Notch pathway loss of function mutant mice results from defects in bile duct morphogenesis rather than cell fate specification.     

Expression of Arabidopsis MCA1 enhanced mechanosensitive channel activity in the Xenopus laevis oocyte plasma membrane

Higher plants sense and respond to osmotic and mechanical stresses such as turgor, touch, flexure and gravity. Mechanosensitive (MS) channels, directly activated by tension in the cell membrane and cytoskeleton, are supposed to be involved in the cell volume regulation under hypotonic conditions and the sensing of these mechanical stresses based on electrophysiological and pharmacological studies. However, limited progress has been achieved in the molecular identification of plant MS channels. Here, we show that MCA1 (mid1-complementing activity 1; a putative mechanosensitive Ca²⁺-permeable channel in Arabidopsis thaliana) increased MS channel activity in the plasma membrane of Xenopus laevis oocytes. The functional and kinetic properties of MCA1 were examined by using a Xenopus laevis oocytes expression system, which showed that MCA1-dependent MS cation currents were activated by hypo-osmotic shock or by membrane stretch produced by pipette suction. Single-channel analyses suggest that MCA1 encodes a possible MS channel with a conductance of 34 pS.     

Tissue architecture in the Caenorhabditis elegans gonad depends on interactions among fibulin-1, type IV collagen and the ADAMTS extracellular protease

Molecules in the extracellular matrix (ECM) regulate cellular behavior in both development and pathology. Fibulin-1 is a conserved ECM protein. The Caenorhabditis elegans ortholog, FBL-1, regulates gonad-arm elongation and expansion by acting antagonistically to GON-1, an ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family protease. The elongation of gonad arms is directed by gonadal distal tip cells (DTCs). Here we report that a dominant mutation in the EMB-9/type IV collagen α1 subunit can compensate for loss of FBL-1 activity in gonadogenesis. A specific amino acid substitution in the noncollagenous 1 (NC1) domain of EMB-9 suppressed the fbl-1 null mutant. FBL-1 was required to maintain wild-type EMB-9 in the basement membrane (BM), whereas mutant EMB-9 was retained in the absence of FBL-1. EMB-9 (either wild type or mutant) localization in the BM enhanced PAT-3/β-integrin expression in DTCs. In addition, overexpression of PAT-3 partially rescued the DTC migration defects in fbl-1 mutants, suggesting that EMB-9 acts in part through PAT-3 to control DTC migration. In contrast to the suppression of fbl-1(tk45), mutant EMB-9 enhanced the gonadal defects of gon-1(e1254), suggesting that it gained a function similar to that of wild-type FBL-1, which promotes DTC migration by inhibiting GON-1. We propose that FBL-1 and GON-1 control EMB-9 accumulation in the BM and promote PAT-3 expression to control DTC migration.     

Development of the cation-induced stacking capacity during the biogenesis of higher plant thylakoids

We studied the capacity of the thylakoid membrane to form grana stacks in the presence of cations, monovalent or divalent, added to N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine “low-salt” disorganized plastids during their greening. Grana stacking was monitored by the yield of heavy subchloroplast fractions separated by differential centrifugation after digitonin disruption of plastids (J. H. Argyroudi-Akoyunoglou, 1976, Arch. Biochem. Biophys., 176, 267–274). Primary thylakoids of the agranal protochloroplasts formed in periodic light do not show the cation-induced stacking capacity of the mature green chloroplast thylakoids. Similarly, the cation effect saturates at lower cation concentrations in mature chloroplasts than in plastids of the early stages of greening. The capacity for cation-induced stacking and for saturation of the effect at low cation concentrations appears gradually after exposure to continuous light and parallel to the appearance of chlorophyll b and the polypeptides of the 25,000–30,000 molecular weight range of lipid-free thylakoids, probably derived from the chlorophyll b-rich chlorophyll protein Complex II. The thylakoid peripheral stroma proteins ribulosediphosphate carboxylase and the coupling factor protein are not involved in the cation-induced stacking, since their removal (H. Strotmann, H. Hesse, and K. Edelmann, 1973, Biochim. Biophys. Acta, 314, 202–210) does not affect the thylakoid aggregation.     

DNA cleavage promoted by trigonal-bipyramidal zinc(II) and copper(II) complexes formed by asymmetric tripodal tetradendate 2-[bis(2-aminoethyl)amino]ethanol

Asymmetric trigonal-bipyramidal Zn(II) complex 1 formed by 2-[bis(2-aminoethyl)amino]ethanol (L) was found to be able to promote the cleavage of supercoiled plasmid DNA pBR322 to the nicked and linear DNA via a hydrolytic manner but only in neutral Tris-HCl buffer, no cleavage was observed in HEPES or NaH₂PO₄/Na₂HPO₄ buffer. However, the copper complex 2 of L, possessing the similar coordination geometry, can only promote DNA cleavage via an oxidative mechanism in the presence of ascorbic acid. ESI-MS study implies that complex 1 exist mainly as [Zn(L)]²⁺/[Zn(L-H)]⁺ in neutral Tris-HCl buffer. Moreover, there is no discriminable species for complex 1 in HEPES or NaH₂PO₄/Na₂HPO₄ buffer. A phosphate activation mechanism via phosphate coordinating to Zn(II) center of [Zn(L)]²⁺/[Zn(L-H)]⁺ to form the stable trigonal-bipyramidal structure is proposed for the hydrolytic cleavage promote by complex 1. For complex 2, the abundance of [Cu(L)Cl]⁺ is higher than that of [Cu(L)]²⁺/[Cu(L-H)]⁺ in Tris-HCl buffer. The lower phosphate binding/activating ability of Cu(II) in complex 2 may be the origin for its incapability to promote the hydrolytic DNA cleavage. However, the readily accessible redox potential of Cu(II) makes complex 2 promote the oxidative DNA cleavage. Although the DNA cleavage promoted by complex 1 has no specificity, trigonal-bipyramidal Zn(II) complexes formed by asymmetric tripodal polyamine with ethoxyl pendent should be a novel potential model for practical artificial nuclease.     

Small GTPase Determinants for the Golgi Processing and Plasmalemmal Expression of Human Ether-a-go-go Related (hERG) K+ Channels

The pro-arrhythmic Long QT syndrome (LQT) is linked to 10 different genes (LQT1–10). Approximately 40% of genotype-positive LQT patients have LQT2, which is characterized by mutations in the human ether-a-go-go related gene (hERG). hERG encodes the voltage-gated K⁺ channel α-subunits that form the pore of the rapidly activating delayed rectifier K⁺ current in the heart. The purpose of this study was to elucidate the mechanisms that regulate the intracellular transport or trafficking of hERG, because trafficking is impaired for about 90% of LQT2 missense mutations. Protein trafficking is regulated by small GTPases. To identify the small GTPases that are critical for hERG trafficking, we coexpressed hERG and dominant negative (DN) GTPase mutations in HEK293 cells. The GTPases Sar1 and ARF1 regulate the endoplasmic reticulum (ER) export of proteins in COPII and COPI vesicles, respectively. Expression of DN Sar1 inhibited the Golgi processing of hERG, decreased hERG current (I_hERG) by 85% (n ≥ 8 cells per group, *, p < 0.01), and reduced the plasmalemmal staining of hERG. The coexpression of DN ARF1 had relatively small effects on hERG trafficking. Surprisingly, the coexpression of DN Rab11B, which regulates the endosomal recycling, inhibited the Golgi processing of hERG, decreased I_hERG by 79% (n ≥ 8 cells per group; *, p < 0.01), and reduced the plasmalemmal staining of hERG. These data suggest that hERG undergoes ER export in COPII vesicles and endosomal recycling prior to being processed in the Golgi. We conclude that hERG trafficking involves a pathway between the ER and endosomal compartments that influences expression in the plasmalemma.The human KCNH2 or ether-a-go-go related gene (hERG)³ encodes the voltage-gated K⁺ channel α-subunits that oligomerize to form the pore of the rapidly activating delayed rectifier K⁺ current (I_Kr) in cardiac myocytes (1–3). Hundreds of hERG mutations are linked to the congenital pro-arrhythmic Type 2 Long QT syndrome (LQT2) and functional studies suggest that these mutations result in a loss of normal hERG K⁺ channel (hERG) function (4, 5). In LQT2, missense mutations are the dominant abnormality and many LQT2 missense mutations reduce hERG K⁺ current (I_hERG) by decreasing the intracellular transport or trafficking of hERG to the Golgi apparatus (Golgi) and the cell surface membrane (plasmalemma) (6). Therefore, disruption of hERG K⁺ channel trafficking appears to be a principal mechanism for disease.Movement of proteins between membrane-bound intracellular compartments is mediated by small transport vesicles, which bud from a donor compartment to fuse with an appropriate acceptor compartment. The trafficking of many transmembrane and secretory proteins between the ER and Golgi compartments is dependent on the small GTPases ADP-ribosylation factor 1 (ARF1) and Sar1, which regulate the formation of coat-associated protein complex I (COPI) and II (COPII) vesicles, respectively (7–19). These small GTPases facilitate the polymerization of transport vesicle protein coats on the donor membrane. Vesicular cargo selection, docking, and fusion to the target membrane are regulated by adaptor proteins, SNARE proteins, and Rab GTPases. To rationally develop novel therapeutic targets that may increase the expression of trafficking-deficient LQT2 mutant channels, the molecular mechanisms that regulate the trafficking of hERG need to be explored. The purpose of this study is to identify transport proteins that regulate the trafficking of wild type (WT) hERG. We used a strategy of testing specific WT GTPases or ones containing dominant negative (DN) mutations to interfere with their function.     

Vacuolar ion channels in the liverwort Conocephalum conicum

As a liverwort Conocephalum conicum belongs to the oldest terrestrial plants¹ and is phylogenetically located between green algae and higher plants. Recent patch-clamp recordings on Conocephalum vacuoles^2,3 demonstrate ion channels very similar to higher plants and clearly different from vacuolar ion channels described in green algae. Here we summarize the features of a vacuolar cation channel and a vacuolar anion channel that both are common in terrestrial plants but are not detected in green algae, and we speculate about the molecular identity of these channels in the liverwort Conocephalum.Key words: vacuole, SV channel, anion channel, Conocephalum conicum, Embryophyta