Isolation of Alpha-Toxin-Deficient Clostridium perfringens Type F from Sewage Influents and Effluents.

Clostridium perfringens is classified into types A to G, and all types produce alpha-toxins; however, C. perfringens type F that is negative for phospholipase C (PLC) activity of alpha-toxin has been isolated from the environment and cases of humans afflicted by food poisoning. This study aimed to elucidate the distribution of PLC-negative C. perfringens type F in sewage influents and effluents. Influents and effluents of two wastewater treatment plants were collected monthly between July 2016 and January 2020 and between August 2018 and January 2020, respectively. Isolation rates of PLC-negative C. perfringens type F from sewage influents and effluents were 38% (33/86) and 22% (8/36), and the numbers of isolates were 43 and 13, respectively. The locus of the enterotoxin gene of all isolates was determined to be in a plasmid with an IS1151 sequence, and multilocus sequence typing revealed that all 17 representative isolates were assigned as sequence type 186. Sequencing of the plc gene of these representative isolates showed that nonsense mutation (p.W98*) causing alpha-toxin deficiency should be responsible for a loss of PLC enzymatic activity. These results suggest that alpha toxin-deficient C. perfringens type F is distributed in living and water environments since sewage influents contain community wastewater, and effluents contaminate the environment. Detection of C. perfringens type F, independent of PLC activity, should be carried out on human and environmental samples. IMPORTANCE Understanding the diversity of biochemical characteristics that may affect the identification of bacteria is essential. C. perfringens is a ubiquitous bacterium found in the environment, humans, and animals and is responsible for infectious disease in the intestine. Although the alpha-toxin of C. perfringens may be used for its detection, variants of the alpha-toxin lacking its activity have been isolated from soil and humans experiencing symptoms of diarrhea. It is valuable to disclose the prevalence of the alpha-toxin variant in the sewage of wastewater treatment plants, as it may reflect the hygienic condition of the community, as it would be a pollution source for the environment. This study shows the persistent existence and genetic characteristics of the alpha-toxin variant in sewage and reveals a lacking mechanism of the alpha-toxin activity and proposes the detection method of C. perfringens, independent of the alpha-toxin activity.

Revisiting the role of phospholipases C in virulence and the lifecycle of Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the agent of human tuberculosis has developed different virulence mechanisms and virulence-associated tools during its evolution to survive and multiply inside the host. Based on previous reports and by analogy with other bacteria, phospholipases C (PLC) of M. tuberculosis were thought to be among these tools. To get deeper insights into the function of PLCs, we investigated their putative involvement in the intracellular lifestyle of M. tuberculosis, with emphasis on phagosomal rupture and virulence, thereby re-visiting a research theme of longstanding interest. Through the construction and use of an M. tuberculosis H37Rv PLC-null mutant (ΔPLC) and control strains, we found that PLCs of M. tuberculosis were not required for induction of phagosomal rupture and only showed marginal, if any, impact on virulence of M. tuberculosis in the cellular and mouse infection models used in this study. In contrast, we found that PLC-encoding genes were strongly upregulated under phosphate starvation and that PLC-proficient M. tuberculosis strains survived better than ΔPLC mutants under conditions where phosphatidylcholine served as sole phosphate source, opening new perspectives for studies on the role of PLCs in the lifecycle of M. tuberculosis.

Ceramide signalling impinges on Sit4p and Hog1p to promote mitochondrial fission and mitophagy in Isc1p-deficient cells.

Mitochondria function as the powerhouses of the cell for energy conversion through the oxidative phosphorylation process. Accumulation of dysfunctional mitochondria promotes a bioenergetic crisis and cell death by apoptosis. Yeast cells lacking Isc1p, an orthologue of mammalian neutral sphingomyelinase type 2, exhibit mitochondrial dysfunction and shortened lifespan associated with the accumulation of specific ceramide species and activation of the PP2A-like protein phosphatase Sit4p and of the Hog1p kinase. Here, we show that isc1Δ cells display hyperactivation of mitophagy that is suppressed by downregulating Sit4p, Hog1p or the TORC1-Sch9p pathway. Notably, isc1Δ cells also have high levels of Dnm1p associated with unbalanced mitochondrial fission, leading to mitochondrial fragmentation, and DNM1 deletion suppressed the oxidative stress sensitivity and shortened lifespan of isc1Δ cells. Moreover, Isc1p and Dnm1p physically interact, suggesting a possible regulatory role for Isc1p in mitochondrial dynamics. Overall, our work demonstrates that Isc1p-mediated ceramide signalling regulates mitophagy and mitochondrial dynamics in yeast with impact on mitochondrial function and lifespan. Since ceramides have been implicated in ageing and diseases associated with mitochondrial dysfunction, our findings suggest that therapeutic strategies targeting ceramide signalling may improve mitochondrial function and human healthspan.

Inositol phosphosphingolipid phospholipase C1 regulates plasma membrane ATPase (Pma1) stability in Cryptococcus neoformans.

Cryptococcus neoformans is a facultative intracellular pathogen, which can replicate in the acidic environment inside phagolysosomes. Deletion of the enzyme inositol-phosphosphingolipid-phospholipase-C (Isc1) makes C. neoformans hypersensitive to acidic pH likely by inhibiting the function of the proton pump, plasma membrane ATPase (Pma1). In this work, we examined the role of Isc1 on Pma1 transport and oligomerization. Our studies showed that Isc1 deletion did not affect Pma1 synthesis or transport, but significantly inhibited Pma1 oligomerization. Interestingly, Pma1 oligomerization could be restored by supplementing the medium with phytoceramide. These results offer insight into the mechanism of intracellular survival of C. neoformans.

Ca-dependent nonsecretory vesicle fusion in a secretory cell.

We have compared Ca-dependent exocytosis in excised giant membrane patches and in whole-cell patch clamp with emphasis on the rat secretory cell line, RBL. Stable patches of 2-4 pF are easily excised from RBL cells after partially disrupting actin cytoskeleton with latrunculin A. Membrane fusion is triggered by switching the patch to a cytoplasmic solution containing 100-200 microM free Ca. Capacitance and amperometric recording show that large secretory granules (SGs) containing serotonin are mostly lost from patches. Small vesicles that are retained (non-SGs) do not release serotonin or other substances detected by amperometry, although their fusion is reduced by tetanus toxin light chain. Non-SG fusion is unaffected by N-ethylmaleimide, phosphatidylinositol-4,5-bis-phosphate (PI(4,5)P(2)) ligands, such as neomycin, a PI-transfer protein that can remove PI from membranes, the PI(3)-kinase inhibitor LY294002 and PI(4,5)P(2), PI(3)P, and PI(4)P antibodies. In patch recordings, but not whole-cell recordings, fusion can be strongly reduced by ATP removal and by the nonspecific PI-kinase inhibitors wortmannin and adenosine. In whole-cell recording, non-SG fusion is strongly reduced by osmotically induced cell swelling, and subsequent recovery after shrinkage is then inhibited by wortmannin. Thus, membrane stretch that occurs during patch formation may be a major cause of differences between excised patch and whole-cell fusion responses. Regarding Ca sensors for non-SG fusion, fusion remains robust in synaptotagmin (Syt) VII-/- mouse embryonic fibroblasts (MEFs), as well as in PLCdelta1, PLC delta1/delta4, and PLCgamma1-/- MEFs. Thus, Syt VII and several PLCs are not required. Furthermore, the Ca dependence of non-SG fusion reflects a lower Ca affinity (K(D) approximately 71 microM) than expected for these C2 domain-containing proteins. In summary, we find that non-SG membrane fusion behaves and is regulated substantially differently from SG fusion, and we have identified an ATP-dependent process that restores non-SG fusion capability after it is perturbed by membrane stretch or cell dilation.

Isc1p plays a key role in hydrogen peroxide resistance and chronological lifespan through modulation of iron levels and apoptosis.

The inositolphosphosphingolipid phospholipase C (Isc1p) of Saccharomyces cerevisiae belongs to the family of neutral sphingomyelinases that generates the bioactive sphingolipid ceramide. In this work the role of Isc1p in oxidative stress resistance and chronological lifespan was investigated. Loss of Isc1p resulted in a higher sensitivity to hydrogen peroxide that was associated with an increase in oxidative stress markers, namely intracellular oxidation, protein carbonylation, and lipid peroxidation. Microarray analysis showed that Isc1p deficiency up-regulated the iron regulon leading to increased levels of iron, which is known to catalyze the production of the highly reactive hydroxyl radicals via the Fenton reaction. In agreement, iron chelation suppressed hydrogen peroxide sensitivity of isc1Delta mutants. Cells lacking Isc1p also displayed a shortened chronological lifespan associated with oxidative stress markers and aging of parental cells was correlated with a decrease in Isc1p activity. The analysis of DNA fragmentation and caspase-like activity showed that Isc1p deficiency increased apoptotic cell death associated with oxidative stress and aging. Furthermore, deletion of Yca1p metacaspase suppressed the oxidative stress sensitivity and premature aging phenotypes of isc1Delta mutants. These results indicate that Isc1p plays an important role in the regulation of cellular redox homeostasis, through modulation of iron levels, and of apoptosis.

Phospholipase C regulation of phosphatidylinositol 3,4,5-trisphosphate-mediated chemotaxis.

Generation of a phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] gradient within the plasma membrane is important for cell polarization and chemotaxis in many eukaryotic cells. The gradient is produced by the combined activity of phosphatidylinositol 3-kinase (PI3K) to increase PI(3,4,5)P(3) on the membrane nearest the polarizing signal and PI(3,4,5)P(3) dephosphorylation by phosphatase and tensin homolog deleted on chromosome ten (PTEN) elsewhere. Common to both of these enzymes is the lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)], which is not only the substrate of PI3K and product of PTEN but also important for membrane binding of PTEN. Consequently, regulation of phospholipase C (PLC) activity, which hydrolyzes PI(4,5)P(2), could have important consequences for PI(3,4,5)P(3) localization. We investigate the role of PLC in PI(3,4,5)P(3)-mediated chemotaxis in Dictyostelium. plc-null cells are resistant to the PI3K inhibitor LY294002 and produce little PI(3,4,5)P(3) after cAMP stimulation, as monitored by the PI(3,4,5)P(3)-specific pleckstrin homology (PH)-domain of CRAC (PH(CRAC)GFP). In contrast, PLC overexpression elevates PI(3,4,5)P(3) and impairs chemotaxis in a similar way to loss of pten. PI3K localization at the leading edge of plc-null cells is unaltered, but dissociation of PTEN from the membrane is strongly reduced in both gradient and uniform stimulation with cAMP. These results indicate that local activation of PLC can control PTEN localization and suggest a novel mechanism to regulate the internal PI(3,4,5)P(3) gradient.

Phospholipase cbeta is critical for T cell chemotaxis.

Chemokines acting through G protein-coupled receptors play an essential role in the immune response. PI3K and phospholipase C (PLC) are distinct signaling molecules that have been proposed in the regulation of chemokine-mediated cell migration. Studies with knockout mice have demonstrated a critical role for PI3K in G(alphai) protein-coupled receptor-mediated neutrophil and lymphocyte chemotaxis. Although PLCbeta is not essential for the chemotactic response of neutrophils, its role in lymphocyte migration has not been clearly defined. We compared the chemotactic response of peripheral T cells derived from wild-type mice with mice containing loss-of-function mutations in both of the two predominant lymphocyte PLCbeta isoforms (PLCbeta2 and PLCbeta3), and demonstrate that loss of PLCbeta2 and PLCbeta3 significantly impaired T cell migration. Because second messengers generated by PLCbeta lead to a rise in intracellular calcium and activation of PKC, we analyzed which of these responses was critical for the PLCbeta-mediated chemotaxis. Intracellular calcium chelation decreased the chemotactic response of wild-type lymphocytes, but pharmacologic inhibition of several PKC isoforms had no effect. Furthermore, calcium efflux induced by stromal cell-derived factor-1alpha was undetectable in PLCbeta2beta3-null lymphocytes, suggesting that the migration defect is due to the impaired ability to increase intracellular calcium. This study demonstrates that, in contrast to neutrophils, phospholipid second messengers generated by PLCbeta play a critical role in T lymphocyte chemotaxis.

Lack of phospholipase C-delta1 induces skin inflammation.

Phospholipase C (PLC) is a key enzyme in phosphoinositide signaling. We previously generated PLC-delta1 knockout (KO) mice and found that these mice showed remarkable hair loss caused by abnormalities in hair follicle structures. Here we show that the skin of PLC-delta1 KO mice displays typical inflammatory phenotypes, including increased dermal cellularity, leukocyte infiltration, and expression of pro-inflammatory cytokines. In addition, exogenously expressed PLC-delta1 attenuates lipopolysaccharide-induced expression of IL-1beta, a pro-inflammatory cytokine, in an enzymatic activity-dependent manner. Furthermore, suppression of skin inflammation by anti-inflammatory reagents cured the epidermal hyperplasia in PLC-delta1 KO mice. Taken together, these results indicate that lack of PLC-delta1 induces skin inflammation and that the epidermal hyperplasia in PLC-delta1 KO mice is caused by skin inflammation. Our results also suggest that PLC-delta1 regulates homeostasis of the immune system in skin.

G alpha(q)-coupled muscarinic acetylcholine receptors enhance nicotinic acetylcholine receptor signaling in Caenorhabditis elegans mating behavior.

In this study, we address why metabotropic and ionotropic cholinergic signaling pathways are used to facilitate motor behaviors. We demonstrate that a G alpha(q)-coupled muscarinic acetylcholine receptor (mAChR) signaling pathway enhances nicotinic acetylcholine receptor (nAChR) signaling to facilitate the insertion of the Caenorhabditis elegans male copulatory spicules into the hermaphrodite during mating. Previous studies showed that ACh (acetylcholine) activates nAChRs on the spicule protractor muscles to induce the attached spicules to extend from the tail. Using the mAChR agonist Oxo M (oxotremorine M), we identified a GAR-3(mAChR)-G alpha(q) pathway that promotes protractor muscle contraction by upregulating nAChR signaling before mating. GAR-3(mAChR) is expressed in the protractor muscles and in the spicule-associated SPC and PCB cholinergic neurons. However, ablation of these neurons or impairing cholinergic transmission reduces drug-induced spicule protraction, suggesting that drug-stimulated neurons directly activate muscle contraction. Behavioral analysis of gar-3 mutants indicates that, in wild-type males, GAR-3(mAChR) expression in the SPC and PCB neurons is required for the male to sustain rhythmic spicule muscle contractions during attempts to breach the vulva. We propose that the GAR-3(mAChR)/G alpha(q) pathway sensitizes the spicule neurons and muscles before and during mating so that the male can respond to hermaphrodite vulva efficiently.

Epac-mediated activation of phospholipase C(epsilon) plays a critical role in beta-adrenergic receptor-dependent enhancement of Ca2+ mobilization in cardiac myocytes.

Recently we demonstrated that PLC(epsilon) plays an important role in beta-adrenergic receptor (betaAR) stimulation of Ca(2+)-induced Ca(2+) release (CICR) in cardiac myocytes. Here we have reported for the first time that a pathway downstream of betaAR involving the cAMP-dependent Rap GTP exchange factor, Epac, and PLC(epsilon) regulates CICR in cardiac myocytes. To demonstrate a role for Epac in the stimulation of CICR, cardiac myocytes were treated with an Epac-selective cAMP analog, 8-4-(chlorophenylthio)-2'-O-methyladenosine-3',5'-monophosphate (cpTOME). cpTOME treatment increased the amplitude of electrically evoked Ca(2+) transients, implicating Epac for the first time in cardiac CICR. This response is abolished in PLC(epsilon)(-/-) cardiac myocytes but rescued by transduction with PLC(epsilon), indicating that Epac is upstream of PLC(epsilon). Furthermore, transduction of PLC(epsilon)(+/+) cardiac myocytes with a Rap inhibitor, RapGAP1, significantly inhibited isoproterenol-dependent CICR. Using a combination of cpTOME and PKA-selective activators and inhibitors, we have shown that betaAR-dependent increases in CICR consist of two independent components mediated by PKA and the novel Epac/(epsilon) pathway. We also show that Epac/PLC(epsilon)-dependent effects on CICR are independent of sarcoplasmic reticulum loading and Ca(2+) clearance mechanisms. These data define a novel endogenous PKA-independent betaAR-signaling pathway through cAMP-dependent Epac activation, Rap, and PLC(epsilon) that enhances intracellular Ca(2+) release in cardiac myocytes.

Disruption of phospholipase Cdelta4 gene modulates the liver regeneration in cooperation with nuclear protein kinase C.

Phospholipase Cdelta4 (PLC delta4) gene has been cloned from the cDNA library of regenerating rat liver. Using PLC delta4 gene-disrupted mice (PLC delta4(-/-)), we studied a role of PLC delta4 during liver regeneration after partial hepatectomy (PH). In PLC delta4(-/-), liver regeneration occurred in an apparently normal way. However, BrdU-indices indicated that PLC delta4 gene disruption delayed the onset of DNA synthesis by 2 h. Noticeably, the BrdU-indices in PLC delta4(+/+) remained rather constant throughout S phase, 25-35%, whereas in PLC delta4(-/-), it fluctuated drastically from 25% at 34 h to 65% at late S, 42 h after PH. This fact showed that PLC delta4 gene disruption caused a higher synchronization of cell proliferation. The mRNA for PLC delta4 in PLC delta4(+/+) appeared at late G1, and the expression continued throughout S phase. PLC activity increased transiently in chromatin at the late G1 and S phases in only PLC delta4(+/+), but not in PLC delta4(-/-). The specific increases in PLC activity well correlated with the transient increases of protein kinase C (PKC) alpha in chromatin of PLC delta4(+/+). PKC epsilon also increased transiently in chromatin from PLC delta4(+/+) at late S. It is concluded that PLC delta4 regulates the liver regeneration in cooperation with nuclear PKC alpha and epsilon.

Gsalpha is involved in sugar perception in Drosophila melanogaster.

In Drosophila melanogaster, gustatory receptor genes (Grs) encode G-protein-coupled receptors (GPCRs) in gustatory receptor neurons (GRNs) and some olfactory receptor neurons. One of the Gr genes, Gr5a, encodes a sugar receptor that is expressed in a subset of GRNs and has been most extensively studied both molecularly and physiologically, but the G-protein alpha subunit (Galpha) that is coupled to this sugar receptor remains unknown. Here, we propose that Gs is the Galpha that is responsible for Gr5a-mediated sugar-taste transduction, based on the following findings: First, immunoreactivities against Gs were detected in a subset of GRNs including all Gr5a-expressing neurons. Second, trehalose-intake is reduced in flies heterozygous for null mutations in DGsalpha, a homolog of mammalian Gs, and trehalose-induced electrical activities in sugar-sensitive GRNs were depressed in those flies. Furthermore, expression of wild-type DGsalpha in sugar-sensitive GRNs in heterozygotic DGsalpha mutant flies rescued those impairments. Third, expression of double-stranded RNA for DGsalpha in sugar-sensitive GRNs depressed both behavioral and electrophysiological responses to trehalose. Together, these findings indicate that DGsalpha is involved in trehalose perception. We suggest that sugar-taste signals are processed through the Gsalpha-mediating signal transduction pathway in sugar-sensitive GRNs in Drosophila.

Phospholipase Cbeta1 modulates pain sensitivity, opioid antinociception and opioid tolerance formation.

Phospholipase C (PLC) activity has been implicated in multiple opioid-induced sequelae. The relevance of PLC-linked pathways to opioid actions is isoform-specific. Chronic morphine augments PLCbeta1 signaling while diminishing that of PLCbeta3. This suggests that PLCbeta1 makes an important contribution to opioid tolerance formation (PNAS 100: 13686-1369, 2003). In the present study, PLCbeta1 knockout animals (-/-) were used to assess the relevance of PLCbeta1 to pain thresholds, morphine antinociception and analgesic tolerance formation. Response latencies to thermal nociceptive stimuli were markedly diminished in -/- animals relative to their wild-type (+/+) and heterozygous (+/-) counterparts; thermal nociceptive thresholds obtained in +/+ and +/- mice did not differ. This suggests that the contribution of PLCbeta1 to thermal pain thresholds requires a critical concentration of PLCbeta1 protein. PLCbeta1 genotype also influenced acute and chronic responsiveness to morphine. Analgesic dose responsiveness and the magnitude of analgesic tolerance formation to morphine were significantly attenuated in -/- vs. +/+ animals. Notably, in contrast to thermal nociceptive thresholds, acute and chronic morphine responsiveness differed significantly only between +/+ and -/- genotypes and not between -/- vs. +/- groups. These data suggest that whereas the contribution of PLCbeta1 to thermal nociceptive response thresholds requires a critical concentration of PLCbeta1 protein, its participation in morphine analgesic and tolerance-producing mechanisms is graded. Importantly, GTPgammaS binding studies revealed that there is no detectable diminution in functional opioid receptors in spinal tissue from -/- animals. This underscores the importance of PLCbeta1 to morphine sequelae that are initiated downstream from the opioid receptor.

PLCbeta2-independent behavioral avoidance of prototypical bitter-tasting ligands.

Using a brief-access taste assay, we show in the present report that although phospholipase C beta2 knockout (PLCbeta2 KO) mice are unresponsive to low- and midrange concentrations of quinine and denatonium, they do significantly avoid licking higher concentrations of these aversive compounds. PLCbeta2 KO mice displayed no concentration-dependent licking of the prototypical sweetener sucrose but were similar to wild-type mice in their responses to citric acid and NaCl, notwithstanding some interesting exceptions. Although these findings confirm an essential role for PLCbeta2 in taste responsiveness to sucrose and to low- to midrange concentrations of quinine and denatonium in mice as previously reported, they importantly suggest that higher concentrations of the latter two compounds, which are bitter to humans, can engage a PLCbeta2-independent taste transduction pathway.

Phospholipase C-gamma 2 is a critical signaling mediator for murine NK cell activating receptors.

Phospholipase C-gamma (PLCgamma) is a key regulator of intracellular Ca(2+) mobilization. Two isoforms of PLCgamma have been identified, PLCgamma1 and PLCgamma2. Previously, in vitro studies indicated that activating NK cell receptors signal through both isoforms. However, PLCgamma2 deficiency alone was sufficient to induce a substantial impairment of NK cell-mediated cytotoxicity in vitro. Why PLCgamma2 is more important than PLCgamma1 for NK cell activation and whether PLCgamma2 is also critical for NK cell development, secretion of IFN-gamma, and clearance of viral infections in vivo is not known. In this study, we report that PLCgamma2 is the predominant isoform expressed in murine NK cells. PLCgamma2 deficiency did not affect NK cell numbers in bone marrow and spleen, but acquisition of Ly49 receptors by NK cells was partially impaired. PLCgamma2-deficient NK cells exhibited a dramatic impairment of cytolytic function and IFN-gamma production upon ligation of activating receptors, whereas they did secrete IFN-gamma in response to cytokines. Consequently, mice lacking PLCgamma2 controlled murine CMV infection substantially less effectively than did wild-type animals, and this defect was most evident in the spleen, where viral clearance mostly depends on NK cell lytic function. These results demonstrate that PLCgamma2 is crucial for development of the NK cell receptor repertoire and signaling of activating NK cell receptors, mediating optimal NK cell function in vivo.

Phospholipase C-beta3 mediates the thrombin-induced Ca2+ response in glial cells.

Phospholipase C-beta (PLC-beta) hydrolyses phosphatidylinositol 4,5-bisphosphate and generates inositol 1,4,5-trisphosphate in response to activation of various G protein-coupled receptors (GPCRs). Using glial cells from knock-out mice lacking either PLC-beta1 [PLC-beta1 (-/-)] or PLC-beta3 [PLC-beta3 (-/-)], we examined which isotype of PLC-beta participated in the cellular signaling events triggered by thrombin. Generation of inositol phosphates (IPs) was enhanced by thrombin in PLC-beta1 (-/-) cells, but was negligible in PLC-beta3 (-/-) cells. Expression of PLC-beta3 in PLC-beta3 (-/-) cells resulted in an increase in pertussis toxin (PTx)-sensitive IPs in response to thrombin as well as to PAR1-specific peptide, while expression of PLC-beta1 in PLC-beta1 (-/-) cells did not have any effect on IP generation. The thrombin-induced [Ca2+]i increase was delayed and attenuated in PLC-beta3 (-/-) cells, but normal in PLC-beta1 (-/-) cells. Pertussis toxin evoked a delayed [Ca2+]i increase in PLC-beta3 (-/-) cells as well as in PLC-beta1 (-/-) cells. These results suggest that activation of PLC-beta3 by pertussis toxin-sensitive G proteins is responsible for the transient [Ca2+]i increase in response to thrombin, whereas the delayed [Ca2+]i increase may be due to activation of some other PLC, such as PLC-beta4, acting via PTx-insensitive G proteins.

PLCgamma1 is essential for early events in integrin signalling required for cell motility.

Cell motility is a critical event in many processes and is underlined by complex signalling interactions. Although many components have been implicated in different forms of cell migration, identification of early key mediators of these events has proved difficult. One potential signalling intermediate, PLCgamma1, has previously been implicated in growth-factor-mediated chemotaxis but its position and roles in more-complex motility events remain poorly understood. This study links PLCgamma1 to early, integrin-regulated changes leading to cell motility. The key role of PLCgamma1 was supported by findings that specific depletion of PLCgamma1 by small interfering (si)RNA, or by pharmacological inhibition, or the absence of this isoform in PLCgamma1(-/-) cells resulted in the failure to form cell protrusions and undergo cell spreading and elongation in response to integrin engagement. This integrin-PLCgamma1 pathway was shown to underlie motility processes involved in morphogenesis of endothelial cells on basement membranes and invasion of cancer cells into such three-dimensional matrices. By combining cellular and biochemical approaches, we have further characterized this signalling pathway. Upstream of PLCgamma1 activity, beta1 integrin and Src kinase are demonstrated to be essential for phosphorylation of PLCgamma1, formation of protein complexes and accumulation of intracellular calcium. Cancer cell invasion and the early morphological changes associated with cell motility were abolished by inhibition of beta1 integrin or Src. Our findings establish PLCgamma1 as a key player in integrin-mediated cell motility processes and identify other critical components of the signalling pathway involved in establishing a motile phenotype. This suggests a more general role for PLCgamma1 in cell motility, functioning as a mediator of both growth factor and integrin-initiated signals.

Botrocetin/VWF-induced signaling through GPIb-IX-V produces TxA2 in an alphaIIbbeta3- and aggregation-independent manner.

Binding of von Willebrand factor (VWF) to the platelet membrane glycoprotein (GP) Ib-IX-V complex initiates a signaling cascade that causes alphaIIbbeta3 activation and platelet aggregation. Previous work demonstrated that botrocetin (bt)/VWF-mediated agglutination activates alphaIIbbeta3 and elicits adenosine triphosphate (ATP) secretion in a thromboxane A2 (TxA2)- and Ca2+-dependent manner. This agglutination-elicited TxA2 production occurs in the absence of ATP secretion. However, the signaling components and signaling network or pathway activated by GPIb-mediated agglutination to cause TxA2 production have not been identified. Therefore, the focus of this study was to elucidate at least part of the signal transduction network or pathway activated by GPIb-mediated agglutination to cause TxA2 production. The phosphatidylinositol 3-kinase (PI3K) selective inhibitor wortmannin, and mouse platelets deficient in Lyn, Src, Syk, Src homology 2 (SH2) domain-containing leukocyte protein 76 (SLP-76), phospholipase Cgamma2 (PLCgamma2), linker for activation of T cells (LAT), or Fc receptor gamma-chain (FcRgamma-chain) were used for these studies. LAT and FcRgamma-chain were found not to be required for agglutination-driven TxA2 production or activation of alphaIIbbeta3, but were required for granule secretion and aggregation. The results also clearly demonstrate that bt/VWF-mediated agglutination-induced TxA2 production is dependent on signaling apparently initiated by Lyn, enhanced by Src, and propagated through Syk, SLP-76, PI3K, PLCgamma2, and protein kinase C (PKC).

A new tyrosine phosphorylation site in PLC gamma 1: the role of tyrosine 775 in immune receptor signaling.

Phospholipase Cgamma (PLCgamma) is a ubiquitous gatekeeper of calcium mobilization and diacylglycerol-mediated events induced by the activation of Ag and growth factor receptors. The activity of PLCgamma is regulated through its controlled membrane translocation and tyrosine (Y) phosphorylation. Four activation-induced tyrosine phosphorylation sites have been previously described (Y472, Y771, Y783, and Y1254), but their specific roles in Ag receptor-induced PLCgamma1 activation are not fully elucidated. Unexpectedly, we found that the phosphorylation of a PLCgamma1 construct with all four sites mutated to phenylalanine was comparable with that observed with wild-type PLCgamma1, suggesting the existence of an unidentified site(s). Sequence alignment with known phosphorylation sites in PLCgamma2 indicated homology of PLCgamma1 tyrosine residue 775 (Y775) with PLCgamma2 Y753, a characterized phosphorylation site. Tyrosine 775 was characterized as a phosphorylation site using phospho-specific anti-Y775 antiserum, and by mutational analysis. Phosphorylation of Y775 did not depend on the other tyrosines, and point mutation of PLCgamma1 Y775, or the previously described Y783, substantially reduced AgR-induced calcium, NF-AT, and AP-1 activation. Mutation of Y472, Y771, and Y1254 had no effect on overall PLCgamma1 phosphorylation or activation. Although the concomitant mutation of Y775 and Y783 abolished downstream PLCgamma1 signaling, these two tyrosines were sufficient to reconstitute the wild-type response in the absence of functional Y472, Y771, and Y1254. These data establish Y775 as a critical phosphorylation site for PLCgamma1 activation and confirm the functional importance of Y783.