Isolation and characterization of temperature-sensitiveplc1 mutants of the yeastSaccharomyces cerevisiae

ThePLC1 gene of the yeastSaccharomyces cerevisiae has been discovered to encode a homolog of mammalian phosphoinositide-specific phospholipase C (PLC). Five temperature-sensitiveplc1 mutants were isolated by in vitro mutagenesis with subsequent plasmid shuffling. All of the amino acid substitutions that caused a temperature-sensitive growth phenotype were located in the X or the Y region, both of which are conserved among PLC isoenzymes. The PLC activity of all products of mutantplc1 genes was dramatically lower than that of the wild-type product, indicating that PLC activity itself is important for cell growth. At the restrictive temperature,plc1 mutant cells ceased growth at random times during the cell cycle, a result that suggests thatPLC1 is required at several or all stages of the cell cycle.     

PLC1 and H2O2 Mediated the Allelopathic Effect of Oridonin on Root Growth in Arabidopsis thaliana

Oridonin is a diterpenoid isolated from medicinal herb Rabdosia rubescens (Hemsl.) Hara (Lamiaceae), which has an allelopathic effect on plants. Phospholipase C (PLC1) and hydrogen peroxide (H₂O₂) are involved in many biotic or abiotic stress responses. Using the 16-day-old seedlings of Arabidopsis thaliana ecotype (WT) and PLC1-deficient mutant (plc1) as materials (treated with 10 μM or 60 μM oridonin for 72 h), the effect of oridonin on root growth regulating by PLC1 and H₂O₂ was investigated. The results showed that the promoting of root growth was about 6.9% at 10 μmol L^?1 oridonin and the inhibiting of root growth was about 19.73% at 60 μmol L^?1 oridonin in WT, the inhibiting of root growth was about 10.5% and 41.2% at 10 mol L^?1 and 60 mol L^?1 oridonin, respectively, in plc1. The expression of ARR1, ARR12, and AHK3 was promoted at low concentrations of oridonin and inhibited at high concentrations in WT, whereas the expression of ARR1 and ARR12 was inhibited with the increase of oridonin concentration in plc1. This suggested that PLC1 was involved in the root growth regulation of oridonin. H₂O₂ was promoted by oridonin with concentration dependence pattern in root cells. Oridonin increased the activity of antioxidant enzymes in both WT and plc1, but the activity of antioxidant enzymes in plc1 was lower than WT. This indicated that PLC1 involved in the activation of antioxidant enzymes promoted by the oridonin. Exogenous CaCl₂ facilitated the accumulation of H₂O₂ in both WT and plc1. And the H₂O₂ of WT was obviously higher than that of plc1. The root growth of WT was inhibited by CaCl₂ with the increase of oridonin. However, there is no effect of CaCl₂ on the root growth in plc1. This reflected that PLC1 positively involved in the regulation of Ca²⁺ on the H₂O₂ and the inhibition effect of Ca²⁺ on the root growth under oridonin treatment. PA promoted the H₂O₂ and suppressed the root growth under oridonin treatment in both WT and plc1. In plc1, PA facilitated the root growth with no oridonin and inhibited the root growth with the increase of oridonin. This reflected that PLC1 positively regulated the promotion effect of PA on the root growth under high oridonin treatment. PLC1 mediated oridonin (10 and 60 mol L^?1) to regulate H₂O₂ levels in A. thaliana seedlings, thereby regulating root tip cell morphology and mitosis. These results demonstrated that PLC1 mediated the low-promotion and high-inhibition effect of oridonin on the root growth in A. thaliana by regulating the concentrations of Ca²⁺ and PA, and further affecting the intracellular H₂O₂ level.

     

Phosphatidylinositol‐specific phospholipase C2 functions in auxin‐modulated root development

Nine phosphatidylinositol‐specific phospholipases C (PLCs) have been identified in the Arabidopsis genome; among the importance of PLC2 in reproductive development is significant. However, the role of PLC2 in vegetative development such as in root growth is elusive. Here, we report that plc2 mutants displayed multiple auxin‐defective phenotypes in root development, including short primary root, impaired root gravitropism, and inhibited root hair growth. The DR5:GUS expression and the endogenous indole‐3‐acetic acid (IAA) content, as well as the responses of a set of auxin‐related genes to exogenous IAA treatment, were all decreased in plc2 seedlings, suggesting the influence of PLC2 on auxin accumulation and signalling. The root elongation of plc2 mutants was less sensitive to the high concentration of exogenous auxins, and the application of 1‐naphthaleneacetic acid or the auxin transport inhibitor N‐1‐naphthylphthalamic acid could rescue the root hair growth of plc2 mutants. In addition, the PIN2 polarity and cycling in plc2 root epidermis cells were altered. These results demonstrate a critical role of PLC2 in auxin‐mediated root development in Arabidopsis, in which PLC2 influences the polar distribution of PIN2.     

Role and mechanism of phosphatidylinositol-specific phospholipase C in survival and virulence of Cryptococcus neoformans

Phospholipase B1 (Plb1) is secreted after release from its glycosylphosphatidylinositol anchor and is implicated in initiation and dissemination of infection of the pathogenic fungus, Cryptococcus neoformans . To investigate the role of phosphatidylinositol-specific phospholipase C (PI-PLC) in Plb1 secretion, we identified two putative PI-PLC-encoding genes in C. neoformans var. grubii ( PLC1 and PLC2 ), and created Δ plc1 and Δ plc2 deletion mutants. In Δ plc1 , which expressed less PI-PLC activity than wild type (WT), three major cryptococcal virulence traits, Plb1 secretion, melanin production and growth at host temperature (37°C) were abolished and absence of Plb1 secretion coincided with Plb1 accumulation in plasma membranes. In addition, Δ plc1 cell walls were defective, as indicated by cell clumping and irregular morphology, slower growth and an inability to activate mitogen-activated protein kinase (MAPK) in the presence of cell wall-perturbing agents. In contrast to Δ plc2 , which was as virulent as WT, Δ plc1 was avirulent in mice and exhibited attenuated killing of Caenorhabditis elegans at 25°C, demonstrating that mechanism(s) independent of the 37°C growth defect contribute to the virulence composite. We conclude that Plc1 is a central regulator of cryptococcal virulence, acting through the protein kinase C/MAPK pathway, that it regulates release of Plb1 from the plasma membrane and is a candidate antifungal drug target.     

Arabidopsis AtPLC2 Is a Primary Phosphoinositide-Specific Phospholipase C in Phosphoinositide Metabolism and the Endoplasmic Reticulum Stress Response

Phosphoinositides represent important lipid signals in the plant development and stress response. However, multiple isoforms of the phosphoinositide biosynthetic genes hamper our understanding of the pivotal enzymes in each step of the pathway as well as their roles in plant growth and development. Here, we report that phosphoinositide-specific phospholipase C2 (AtPLC2) is the primary phospholipase in phosphoinositide metabolism and is involved in seedling growth and the endoplasmic reticulum (ER) stress responses in Arabidopsis thaliana. Lipidomic profiling of multiple plc mutants showed that the plc2-1 mutant increased levels of its substrates phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, suggesting that the major phosphoinositide metabolic pathway is impaired. AtPLC2 displayed a distinct tissue expression pattern and localized at the plasma membrane in different cell types, where phosphoinositide signaling occurs. The seedlings of plc2-1 mutant showed growth defect that was complemented by heterologous expression of AtPLC2, suggesting that phosphoinositide-specific phospholipase C activity borne by AtPLC2 is required for seedling growth. Moreover, the plc2-1 mutant showed hypersensitive response to ER stress as evidenced by changes in relevant phenotypes and gene expression profiles. Our results revealed the primary enzyme in phosphoinositide metabolism, its involvement in seedling growth and an emerging link between phosphoinositide and the ER stress response.     

Arabidopsis PLC2 is involved in auxin‐modulated reproductive development

Phospholipase C (PLC) is an enzyme that plays crucial roles in various signal transduction pathways in mammalian cells. However, the role of PLC in plant development is poorly understood. Here we report involvement of PLC2 in auxin‐mediated reproductive development in Arabidopsis. Disruption of PLC2 led to sterility, indicating a significant role for PLC2 in reproductive development. Development of both male and female gametophytes was severely perturbed in plc2 mutants. Moreover, elevated auxin levels were observed in plc2 floral tissues, suggesting that the infertility of plc2 plants may be associated with increased auxin concentrations in the reproductive organs. We show that expression levels of the auxin reporters DR5:GUS and DR5:GFP were elevated in plc2 anthers and ovules. In addition, we found that expression of the auxin biosynthetic YUCCA genes was increased in plc2 plants. We conclude that PLC2 is involved in auxin biosynthesis and signaling, thus modulating development of both male and female gametophytes in Arabidopsis.     

Phosphatidylinositol‐hydrolyzing phospholipase C4 modulates rice response to salt and drought

Phosphatidylinositol‐specific phospholipase C (PI‐PLC) is involved in stress signalling but its signalling function remains largely unknown in crop plants. Here, we report that the PI‐PLC4 from rice (Oryza sativa cv), OsPLC4, plays a positive role in osmotic stress response. Two independent knockout mutants, plc4‐1 and plc4‐2, exhibited decreased seedling growth and survival rate whereas overexpression of OsPLC4 improved survival rate under high salinity and water deficiency, compared with wild type (WT). OsPLC4 hydrolyses PI, phosphatidylinositol 4‐phosphate (PI4P), and phosphatidylinositol‐4,5‐bisphosphate (PIP₂) to generate diacylglycerol (DAG) in vitro. Knockout of OsPLC4 attenuated salt‐induced increase of phosphatidic acid (PA) whereas overexpression of OsPLC4 decreased the level of PI4P and PIP₂ under salt treatment. Applications of DAG or PA restored the growth defect of plc4‐1 to WT but DAG kinase inhibitor 1 blocked the complementary effect of DAG in plc4‐1 under salt stress. In addition, the loss of OsPLC4 compromised the increase of inositol triphosphate and free cytoplasmic Ca²⁺ ([Ca²⁺]_cyt) and inhibited the induction of genes involved in Ca²⁺ sensor and osmotic stress response to salt stress. The results indicate that OsPLC4 modulates the activity of two signalling pathways, PA and Ca²⁺, to affect rice seedling response to osmotic stress.     

Phosphoinositide-specific phospholipase C forms a complex with 14-3-3 proteins and is involved in expression of UV resistance in fission yeast

The fission yeast plc1 ⁺ gene encodes phosphoinositide-specific phospholipase C. The two- hybrid interaction assay with plexA-plc1 ⁺ as a bait revealed that Plc1p interacted with the 14-3-3 proteins Rad24p and Rad25p. Formation of a complex containing Plc1p and Rad24p in vivo was confirmed by an immunological method. As predicted from the fact that rad24 null mutant cells are hypersensitive to UV irradiation, plc1 null mutant cells were almost as sensitive to UV irradiation as rad24 null mutant cells. In addition, deletion of rad24 in the plc1 null mutant cells did not enhance the UV sensitivity, indicating that plc1 ⁺ and rad24 ⁺ belong to the same epistasis group with respect to UV sensitivity. Whereas Rad24p has been reported to be involved in the DNA damage checkpoint pathway, the delay to mitosis after UV irradiation was not defective either in rad24 null mutant cells or in plc1 null mutant cells in our analysis. Thus, Plc1p is responsible for resistance to UV irradiation, but not for the DNA damage checkpoint pathway, in cooperation with 14-3-3 proteins.     

Thyroxine signal transduction in liver cells involves phospholipase C and phospholipase D activation. Genomic independent action of thyroid hormone

Background

Many eukaryotes, including plants and fungi make spores that resist severe environmental stress. The micro-organism Dictyostelium contains a single phospholipase C gene (PLC); deletion of the gene has no effect on growth, cell movement and differentiation. In this report we show that PLC is essential to sense the environment of food-activated spores.

Results

Plc-null spores germinate at alkaline pH, reduced temperature or increased osmolarity, conditions at which the emerging amoebae can not grow. In contrast, food-activated wild-type spores return to dormancy till conditions in the environment allow growth. The analysis of inositol 1,4,5-trisphosphate (IP₃) levels and the effect of added IP₃ uncover an unexpected mechanism how PLC regulates spore germination: i) deletion of PLC induces the enhanced activity of an IP₅ phosphatase leading to high IP₃ levels in plc-null cells; ii) in wild-type spores unfavourable conditions inhibit PLC leading to a reduction of IP₃ levels; addition of exogenous IP₃ to wild-type spores induces germination at unfavourable conditions; iii) in plc-null spores IP₃ levels remain high, also at unfavourable environmental conditions.

Conclusions

The results imply that environmental conditions regulate PLC activity and that IP₃ induces spore germination; the uncontrolled germination of plc-null spores is not due to a lack of PLC activity but to the constitutive activation of an alternative IP₃-forming pathway.     

Spermine stimulation of phospholipase C fromCatharanthus roseus transformed roots

Polyamines (Pas) are aliphatic amines that are ubiquitous in all living organisms and regulate a broad spectrum of physiological processes. It has been suggested that they can act through a signal transduction pathway. Using Catharanthus roseus hairy roots as a model we determined the levels of Pas throughout a culture cycle. We found that there is a peak in the intracellular concentration of Pas during the first six days of culture. The effect of Pas on phospholipase C (PLC) activity was also investigated. Putrescine, spermidine and spermine were added in vitro to the PLC assay. Putrescine did not modify PLC activity; spermidine inhibited the enzyme but at very high, non-physiological concentrations; and spermine increased the PLC activity four-fold at physiological concentrations. Our results suggest that spermine could regulate root growth by regulating the PLC signal transduction mechanism.     

Linking phospholipase C isoforms with differentiation function in human vascular smooth muscle cells

The phosphoinositol-phospholipase C (PLC) family of enzymes consists of a number of isoforms, each of which has different cellular functions. PLCγ₁ is primarily linked to tyrosine kinase transduction pathways, whereas PLCδ₁ has been associated with a number of regulatory proteins, including those controlling the cell cycle. Recent studies have shown a central role of PLC in cell organisation and in regulating a wide array of cellular responses. It is of importance to define the precise role of each isoform, and how this changes the functional outcome of the cell. Here we investigated differences in PLC isoform levels and activity in relation to differentiation of human and rat vascular smooth muscle cells. Using Western blotting and PLC activity assay, we show that PLCδ₁ and PLCγ₁ are the predominant isoforms in randomly cycling human vascular smooth muscle cells (HVSMCs). Growth arrest of HVSMCs for seven days of serum deprivation was consistently associated with increases in PLCδ₁ and SM α-actin, whereas there were no changes in PLCγ₁ immuno-reactivity. Organ culture of rat mesenteric arteries in serum free media (SFM), a model of de-differentiation, led to a loss of contractility as well as a loss of contractile proteins (SM α-actin and calponin) and PLCδ₁, and no change in PLCγ₁ immuno-reactivity. Taken together, these data indicate that PLCδ₁ is the predominant PLC isoform in vascular smooth muscle, and confirm that PLCδ₁ expression is affected by conditions that affect the cell cycle, differentiation status and contractile function.     

Constancy of Uptake During the Cell Cycle in Escherichia coli

Rates of uptake of several labeled compounds were measured during the cell cycle for three strains of Escherichia coli in balanced growth. Uptake rates were constant during more than the first two-thirds of the cycle, or reasonably so, for all of these compounds: glycine, leucine, glucose, acetate, phosphate, sulfate, and thymidine. When added de novo, uptake of glycine and leucine were not constant, but appeared to be proportional to mean cell volume. These results are in agreement with the finding that cell sizes increase linearly during most of the cell cycle for E. coli. They support the hypothesis, for cultures in balanced growth, that linear growth during the cell cycle is due to constant rates of uptake of all major growth factors. They also support the interpretation that uptake is limited by the presence of a constant number of functional binding or accumulation sites for these growth factors.     

Characterization of Plasmodium falciparum cdc2-related kinase and the effects of a CDK inhibitor on the parasites in erythrocytic schizogony

The cell cycle of Plasmodium is unique among major eukaryotic cell cycle models. Cyclin-dependent kinases (CDKs) are thought to be the key molecular switches that regulate cell cycle progression in the parasite. However, little information is available about Plasmodium CDKs. The present study was performed to investigate the effects of a CDK inhibitor, olomoucine, on the erythrocytic growth of Plasmodium falciparum. This agent inhibited the growth of the parasite at the trophozoite/schizont stage. Furthermore, we characterized the Plasmodium CDK homolog, P. falciparum cdc2-related kinase-1 (Pfcrk-1), which is a potential target of olomoucine. We synthesized a functional kinase domain of Pfcrk-1 as a GST fusion protein using a wheat germ protein expression system, and examined its phosphorylation activity. The activity of this catalytic domain was higher than that of GST-GFP control, but the same as that of a kinase-negative mutant of Pfcrk-1. After the phosphatase treatment, the labeling of [γ-³²P]ATP was abolished. Recombinant human cyclin proteins were added to these kinase reactions, but there were no differences in activity. This report provides important information for the future investigation of Plasmodium CDKs.     

Genetic evidence for a role of phospholipase C at the budding yeast kinetochore

Chromosome segregation during mitosis requires kinetochores, specialized organelles that mediate chromosome attachment to spindle microtubules. We have shown previously that in budding yeast, Plc1p (phosphoinositide-specific phospholipase C) localizes to centromeric loci, associates with the kinetochore proteins Ndc10p and Cep3p, and affects the function of kinetochores. Deletion of PLC1 results in nocodazole sensitivity, mitotic delay, and a higher frequency of chromosome loss. We report here that despite the nocodazole sensitivity of plc1Delta cells, Plc1p is not required for the spindle checkpoint. However, plc1Delta cells require a functional BUB1/BUB3-dependent spindle checkpoint for viability. PLC1 displays strong genetic interactions with genes encoding components of the inner kinetochore, including NDC10, SKP1, MIF2, CEP1, CEP3, and CTF13. Furthermore, plc1Delta cells display alterations in chromatin structure in the core centromere. Chromatin immunoprecipitation experiments indicate that Plc1p localizes to centromeric loci independently of microtubules, and accumulates at the centromeres during G(2)/M stage of cell cycle. These results are consistent with the view that Plc1p affects kinetochore function, possibly by modulating the structure of centromeric chromatin.     

Effects of environmental stresses on the cell cycle of two marine phytoplankton species

Cell cycle phase durations of cultures of Hymenomonas carterae Braarud and Fagerl, a coccolithophore, and Thalassiosira weissflogii Grun., a centric diatom, in temperature-, light- or nitrogen-limited balanced growth were determined using flow cytometry. Suboptimal temperature caused increases in the duration of all phases of the cell cycle (though not equally) in both species, and the increased generation time of nitrogen-limited cells of both species was due almost wholly to expansion of G₁ phase. In H. carterae light limitation caused only G₁ phase to expand, but in T. weissflogii both G₂ + M and G₁ were affected. These results are discussed in relation to cell division phasing patterns of these two species and to models of phytoplankton growth. Simultaneous measurements of protein and DNA on individual cells indicated that under all conditions, the protein content of cells in G₁ was a constant proportion of that of G₂ + M cells. Simultaneous measurements of RNA and protein on each cell indicated that the amounts of these two cell constituents were always tightly correlated. Under conditions of nitrogen limitation both protein and RNA per cell decreased to less than one-third of the levels found in nonlimited cells. This indicates, at least for nitrogen-replete cells, that neither protein nor RNA levels are likely to act as the trigger for cell cycle progression. Strict control by cell size is also unlikely since mean cell volume decreased as growth rates were limited by light and nitrogen supply, but increased with decreasing temperature.     

Phospholipase C in rabbit thymocytes: subcellular distribution and influences of calcium and GTPγS on the substrate dependence of cytosolic and plasma membrane-associated phospholipase C

The subcellular distribution of phospholipase C (PLC) activity in rabbit thymocytes was examined by measuring the enzyme's activity in different subcellular fractions. PLC activity was determined using exogenously added [³H]PIP₂ as substrate. Approx. 80% of the activity of the cell homogenate was found in the cytosolic fraction. A minor portion of PLC activity was attached to the particulate fraction. This membrane-associated PLC activity was found to be predominantly bound to the plasma membrane. Both PIP₂-cleaving PLCs (the PLC associated with the plasma membrane and the PLC in the cytosol) exhibited maximum activity at pH 5. GTPγS stimulated the cytosolic and the membrane-bound PLC. As revealed by computer analysis of the substrate dependence of both basal and GTPγS-stimulated PLC activity, GTPγS enhanced the V_max of the enzymes. Calcium, at a concentration of 1 mM, decreased PLC activity, as compared to a calcium concentration of 100 nM. The characteristics increase in V_max induced by GTPγS was observed at a concentration of 1 mM calcium and was similar to that at 100 nM. These data suggest that the stimulatory effect of GTPγS is not due to an increased affinity of PLCs to calcium.     

A frequent PLCγ1 mutation in adult T-cell leukemia/lymphoma determines functional properties of the malignant cells

BackgroundDevelopment of adult T-cell leukemia/lymphoma (ATL) involves human T-cell leukemia virus type 1 (HTLV-1) infection and accumulation of somatic mutations. The most frequently mutated gene in ATL (36 % of cases) is phospholipase C gamma1 (PLCG1). PLCG1 is also frequently mutated in other T-cell lymphomas. However, the functional consequences of the PLCG1 mutations in cancer cells have not been characterized.MethodsWe compared the activity of the wild-type PLCγ1 with that of a mutant carrying a hot-spot mutation of PLCγ1 (S345F) observed in ATL, both in cells and in cell-free assays. To analyse the impact of the mutation on cellular properties, we quantified cellular proliferation, aggregation, chemotaxis and apoptosis by live cell-imaging in an S345F⁺ ATL-derived cell line (KK1) and a KK1 cell line in which we reverted the mutation to the wild-type sequence using CRISPR/Cas9 and homology-directed repair.FindingsThe PLCγ1 S345F mutation results in an increase of basal PLC activity in vitro and in different cell types. This higher basal activity is further enhanced by upstream signalling. Reversion of the S345F mutation in the KK1 cell line resulted in reduction of the PLC activity, lower rates of proliferation and aggregation, and a marked reduction in chemotaxis towards CCL22. The PLCγ1-pathway inhibitors ibrutinib and ritonavir reduced both the PLC activity and the tested functions of KK1 cells.InterpretationConsistent with observations from clinical studies, our data provide direct evidence that activated variants of the PLCγ1 enzyme contribute to the properties of the malignant T-cell clone in ATL.FundingMRC (UK) Project Grant (P028160).     

Developmental roles of Drosophila tRNA processing endonuclease RNase Z as revealed with a conditional rescue system

Drosophila RNase Z^L (dRNaseZ) belongs to a family of endoribonucleases with a major role in tRNA 3′-end processing. The biochemical function of RNase Z^L is conserved from yeast to human. Here we present a study of its biological function during Drosophila development. In flies, dRNaseZ provides a non-redundant function, as the RNZ^ED24 knockout (KO) mutation causes early larval lethality. Mosaic and conditional rescue techniques were employed to determine dRNaseZ requirements at later stages. We found that dRNaseZ activity is essential for all phases of fly development that involve cell division, including growth of adult tissue progenitors during larval and metamorphic stages, and gametogenesis in adults. At the cellular level, two major phenotypes were identified—cell growth deficiency in endoreplicating tissues and cell cycle arrest in mitotic tissues. While cell growth and proliferation are both dependant on protein synthesis, the two phenotypes displayed reliance on different dRNaseZ functions. We found that dRNaseZ KO completely blocks tRNA maturation without diminishing the abundance of mature tRNA molecules. Our data indicate that growth arrest of endoreplicating cells is primarily attributed to the relocation of the pool of mature tRNAs into the nuclei causing a decrease in translation efficiency. Mitotically dividing cells appear to be less dependent on translation machinery as they maintain their normal size when deprived of dRNaseZ activity, but rather display a cell cycle arrest at the G₂–M transition.