Maintenance of hormone-sensitive phosphoinositide pools in the plasma membrane requires phosphatidylinositol 4-kinase IIIalpha

Type III phosphatidylinositol (PtdIns) 4-kinases (PI4Ks) have been previously shown to support plasma membrane phosphoinositide synthesis during phospholipase C activation and Ca²⁺ signaling. Here, we use biochemical and imaging tools to monitor phosphoinositide changes in the plasma membrane in combination with pharmacological and genetic approaches to determine which of the type III PI4Ks (α or β) is responsible for supplying phosphoinositides during agonist-induced Ca²⁺ signaling. Using inhibitors that discriminate between the α- and β-isoforms of type III PI4Ks, PI4KIIIα was found indispensable for the production of phosphatidylinositol 4-phosphate (PtdIns4P), phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂], and Ca²⁺ signaling in angiotensin II (AngII)-stimulated cells. Down-regulation of either the type II or type III PI4K enzymes by small interfering RNA (siRNA) had small but significant effects on basal PtdIns4P and PtdIns(4,5)P₂ levels in ³²P-labeled cells, but only PI4KIIIα down-regulation caused a slight impairment of PtdIns4P and PtdIns(4,5)P₂ resynthesis in AngII-stimulated cells. None of the PI4K siRNA treatments had a measurable effect on AngII-induced Ca²⁺ signaling. These results indicate that a small fraction of the cellular PI4K activity is sufficient to maintain plasma membrane phosphoinositide pools, and they demonstrate the value of the pharmacological approach in revealing the pivotal role of PI4KIIIα enzyme in maintaining plasma membrane phosphoinositides.     

H2O2 generation is decreased by calcium in isolated brain mitochondria

Release of H(2)O(2) in response to Ca(2+) loads (1-100 microM) was investigated using Amplex red fluorescent assay in isolated guinea-pig brain mitochondria respiring on glutamate plus malate or succinate. In mitochondria challenged with Ca(2+) (10 microM), in the absence of adenine nucleotides and inhibitors of the respiratory chain, the rate of H(2)O(2) release, taken as an indication of H(2)O(2) production, was decreased by 21.8+/-1.6% in the presence of NADH-linked substrates and by 86.5+/-1.8% with succinate. Parallel with this, a Ca(2+)-induced loss in NAD(P)H fluorescence, sustained depolarization, decrease in fluorescent light scattering signal and in calcein fluorescence were detected indicating an increased permeability and swelling of mitochondria, which were prevented by ADP (2 mM). In the presence of ADP H(2)O(2) release from mitochondria was decreased, but Ca(2+) no longer influenced the generation of H(2)O(2). We suggest that the decreased H(2)O(2) generation induced by Ca(2+) is related to depolarization and NAD(P)H loss resulting from a non-specific permeability increase of the mitochondrial inner membrane.     

Sox-9 and cDachsund-2 expression in the developing chick telencephalon

We have created a transgenic mouse line that expresses Cre recombinase under the control of the novel mouse promoter/enhancer D6. We describe the expression pattern of D6-Cre in a Gtrosa26 reporter background as assayed by LacZ activity. The enhancer activity starts at 10.5 days post-coitum in the telencephalon and is at the later embryonic stages highly restricted to the hippocampus and the neocortex. In adult mice D6-derived cells are found in cortical layers II–VI, in the granular cells of the dentate gyrus and in hippocampal fields CA1–CA3. D6-Cre activity is also detected in the ependymal and subependymal zone of the lateral ventricles which is known to harbor neural stem cells.     

Validation of prostate cancer risk-related loci identified from genome-wide association studies using family-based association analysis: evidence from the International Consortium for Prostate Cancer Genetics (ICPCG)

Multiple prostate cancer (PCa) risk-related loci have been discovered by genome-wide association studies (GWAS) based on case-control designs. However, GWAS findings may be confounded by population stratification if cases and controls are inadvertently drawn from different genetic backgrounds. In addition, since these loci were identified in cases with predominantly sporadic disease, little is known about their relationships with hereditary prostate cancer (HPC). The association between seventeen reported PCa susceptibility loci was evaluated with a family-based association test using 1,979 hereditary PCa families of European descent collected by members of the International Consortium for Prostate Cancer Genetics, with a total of 5,730 affected men. The risk alleles for 8 of the 17 loci were significantly over-transmitted from parents to affected offspring, including SNPs residing in 8q24 (regions 1, 2 and 3), 10q11, 11q13, 17q12 (region 1), 17q24 and Xp11. In subgroup analyses, three loci, at 8q24 (regions 1 and 2) plus 17q12, were significantly over-transmitted in hereditary PCa families with five or more affected members, while loci at 3p12, 8q24 (region 2), 11q13, 17q12 (region 1), 17q24 and Xp11 were significantly over-transmitted in HPC families with an average age of diagnosis at 65?years or less. Our results indicate that at least a subset of PCa risk-related loci identified by case-control GWAS are also associated with disease risk in HPC families.     

Measuring hydrogen exchange in proteins by selective water saturation in 1H–15N SOFAST/BEST-type experiments: advantages and limitations

HET^ex-SOFAST NMR (Schanda et al. in J Biomol NMR 33:199–211, 2006) has been proposed some years ago as a fast and sensitive method for semi-quantitative measurement of site-specific amide-water hydrogen exchange effects along the backbone of proteins. Here we extend this concept to BEST readout sequences that provide a better resolution at the expense of some loss in sensitivity. We discuss the theoretical background and implementation of the experiment, and demonstrate its performance for an intrinsically disordered protein, 2 well folded globular proteins, and a transiently populated folding intermediate state. We also provide a critical evaluation of the level of accuracy that can be obtained when extracting quantitative exchange rates from HET^ex NMR measurements.     

Dependence of STIM1/Orai1-mediated Calcium Entry on Plasma Membrane Phosphoinositides

Recent studies identified two main components of store-operated calcium entry (SOCE): the endoplasmic reticulum-localized Ca²⁺ sensor protein, STIM1, and the plasma membrane (PM)-localized Ca²⁺ channel, Orai1/CRACM1. In the present study, we investigated the phosphoinositide dependence of Orai1 channel activation in the PM and of STIM1 movements from the tubular to PM-adjacent endoplasmic reticulum regions during Ca²⁺ store depletion. Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) levels were changed either with agonist stimulation or by chemically induced recruitment of a phosphoinositide 5-phosphatase domain to the PM, whereas PtdIns4P levels were decreased by inhibition or down-regulation of phosphatidylinositol 4-kinases (PI4Ks). Agonist-induced phospholipase C activation and PI4K inhibition, but not isolated PtdIns(4,5)P₂ depletion, substantially reduced endogenous or STIM1/Orai1-mediated SOCE without preventing STIM1 movements toward the PM upon Ca²⁺ store depletion. Patch clamp analysis of cells overexpressing STIM1 and Orai1 proteins confirmed that phospholipase C activation or PI4K inhibition greatly reduced I_CRAC currents. These results suggest an inositide requirement of Orai1 activation but not STIM1 movements and indicate that PtdIns4P rather than PtdIns(4,5)P₂ is a likely determinant of Orai1 channel activity.Store-operated Ca²⁺ entry (SOCE)³ is a ubiquitous Ca²⁺ entry pathway that is regulated by the Ca²⁺ content of the endoplasmic reticulum (ER) (1). SOCE has been identified as the major route of Ca²⁺ entry during activation of cells of the immune system such as T cells and mast cells (2, 3), and it is also present and functionally important in other cells such as platelets (4) and developing myotubes (5). The long awaited mechanism of how the ER luminal Ca²⁺ content is sensed and the information transferred to the plasma membrane (PM) has been clarified recently after identification of the ER Ca²⁺ sensor proteins STIM1 and -2 (6, 7) and the PM Ca²⁺ channels Orai1, -2, and -3 (8–10). According to current views, a decrease in the ER Ca²⁺ concentration is sensed by the luminal EF-hand of the single-transmembrane STIM proteins causing their multimerization. This oligomerization occurs in the tubular ER, where it promotes the interaction of the cytoplasmic C termini of STIM with PM components and association with the PM-localized Orai channels, causing both their clustering and activation in the PM (reviewed recently in Refs. 11–13). Analysis of the interacting domains within the STIM1 and Orai1 proteins suggests that the cytoplasmic domain of STIM1 is necessary and sufficient to activate Orai1 (14), whereas the latter requires its C-terminal membrane-adjacent cytoplasmic tail to be fully activated by the STIM proteins (15, 16). Both STIM1 and -2 contain a polybasic segment in their C termini, and such regions are often responsible for the PM localization of proteins (mostly of the small GTP-binding protein class) via interaction with anionic phospholipids such as phosphatidylserine or PtdIns(4,5)P₂ (17). However, the role of this domain in STIM1 function(s) remains controversial. Deletion of the polybasic tail is reported to prevent PM association but not clustering of STIM1 upon ER store depletion (18). In other studies, truncated STIM1 lacking the polybasic domain shows only slightly altered activation (15) or inactivation (19) kinetics without major defects in supporting Orai1-mediated Ca²⁺ influx. The most recent studies identify the minimal Orai1 activation domain in STIM1 (20, 21) and find that the polybasic domain is not essential for this function but makes electrostatic interaction with classical transient receptor potential channels (22).PM phosphoinositides have been widely reported as regulators of the activity of several ion channels and transporters (23). However, only a few studies have addressed the inositide requirement of SOCE and none specifically that of the Orai1-mediated Ca²⁺ entry process. Sensitivity of SOCE to phosphatidylinositol 3-kinases (PI3K) inhibitors has been reported, but this required concentrations that suggested inhibition of targets other than PI3Ks, possibly myosin light chain kinase or the type-III PI4Ks (4, 24–26). Here we have described studies addressing the role of PM phosphoinositides in STIM1 movements as well as in Orai1 channel gating. Our results show that phosphoinositides do not have a major role in the prominent reorganization of STIM1 after Ca²⁺ store depletion but suggest a function of PtdIns4P rather than PtdIns(4,5)P₂ in supporting the Orai1-mediated Ca²⁺ entry process.     

Multipotent neuroblasts generate a biochemical neuroarchitecture in the central complex of the grasshopper Schistocerca gregaria

We have examined the developmental expression of the neuromodulators locustatachykinin, leucokinin-1, allatostatin and serotonin in a subset of lineages (Y, Z) of the central complex in the brain of the grasshopper Schistocerca gregaria. First, we show that all these neuromodulators are expressed in the same lineages during embryogenesis. The neuroblasts generating these lineages are therefore biochemically multipotent. Second, the neurons expressing the different neuromodulators are found clustered at stereotypic locations in their respective lineages. Locustatachykinin and leucokinin-1 map to the apical region of the lineage, allatostatin medially and serotonin to the base of the lineage. Since the location in these lineages translates into their birth order, we have been able ontogenetically to analyse their biochemical expression patterns. The age-profile within a lineage reveals that locustatachykinin- and leucokinin-1-expressing neurons are born first, then allatostatin neurons and finally serotoninergic neurons. Co-expression has been tested for serotonin with locustatachykin, leucokinin-1 or allatostatin and is negative but is positive for locustatachykinin and leucokinin-1, consistent with the stereotypic location of cells in the lineages. The delay between the birth of a neuron and the expression of its neuromodulator is stereotypic for each substance. Combined with a known birth date, this delay translates into a developmental expression pattern for the central complex itself.     

Transposon-induced symbiotic mutants of Bradyrhizobium japonicum: isolation of two gene regions essential for nodulation

Summary Two strains of the soybean endosymbiont Bradyrhizobium japonicum, USDA 110 and 61 A101 C, were mutagenized with transposon Tn5. After plant infection tests of a total of 6,926 kanamycin and streptomycin resistant transconjugants, 25 mutants were identified that are defective in nodule formation (Nod^-) or nitrogen fixation (Fix^-). Seven Nod^- mutants were isolated from strain USDA 110 and from strain 61 A101 C, 4 Nod^- mutants and 14 Fix^- mutants were identified. Subsequent auxotrophic tests on these symbiotically defective mutants identified 4 His^- Nod^- mutants of USDA 110. Genomic Southern analysis of the 25 mutants revealed that each of them carried a single copy of Tn5 integrated in the genome. Three 61 A101 C Fix^- mutants were found to have vector DNA co-integrated along with Tn5 in the genome. Two independent DNA regions flanking Tn5 were cloned from the three nonauxotrophic Nod^- mutants and one His^-Nod^- mutant of USDA 110. Homogenotization of the cloned fragments into wild-type strain USDA 110 and subsequent nodulation assay of the resulting homogenotes confirmed that the Tn5 insertion was responsible for the Nod^- phenotype. Partial EcoR1 restriction enzyme maps around the Tn5 insertion sites were generated. Hybridization of these cloned regions to the previously cloned nod regions of R. meliloti and nif and nod regions of B. japonicum USDA 110 showed no homology, suggesting that these regions represent new symbiotic clusters of B. japonicum.