Vertebrate body plans have a conserved left-right (LR) asymmetry manifested in the position and anatomy of the heart, visceral organs, and brain. Recent studies have suggested that LR asymmetry is established by asymmetric Ca2+ signaling resulting from cilia-driven flow of extracellular fluid across the node. We report here that inositol 1,3,4,5,6-pentakisphosphate 2-kinase (Ipk1), which generates inositol hexakisphosphate, is critical for normal LR axis determination in zebrafish. Zebrafish embryos express ipk1 symmetrically during gastrulation and early segmentation. ipk1 knockdown by antisense morpholino oligonucleotide injection randomized LR-specific gene expression and organ placement, effects that were associated with reduced intracellular Ca2+ flux in cells surrounding the ciliated Kupffer's vesicle, a structure analogous to the mouse node. Our data suggest that the pathway for inositol hexakisphosphate production is a key regulator of asymmetric Ca(2+) flux during LR specification.

Functional peripheral mature follicular B (FoB) lymphocytes are thought to develop from immature transitional cells in a BCR-dependent manner. We have previously shown that BCR cross-linking in vitro results in death of early transitional (T1) B cells, whereas late transitional (T2) B cells survive and display phenotypic characteristics of mature FoB cells. We now demonstrate that diacylglycerol (DAG), a lipid second messenger implicated in cell survival and differentiation, is produced preferentially in T2 compared with T1 B cells upon BCR cross-linking. Consistently, inositol 1,4,5-triphosphate is also produced preferentially in T2 compared with T1 B cells. Unexpectedly, the initial calcium peak appears similar in both T1 and T2 B cells, whereas sustained calcium levels are higher in T1 B cells. Pretreatment with 2-aminoethoxydiphenylborate, an inhibitor of inositol 1,4,5-triphosphate receptor-mediated calcium release, and verapamil, an inhibitor of L-type calcium channels, preferentially affects T1 B cells, suggesting that distinct mechanisms regulate calcium mobilization in each of the two transitional B cell subsets. Finally, BCR-mediated DAG production is dependent upon Bruton’s tyrosine kinase and phospholipase C-γ2, enzymes required for the development of FoB from T2 B cells. These results suggest that calcium signaling in the absence of DAG-mediated signals may lead to T1 B cell tolerance, whereas the combined action of DAG and calcium signaling is necessary for survival and differentiation of T2 into mature FoB lymphocytes.

Cilia project from cells as membranous extensions, with microtubule structural cores assembling from basal bodies by intraflagellar transport (IFT). Here, we report a ciliary role for the inositol 1,3,4,5,6-pentakisphosphate 2-kinase (Ipk1) that generates inositol hexakisphosphate. In zebrafish embryos, reducing Ipk1 levels inhibited ciliary beating in Kupffer's vesicle and decreased ciliary length in the spinal canal, pronephric ducts, and Kupffer's vesicle. Electron microscopy showed that ciliary axonemal structures were not grossly altered. However, coincident knockdown of Ipk1 and IFT88 or IFT57 had synergistic perturbations. With GFP-Ipk1 enriched in centrosomes and basal bodies, we propose that Ipk1 plays a previously uncharacterized role in ciliary function.cilia ͉ inositol hexakisphosphate ͉ kidney development ͉ left-right asymmetry ͉ zebrafish

Inositol phosphate (IP) kinases constitute an emerging class of cellular kinases linked to multiple cellular activities. Here, we report a previously uncharacterized cellular function in Hedgehog (Hh) signaling for the IP kinase designated inositol hexakisphosphate kinase-2 (IP6K2) that produces diphosphoryl inositol phosphates (PP-IPs). In zebrafish embryos, IP6K2 activity was required for normal development of craniofacial structures, somites, and neural crest cells. ip6k2 depletion in both zebrafish and mammalian cells also inhibited Hh target gene expression. Inhibiting IP 6 kinase activity using N(2)-(m-(trifluoromethy)lbenzyl) N(6)-(p-nitrobenzyl) purine (TNP) resulted in altered Hh signal transduction. In zebrafish, restoring IP6K2 levels with exogenous ip6k2 mRNA reversed the effects of IP6K2 depletion. Furthermore, overexpression of ip6k2 in mammalian cells enhanced the Hh pathway response, suggesting IP6K2 is a positive regulator of Hh signaling. Perturbations from IP6K2 depletion or TNP were reversed by overexpressing smoM2, gli1, or ip6k2. Moreover, the inhibitory effect of cyclopamine was reversed by overexpressing ip6k2. This identified roles for the inositol kinase pathway in early vertebrate development and tissue morphogenesis, and in Hh signaling. We propose that IP6K2 activity is required at the level or downstream of Smoothened but upstream of the transcription activator Gli1. At the cell membrane, lipid phosphoinositides are essential signaling mediators (5). Activation of phospholipase C generates inositol 1,4,5-trisphosphate (IP 3 ) and 1,2-diacylglycerol as second messenger intracellular signals. Soluble IP 3 molecule is a starting point for metabolism by inositol phosphate (IP) kinases and phosphatases. The resulting IP ensemble includes inositol tetrakisphosphate (IP 4 ) isomers, inositol 1,3,4,5,6-pentakisphosphate (IP 5 ), inositol hexakisphosphate (IP 6 ), and diphosphoryl inositol phosphate (PP-IP) isomers (e.g., PP-IP 4 , IP 7 ) (6, 7). Different IPs regulate vital functions, including transcription, mRNA export, RNA editing, translation, and ciliogenesis (6, 7).The IP 6 kinases (IP6K) specifically produce diphosphoryl isomers, including PP-IP 4 , PP-IP 5 , PP 2 -IP 3 , and PP 2 -IP 4 (8, 9). Loss of IP6K activity results in different cellular defects, including altered vesicular endocytosis and telomere length in yeast, perturbed chemotaxis in Dictyostelium, and inhibited insulin exocytosis in pancreatic β-cells (9-12). Of the three documented IP6K family members, the IP6K2 isoform is linked to cell growth regulation and apoptosis in mammalian cells (13). However, IP6K2 roles in development and signaling in multicellular organisms are not fully understood.Here we report that IP6K2 is required for development of craniofacial skeleton, somites, and slow muscle fibers in zebrafish. Moreover, loss of IP6K2 perturbs neural crest cell (NCC) development and migration, and inhibits Hh target gene expression. NCCs are migratory cells that detach from the embryonic neural epithelium along t...

The Candida albicans homologues of the most studied Saccharomyces cerevisiae stationaryphase genes, SNO1 and SNZ1, were used to test the hypothesis that, within a biofilm, some cells reach stationary phase within continuously fed, as well as static, C. albicans biofilms grown on dental acrylic. The authors first studied the expression patterns of these two genes in planktonic growth conditions. Using real-time RT-PCR (RT-RTPCR), increased peak expression of both SNZ1 and SNO1 was observed at 5 and 6 days, respectively, in C. albicans grown in suspension culture. SNZ1-yellow fluorescent protein (YFP) and SNO1-YFP were constructed to study expression at the cellular level and protein localization in C. albicans. Snz1p-YFP and Sno1p-YFP localized to the cytoplasm with maximum expression (>90 %) at 5 and 6 days, respectively, in planktonic conditions. When yeast growth was reinitiated, loss of fluorescence began immediately. Germ tubes and hyphae were non-fluorescent. Pseudohyphae began appearing at 9 days in planktonic yeast culture and expressed each protein by 11 days; however, the cells budding from pseudohyphae were not fluorescent. Biofilm was formed in vitro under either static or continuously fed conditions. Increased expression of the two genes was shown by RT-RTPCR, beginning by day 3 and increasing through to day 15 (continuously fed biofilm). Only the bottommost layer of acrylic-adhered cells in the biofilm showed 25 and 40 % fluorescence at 6 and 15 days, respectively. These observations suggest that only a few cells in C. albicans biofilms express genes associated with the planktonic stationary phase and that these are found at the bottom of the biofilm adhered to the surface.

The inositol 1,3,4,5,6-pentakisphosphate (IP 5 ) 2-kinase (Ipk1) catalyzes the production of inositol hexakisphosphate (IP 6 ) in eukaryotic cells. Previous studies have shown that IP 6 is required for efficient nuclear mRNA export in the budding yeast Saccharomyces cerevisiae. Here, we report the first functional analysis of ipk1 ؉ in Schizosaccharomyces pombe. S. pombe Ipk1 (SpIpk1) is unique among Ipk1 orthologues in that it harbors a novel amino (N)-terminal domain with coiled-coil structural motifs similar to those of BAR (Bin-amphiphysin-Rvs) domain proteins. Mutants with ipk1 ؉ deleted (ipk1⌬) had mRNA export defects as well as pleiotropic defects in polarized growth, cell morphology, endocytosis, and cell separation. The SpIpk1 catalytic carboxy-terminal domain was required to rescue these defects, and the mRNA export block was genetically linked to SpDbp5 function and, likely, IP 6 production. However, the overexpression of the Nterminal domain alone also inhibited these functions in wild-type cells. This revealed a distinct noncatalytic function for the N-terminal domain. To test for connections with other inositol polyphosphates, we also analyzed whether the loss of asp1 ؉ function, encoding an IP 6 kinase downstream of Ipk1, had an effect on ipk1⌬ cells. The asp1⌬ mutant alone did not block mRNA export, and its cell morphology, polarized growth, and endocytosis defects were less severe than those of ipk1⌬ cells. Moreover, ipk1⌬ asp1⌬ double mutants had altered inositol polyphosphate levels distinct from those of the ipk1⌬ mutant. This suggested novel roles for asp1 ؉ upstream of ipk1 ؉ . We propose that IP 6 production is a key signaling linchpin for regulating multiple essential cellular processes.Inositol polyphosphates (IPs) constitute an emerging class of signaling molecules that regulate multiple cellular activities including chromatin remodeling and transcription, mRNA export, telomere length regulation, RNA editing, exocytosis, ciliary beating and length maintenance, and translation (8,21,32,48,51,56,59,(70)(71)(72). IP production is initiated with the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) by phospholipase C (PLC), producing diacylglycerol and soluble inositol 1,4,5-trisphosphate (IP 3 ). IP 3 is sequentially phosphorylated by the coordinated actions of specific kinases to produce more highly phosphorylated IP molecules, including inositol 1,3,4,5-tetrakisphosphate (IP 4 ), inositol 1,3, 4,5,6-pentakisphosphate (IP 5 ), inositol hexakisphosphate (IP 6 ), and inositol pyrophosphate isomers (e.g., PP-IP 4 and IP 7 ) (2,24,31,41,43,55,71). The perturbation of IP synthesis pathways is linked to defects in nutrient homeostasis in fungi (30, 41, 43) and developmental defects in vertebrates (16,50,51,63). In mammalian tissue culture cells, the total cellular IP pool undergoes complex changes during transit through the cell cycle, with IP 5 , IP 6 , and IP 7 being most abundant in G 1 phase, decreasing during S phase, and rising again during G 2 /M phase (4). However, a function...

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