Polarized PtdIns(4,5)P2 distribution mediated by a voltage-sensing phosphatase (VSP) regulates sperm motility.
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ABSTRACT:
SUBMITTER: Kawai T
PROVIDER: S-EPMC6925991 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Voltage-sensing phosphatases (VSPs) consist of a voltage-sensor domain and a cytoplasmic region with remarkable sequence similarity to phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor phosphatase. VSPs dephosphorylate the 5' position of the inositol ring of both phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] upon voltage depolarization. However, it is unclear whether VSPs also have 3' phosphatase activity. To gain insights into this question, we performed in vitro assays of phosphatase activities of Ciona intestinalis VSP (Ci-VSP) and transmembrane phosphatase with tensin homology (TPTE) and PTEN homologous inositol lipid phosphatase (TPIP; one human ortholog of VSP) with radiolabeled PI(3,4,5)P(3). TLC assay showed that the 3' phosphate of PI(3,4,5)P(3) was not dephosphorylated, whereas that of phosphatidylinositol 3,4-bisphosphate [PI(3,4)P(2)] was removed by VSPs. Monitoring of PI(3,4)P(2) levels with the pleckstrin homology (PH) domain from tandem PH domain-containing protein (TAPP1) fused with GFP (PH(TAPP1)-GFP) by confocal microscopy in amphibian oocytes showed an increase of fluorescence intensity during depolarization to 0 mV, consistent with 5' phosphatase activity of VSP toward PI(3,4,5)P(3). However, depolarization to 60 mV showed a transient increase of GFP fluorescence followed by a decrease, indicating that, after PI(3,4,5)P(3) is dephosphorylated at the 5' position, PI(3,4)P(2) is then dephosphorylated at the 3' position. These results suggest that substrate specificity of the VSP changes with membrane potential.
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Project description:Voltage-sensing phosphatase (Vsp) is a unique membrane protein that translates membrane electrical activities into the changes of phosphoinositide profiles. Vsp orthologs from various species have been intensively investigated toward their biophysical properties, primarily using a heterologous expression system. In contrast, the physiological role of Vsp in native tissues remains largely unknown. Here we report that zebrafish Vsp (Dr-Vsp), encoded by tpte gene, is functionally expressed on the endomembranes of lysosome-rich enterocytes (LREs) that mediate dietary protein absorption via endocytosis in the zebrafish mid-intestine. Dr-Vsp-deficient LREs were remarkably defective in forming endosomal vacuoles after initial uptake of dextran and mCherry. Dr-Vsp-deficient zebrafish exhibited growth restriction and higher mortality during the critical period when zebrafish larvae rely primarily on exogenous feeding via intestinal absorption. Furthermore, our comparative study on marine invertebrate Ciona intestinalis Vsp (Ci-Vsp) revealed co-expression with endocytosis-associated genes in absorptive epithelial cells of the Ciona digestive tract, corresponding to zebrafish LREs. These findings signify a crucial role of Vsp in regulating endocytosis-dependent nutrient absorption in specialized enterocytes across animal species.
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Project description:Voltage-sensing phosphatase (VSP) consists of a voltage sensor domain (VSD) and a cytoplasmic catalytic region (CCR), which is similar to phosphatase and tensin homolog (PTEN). How the VSD regulates the innate enzyme component of VSP remains unclear. Here, we took a combined approach that entailed the use of electrophysiology, fluorometry, and structural modeling to study the electrochemical coupling in Ciona intestinalis VSP. We found that two hydrophobic residues at the lowest part of S4 play an essential role in the later transition of VSD-CCR coupling. Voltage clamp fluorometry and disulfide bond locking indicated that S4 and its neighboring linker move as one helix (S4-linker helix) and approach the hydrophobic spine in the CCR, a structure located near the cell membrane and also conserved in PTEN. We propose that the hydrophobic spine operates as a hub for translating an electrical signal into a chemical one in VSP.
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Project description:The tumor suppressor PTEN dephosphorylates PtdIns(3,4,5)P3 into PtdIns(4,5)P2 Here, we make the unexpected discovery that in Drosophila melanogaster PTEN reduces PtdIns(4,5)P2 levels on endosomes, independently of its phosphatase activity. This new PTEN function requires the enzymatic action of dPLCXD, an atypical phospholipase C. Importantly, we discovered that this novel PTEN/dPLCXD pathway can compensate for depletion of dOCRL, a PtdIns(4,5)P2 phosphatase. Mutation of OCRL1, the human orthologue of dOCRL, causes oculocerebrorenal Lowe syndrome, a rare multisystemic genetic disease. Both OCRL1 and dOCRL loss have been shown to promote accumulation of PtdIns(4,5)P2 on endosomes and cytokinesis defects. Here, we show that PTEN or dPLCXD overexpression prevents these defects. In addition, we found that chemical activation of this pathway restores normal cytokinesis in human Lowe syndrome cells and rescues OCRL phenotypes in a zebrafish Lowe syndrome model. Our findings identify a novel PTEN/dPLCXD pathway that controls PtdIns(4,5)P2 levels on endosomes. They also point to a potential new strategy for the treatment of Lowe syndrome.
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Project description:Elucidation of the role of PtdIns(4,5)P(2) in epithelial function has been hampered by the inability to selectively manipulate the cellular content of this phosphoinositide. Here we report that SigD, a phosphatase derived from Salmonella, can effectively hydrolyze PtdIns(4,5)P(2), generating PtdIns(5)P. When expressed by microinjecting cDNA into epithelial cells forming confluent monolayers, wild-type SigD induced striking morphological and functional changes that were not mimicked by a phosphatase-deficient SigD mutant (C462S). Depletion of PtdIns(4,5)P(2) in intact SigD-injected cells was verified by detachment from the membrane of the pleckstrin homology domain of phospholipase Cdelta, used as a probe for the phosphoinositide by conjugation to green fluorescent protein. Single-cell measurements of cytosolic pH indicated that the Na(+)/H(+) exchange activity of epithelia was markedly inhibited by depletion of PtdIns(4,5)P(2). Similarly, anion permeability, measured using two different halide-sensitive probes, was depressed in cells expressing SigD. Depletion of PtdIns(4,5)P(2) was associated with marked alterations in the actin cytoskeleton and its association with the plasma membrane. The junctional complexes surrounding the injected cells gradually opened and the PtdIns(4,5)P(2)-depleted cells eventually detached from the monolayer, which underwent rapid restitution. Similar observations were made in intestinal and renal epithelial cultures. In addition to its effects on phosphoinositides, SigD has been shown to convert inositol 1,3,4,5,6-pentakisphosphate (IP(5)) into inositol 1,4,5,6-tetrakisphosphate (IP(4)), and the latter has been postulated to mediate the diarrhea caused by Salmonella. However, the effects of SigD on epithelial cells were not mimicked by microinjection of IP(4). In contrast, the cytoskeletal and ion transport effects were replicated by hydrolyzing PtdIns(4,5)P(2) with a membrane-targeted 5-phosphatase or by occluding the inositide using high-avidity tandem PH domain constructs. We therefore suggest that opening of the tight junctions and inhibition of Na(+)/H(+) exchange caused by PtdIns(4,5)P(2) hydrolysis combine to account, at least in part, for the fluid loss observed during Salmonella-induced diarrhea.
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Project description:NOXO1β [NOXO1 (Nox organizer 1) β] is a cytosolic protein that, in conjunction with NOXA1 (Nox activator 1), regulates generation of reactive oxygen species by the NADPH oxidase 1 (Nox1) enzyme complex. NOXO1β is targeted to membranes through an N-terminal PX (phox homology) domain. We have used NMR spectroscopy to solve the structure of the NOXO1β PX domain and surface plasmon resonance (SPR) to assess phospholipid specificity. The solution structure of the NOXO1β PX domain shows greatest similarity to that of the phosphatidylinositol 3-kinase-C2α PX domain with regard to the positions and types of residues that are predicted to interact with phosphatidylinositol phosphate (PtdInsP) head groups. SPR experiments identify PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3) as preferred targets of NOXO1β PX. These findings contrast with previous lipid overlay experiments showing strongest binding to monophosphorylated PtdInsP and phosphatidylserine. Our data suggest that localized membrane accumulation of PtdIns(4,5)P(2) or PtdIns(3,4,5)P(2) may serve to recruit NOXO1β and activate Nox1.
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Project description:Angiogenesis is a coordinated sequence of cellular responses that result in the outgrowth of new blood vessels. The angiogenic program is regulated by extracellular factors, whose input is integrated at least in part at the level of signal transduction pathways driven by phosphoinositide 3 kinase (PI3K) and phospholipase Cgamma (PLCgamma). Using an in vitro angiogenesis model, we discovered that PI3K was essential for tube formation, whereas PLCgamma promoted regression. The underlying mechanism by which PLCgamma antagonized tube formation appeared to be by competing with PI3K for their common substrate, phosphatidylinositol-4,5-bisphosphate. These studies are the first to identify signaling enzymes involved with vessel regression, and reveal that the angiogenic program can be coordinated by the availability of a membrane lipid.
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Project description:The Ciona intestinalis voltage-sensing phosphatase (Ci-VSP) is a membrane protein containing a voltage-sensing domain (VSD) that is homologous to VSDs from voltage-gated ion channels responsible for cellular excitability. Previously published crystal structures of Ci-VSD in putative resting and active conformations suggested a helical-screw voltage sensing mechanism in which the S4 helix translocates and rotates to enable exchange of salt-bridge partners, but the microscopic details of the transition between the resting and active conformations remained unknown. Here, by combining extensive molecular dynamics simulations with a recently developed computational framework based on dynamical operators, we elucidate the microscopic mechanism of the resting-active transition at physiological membrane potential. Sparse regression reveals a small set of coordinates that distinguish intermediates that are hidden from electrophysiological measurements. The intermediates arise from a noncanonical helical-screw mechanism in which translocation, rotation, and side-chain movement of the S4 helix are only loosely coupled. These results provide insights into existing experimental and computational findings on voltage sensing and suggest ways of further probing its mechanism.
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Project description:Voltage-sensing phosphatases (VSPs) are transmembrane proteins that couple changes in membrane potential to hydrolysis of inositol signaling lipids. VSPs catalyze the dephosphorylation of phosphatidylinositol phosphates (PIPs) that regulate diverse aspects of cell membrane physiology including cell division, growth and migration. VSPs are highly conserved among chordates, and their RNA transcripts have been detected in the adult and embryonic stages of frogs, fish, chickens, mice and humans. However, the subcellular localization and biological function of VSP remains unknown. Using reverse transcriptase-PCR (RT-PCR), we show that both Xenopus laevis VSPs (Xl-VSP1 and Xl-VSP2) mRNAs are expressed in early embryos, suggesting that both Xl-VSPs are involved in early tadpole development. To understand which embryonic tissues express Xl-VSP mRNA, we used in situ hybridization (ISH) and found Xl-VSP mRNA in both the brain and kidney of NF stage 32-36 embryos. By Western blot analysis with a VSP antibody, we show increasing levels of Xl-VSP protein in the developing embryo, and by immunohistochemistry (IHC), we demonstrate that Xl-VSP protein is specifically localized to the apical membrane of both embryonic and adult kidney tubules. We further characterized the catalytic activity of both Xl-VSP homologs and found that while Xl-VSP1 catalyzes 3- and 5-phosphate removal, Xl-VSP2 is a less efficient 3-phosphatase with different substrate specificity. Our results suggest that Xl-VSP1 and Xl-VSP2 serve different functional roles and that VSPs are an integral component of voltage-dependent PIP signaling pathways during vertebrate kidney tubule development and function.
| S-EPMC6456211 | biostudies-literature
Project description:In the voltage-sensing phosphatase Ci-VSP, a voltage-sensing domain (VSD) controls a lipid phosphatase domain (PD). The mechanism by which the domains are allosterically coupled is not well understood. Using an in vivo assay, we found that the interdomain linker that connects the VSD to the PD is essential for coupling the full-length protein. Biochemical assays showed that the linker is also needed for activity in the isolated PD. We also identified a late step of VSD motion in the full-length protein that depends on the linker. Notably, we found that this VSD motion requires PI(4,5)P2, a substrate of Ci-VSP. These results suggest that the voltage-driven motion of the VSD turns the enzyme on by rearranging the linker into an activated conformation, and that this activated conformation is stabilized by PI(4,5)P2. We propose that Ci-VSP activity is self-limited because its decrease of PI(4,5)P2 levels decouples the VSD from the enzyme.
| S-EPMC2857593 | biostudies-literature