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The deficiency of PIP2 5-phosphatase in Lowe syndrome affects actin polymerization.

ABSTRACT: Lowe syndrome is a rare X-linked disorder characterized by bilateral congenital cataracts, renal Fanconi syndrome, and mental retardation. Lowe syndrome results from mutations in the OCRL1 gene, which encodes a phosphatidylinositol 4,5 bisphosphate 5-phosphatase located in the trans-Golgi network. As a first step in identifying the link between ocrl1 deficiency and the clinical disorder, we have identified a reproducible cellular abnormality of the actin cytoskeleton in fibroblasts from patients with Lowe syndrome. The cellular abnormality is characterized by a decrease in long actin stress fibers, enhanced sensitivity to actin depolymerizing agents, and an increase in punctate F-actin staining in a distinctly anomalous distribution in the center of the cell. We also demonstrate an abnormal distribution of two actin-binding proteins, gelsolin and alpha-actinin, proteins regulated by both PIP(2) and Ca(+2) that would be expected to be altered in Lowe cells. Actin polymerization plays a key role in the formation, maintenance, and proper function of tight junctions and adherens junctions, which have been demonstrated to be critical in renal proximal tubule function, and in the differentiation of the lens. These findings point to a general mechanism to explain how this PIP(2) 5-phosphatase deficiency might produce the Lowe syndrome phenotype.

SUBMITTER: Suchy SF

PROVIDER: S-EPMC378584 | biostudies-literature | 2002 Dec

REPOSITORIES: biostudies-literature

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The deficiency of PIP2 5-phosphatase in Lowe syndrome affects actin polymerization.

Suchy Sharon F SF Nussbaum Robert L RL

American journal of human genetics 20021111 6

Lowe syndrome is a rare X-linked disorder characterized by bilateral congenital cataracts, renal Fanconi syndrome, and mental retardation. Lowe syndrome results from mutations in the OCRL1 gene, which encodes a phosphatidylinositol 4,5 bisphosphate 5-phosphatase located in the trans-Golgi network. As a first step in identifying the link between ocrl1 deficiency and the clinical disorder, we have identified a reproducible cellular abnormality of the actin cytoskeleton in fibroblasts from patients ...[more]

PMID: 12428211

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Project description:Listeria monocytogenes is a bacterial pathogen that induces its own entry into a broad range of mammalian cells through interaction of the bacterial surface protein InlB with the cellular receptor Met, promoting an actin polymerization/depolymerization process that leads to pathogen engulfment. Phosphatidylinositol bisphosphate (PI[4,5]P(2)) and trisphosphate (PI[3,4,5]P(3)) are two major phosphoinositide species that function as molecular scaffolds, recruiting cellular effectors that regulate actin dynamics during L. monocytogenes infection. Because the phosphatidylinositol 5'-phosphatase OCRL dephosphorylates PI(4,5)P(2) and to a lesser extent PI(3,4,5)P(3), we investigated whether this phosphatase modulates cell invasion by L. monocytogenes. Inactivation of OCRL by small interfering RNA (siRNA) leads to an increase in the internalization levels of L. monocytogenes in HeLa cells. Interestingly, OCRL depletion does not increase but rather decreases the surface expression of the receptor Met, suggesting that OCRL controls bacterial internalization by modulating signaling cascades downstream of Met. Immuno-fluorescence microscopy reveals that endogenous and overexpressed OCRL are present at L. monocytogenes invasion foci; live-cell imaging additionally shows that actin depolymerization coincides with EGFP-OCRL-a accumulation around invading bacteria. Together, these observations suggest that OCRL promotes actin depolymerization during L. monocytogenes infection; in agreement with this hypothesis, OCRL depletion leads to an increase in actin, PI(4,5)P(2), and PI(3,4,5)P(3) levels at bacterial internalization foci. Furthermore, in cells knocked down for OCRL, transfection of enzymatically active EGFP-OCRL-a (but not of a phosphatase-dead enzyme) decreases the levels of intracellular L. monocytogenes and of actin associated with invading bacteria. These results demonstrate that through its phosphatase activity, OCRL restricts L. monocytogenes invasion by modulating actin dynamics at bacterial internalization sites.

Project description:Background:Lowe syndrome (LS) is a rare genetic disorder caused by loss of function mutations in the X-linked gene, OCRL, which codes for inositol polyphosphate 5-phosphatase. LS is characterized by the triad of congenital cataracts, neurodevelopmental impairment (primarily intellectual and developmental disabilities [IDD]), and renal proximal tubular dysfunction. Studies carried out over the years have shown that hypomorphic mutations in OCRL adversely affect endosome recycling and actin polymerization in kidney cells and patient-derived fibroblasts. The renal problem has been traced to an impaired recycling of megalin, a multi-ligand receptor that plays a key role in the reuptake of lipoproteins, amino acids, vitamin-binding proteins, and hormones. However, the neurodevelopmental aspects of the disorder have been difficult to study because the mouse knockout (KO) model does not display LS-related phenotypes. Fortunately, the discovery of induced pluripotent stem (iPS) cells has provided an opportunity to grow patient-specific neurons, which can be used to model neurodevelopmental disorders in vitro, as demonstrated in the many studies that have been published in the past few years in autism spectrum disorders (ASD), schizophrenia (SZ), bipolar disorder (BD), and IDD. Methods:We now report the first findings in neurons and neural progenitor cells (NPCs) generated from iPS cells derived from patients with LS and their typically developing male siblings, as well as an isogenic line in which the OCRL gene has been incapacitated by a null mutation generated using CRISPR-Cas9 gene editing. Results:We show that neuronal cells derived from patient-specific iPS cells containing hypomorphic variants are deficient in their capacity to produce F-filamentous actin (F-actin) fibers. Abnormalities were also found in the expression of WAVE-1, a component of the WAVE regulatory complex (WRC) that regulates actin polymerization. Curiously, neuronal cells carrying the engineered OCRL null mutation, in which OCRL protein is not expressed, did not show similar defects in F-actin and WAVE-1 expression. This is similar to the apparent lack of a phenotype in the mouse Ocrl KO model, and suggests that in the complete absence of OCRL protein, as opposed to producing a dysfunctional protein, as seen with the hypomorphic variants, there is partial compensation for the F-actin/WAVE-1 regulating function of OCRL. Conclusions:Alterations in F-actin polymerization and WRC have been found in a number of genetic subgroups of IDD and ASD. Thus, LS, a very rare genetic condition, is linked to a more expansive family of genes responsible for neurodevelopmental disorders that have shared pathogenic features.

Project description:BackgroundFertilization of echinoderm eggs is accompanied by dynamic changes of the actin cytoskeleton and by a drastic increase of cytosolic Ca(2+). Since the plasma membrane-enriched phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) serves as the precursor of inositol 1,4,5 trisphosphate (InsP(3)) and also regulates actin-binding proteins, PIP2 might be involved in these two processes.Methodology/principal findingsIn this report, we have studied the roles of PIP2 at fertilization of starfish eggs by using fluorescently tagged pleckstrin homology (PH) domain of PLC-?1, which has specific binding affinity to PIP2, in combination with Ca(2+) and F-actin imaging techniques and transmission electron microscopy. During fertilization, PIP2 increased at the plasma membrane in two phases rather than continually decreasing. The first increase was quickly followed by a decrease about 40 seconds after sperm-egg contact. However, these changes took place only after the Ca(2+) wave had already initiated and propagated. The fertilized eggs then displayed a prolonged increase of PIP2 that was accompanied by the appearance of numerous spikes in the perivitelline space during the elevation of the fertilization envelope (FE). These spikes, protruding from the plasma membrane, were filled with microfilaments. Sequestration of PIP2 by RFP-PH at higher doses resulted in changes of subplasmalemmal actin networks which significantly delayed the intracellular Ca(2+) signaling, impaired elevation of FE, and increased occurrences of polyspermic fertilization.Conclusions/significanceOur results suggest that PIP2 plays comprehensive roles in shaping Ca(2+) waves and guiding structural and functional changes required for successful fertilization. We propose that the PIP2 increase and the subsequent formation of actin spikes not only provide the mechanical supports for the elevating FE, but also accommodate increased membrane surfaces during cortical granule exocytosis.

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The deficiency of PIP2 5-phosphatase in Lowe syndrome affects actin polymerization.

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The deficiency of PIP2 5-phosphatase in Lowe syndrome affects actin polymerization.

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