The SH3/PH Domain Protein AgBoi1/2 Collaborates with the Rho-Type GTPase
AgRho3 To Prevent Nonpolar Growth at Hyphal Tips of Ashbya
gossypii

ABSTRACT Unlike most other cells, hyphae of filamentous fungi permanently elongate and lack nonpolar growth phases. We identified AgBoi1/2p in the filamentous ascomycete Ashbya gossypii as a component required to prevent nonpolar growth at hyphal tips. Strains lacking AgBoi1/2p frequently show spherical enlargement at hyphal tips with concomitant depolarization of actin patches and loss of tip-located actin cables. These enlarged tips can repolarize and resume hyphal tip extension in the previous polarity axis. AgBoi1/2p permanently localizes to hyphal tips and transiently to sites of septation. Only the tip localization is important for sustained elongation of hyphae. In a yeast two-hybrid experiment, we identified the Rho-type GTPase AgRho3p as an interactor of AgBoi1/2p. AgRho3p is also required to prevent nonpolar growth at hyphal tips, and strains deleted for both AgBOI1/2 and AgRHO3 phenocopied the respective single-deletion strains, demonstrating that AgBoi1/2p and AgRho3p function in a common pathway. Monitoring the polarisome of growing hyphae using AgSpa2p fused to the green fluorescent protein as a marker, we found that polarisome disassembly precedes the onset of nonpolar growth in strains lacking AgBoi1/2p or AgRho3p. AgRho3p locked in its GTP-bound form interacts with the Rho-binding domain of the polarisome-associated formin AgBni1p, implying that AgRho3p has the capacity to directly activate formin-driven actin cable nucleation. We conclude that AgBoi1/2p and AgRho3p support polarisome-mediated actin cable formation at hyphal tips, thereby ensuring permanent polar tip growth.

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A PAK-like protein kinase is required for maturation of young hyphae and septation in the filamentous ascomycete Ashbya gossypii

Journal of Cell Science ◽

10.1242/jcs.113.24.4563 ◽

2000 ◽

Vol 113 (24) ◽

pp. 4563-4575 ◽

Cited By ~ 3

Author(s):

Y. Ayad-Durieux ◽

P. Knechtle ◽

S. Goff ◽

F. Dietrich ◽

P. Philippsen

Keyword(s):

Protein Kinase ◽

Filamentous Fungi ◽

Fluorescent Protein ◽

Ashbya Gossypii ◽

Polarized Growth ◽

Fast Growing ◽

Filamentous Ascomycete ◽

Green Fluorescent ◽

Actin Cables ◽

Developmental Switch

Filamentous fungi grow by hyphal extension, which is an extreme example of polarized growth. In contrast to yeast species, where polarized growth of the tip of an emerging bud is temporally limited, filamentous fungi exhibit constitutive polarized growth of the hyphal tip. In many fungi, including Ashbya gossypii, polarized growth is reinforced by a process called hyphal maturation. Hyphal maturation refers to the developmental switch from slow-growing hyphae of young mycelium to fast-growing hyphae of mature mycelium. This process is essential for efficient expansion of mycelium. We report for the first time on the identification and characterization of a fungal gene important for hyphal maturation. This novel A. gossypii gene encodes a presumptive PAK (p21-activated kinase)-like kinase. Its closest homolog is the S. cerevisiae Cla4 protein kinase; the A. gossypii protein is therefore called AgCla4p. Agcla4 deletion strains are no longer able to perform the developmental switch from young to mature hyphae, and GFP (green fluorescent protein)-tagged AgCla4p localizes with much higher frequency in mature hyphal tips than in young hyphal tips. Both results support the importance of AgCla4p in hyphal maturation. AgCla4p is also required for septation, indicated by the inability of Agcla4 deletion strains to properly form actin rings and chitin rings. Despite the requirement of AgCla4p for the development of fast-growing hyphae, AgCla4p is not necessary for actin polarization per se, because tips enriched in cortical patches and hyphae with a fully developed network of actin cables can be seen in Agcla4 deletion strains. The possibility that AgCla4p may be involved in regulatory mechanisms that control the dynamics of the actin patches and/or actin cables is discussed.

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A Ras-like GTPase Is Involved in Hyphal Growth Guidance in the Filamentous Fungus Ashbya gossypii

Molecular Biology of the Cell ◽

10.1091/mbc.e04-02-0104 ◽

2004 ◽

Vol 15 (10) ◽

pp. 4622-4632 ◽

Cited By ~ 54

Author(s):

Yasmina Bauer ◽

Philipp Knechtle ◽

Jürgen Wendland ◽

Hanspeter Helfer ◽

Peter Philippsen

Keyword(s):

Fluorescent Protein ◽

Hyphal Growth ◽

Time Lapse ◽

Growth Direction ◽

Ashbya Gossypii ◽

Growth Guidance ◽

Characteristic Features ◽

Green Fluorescent ◽

Hyphal Tip

Characteristic features of morphogenesis in filamentous fungi are sustained polar growth at tips of hyphae and frequent initiation of novel growth sites (branches) along the extending hyphae. We have begun to study regulation of this process on the molecular level by using the model fungus Ashbya gossypii. We found that the A. gossypii Ras-like GTPase Rsr1p/Bud1p localizes to the tip region and that it is involved in apical polarization of the actin cytoskeleton, a determinant of growth direction. In the absence of RSR1/BUD1, hyphal growth was severely slowed down due to frequent phases of pausing of growth at the hyphal tip. During pausing events a hyphal tip marker, encoded by the polarisome component AgSPA2, disappeared from the tip as was shown by in vivo time-lapse fluorescence microscopy of green fluorescent protein-labeled AgSpa2p. Reoccurrence of AgSpa2p was required for the resumption of hyphal growth. In the Agrsr1/bud1Δ deletion mutant, resumption of growth occurred at the hyphal tip in a frequently uncoordinated manner to the previous axis of polarity. Additionally, hyphal filaments in the mutant developed aberrant branching sites by mislocalizing AgSpa2p thus distorting hyphal morphology. These results define AgRsr1p/Bud1p as a key regulator of hyphal growth guidance.

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From Function to Shape: A Novel Role of a Formin in Morphogenesis of the Fungus Ashbya gossypii

Molecular Biology of the Cell ◽

10.1091/mbc.e05-06-0479 ◽

2006 ◽

Vol 17 (1) ◽

pp. 130-145 ◽

Cited By ~ 52

Author(s):

Hans-Peter Schmitz ◽

Andreas Kaufmann ◽

Michael Köhli ◽

Pierre Philippe Laissue ◽

Peter Philippsen

Keyword(s):

Giant Cells ◽

Polar Growth ◽

Ashbya Gossypii ◽

Essential Factor ◽

Wild Type ◽

Actin Cable ◽

Lateral Branches ◽

Actin Cables ◽

Two Hybrid

Morphogenesis of filamentous ascomycetes includes continuously elongating hyphae, frequently emerging lateral branches, and, under certain circumstances, symmetrically dividing hyphal tips. We identified the formin AgBni1p of the model fungus Ashbya gossypii as an essential factor in these processes. AgBni1p is an essential protein apparently lacking functional overlaps with the two additional A. gossypii formins that are nonessential. Agbni1 null mutants fail to develop hyphae and instead expand to potato-shaped giant cells, which lack actin cables and thus tip-directed transport of secretory vesicles. Consistent with the essential role in hyphal development, AgBni1p locates to tips, but not to septa. The presence of a diaphanous autoregulatory domain (DAD) indicates that the activation of AgBni1p depends on Rho-type GTPases. Deletion of this domain, which should render AgBni1p constitutively active, completely changes the branching pattern of young hyphae. New axes of polarity are no longer established subapically (lateral branching) but by symmetric divisions of hyphal tips (tip splitting). In wild-type hyphae, tip splitting is induced much later and only at much higher elongation speed. When GTP-locked Rho-type GTPases were tested, only the young hyphae with mutated AgCdc42p split at their tips, similar to the DAD deletion mutant. Two-hybrid experiments confirmed that AgBni1p interacts with GTP-bound AgCdc42p. These data suggest a pathway for transforming one axis into two new axes of polar growth, in which an increased activation of AgBni1p by a pulse of activated AgCdc42p stimulates additional actin cable formation and tip-directed vesicle transport, thus enlarging and ultimately splitting the polarity site.

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Phosphatidylinositol-4,5-Bisphosphate-Rich Plasma Membrane Patches Organize Active Zones of Endocytosis and Ruffling in Cultured Adipocytes

Molecular and Cellular Biology ◽

10.1128/mcb.24.20.9102-9123.2004 ◽

2004 ◽

Vol 24 (20) ◽

pp. 9102-9123 ◽

Cited By ~ 54

Author(s):

Shaohui Huang ◽

Larry Lifshitz ◽

Varsha Patki-Kamath ◽

Richard Tuft ◽

Kevin Fogarty ◽

...

Keyword(s):

Plasma Membrane ◽

Large Scale ◽

Plasma Membranes ◽

Fluorescent Protein ◽

Dimensional Space ◽

Three Dimensional ◽

Actin Dynamics ◽

Ph Domain ◽

Membrane Ruffling ◽

Green Fluorescent

ABSTRACT A major regulator of endocytosis and cortical F-actin is thought to be phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] present in plasma membranes. Here we report that in 3T3-L1 adipocytes, clathrin-coated membrane retrieval and dense concentrations of polymerized actin occur in restricted zones of high endocytic activity. Ultrafast-acquisition and superresolution deconvolution microscopy of cultured adipocytes expressing an enhanced green fluorescent protein- or enhanced cyan fluorescent protein (ECFP)-tagged phospholipase Cδ1 (PLCδ1) pleckstrin homology (PH) domain reveals that these zones spatially coincide with large-scale PtdIns(4,5)P2-rich plasma membrane patches (PRMPs). PRMPs exhibit lateral dimensions exceeding several micrometers, are relatively stationary, and display extensive local membrane folding that concentrates PtdIns(4,5)P2 in three-dimensional space. In addition, a higher concentration of PtdIns(4,5)P2 in the membranes of PRMPs than in other regions of the plasma membrane can be detected by quantitative fluorescence microscopy. Vesicular structures containing both clathrin heavy chains and PtdIns(4,5)P2 are revealed immediately beneath PRMPs, as is dense F actin. Blockade of PtdIns(4,5)P2 function in PRMPs by high expression of the ECFP-tagged PLCδ1 PH domain inhibits transferrin endocytosis and reduces the abundance of cortical F-actin. Membrane ruffles induced by the expression of unconventional myosin 1c were also found to localize at PRMPs. These results are consistent with the hypothesis that PRMPs organize active PtdIns(4,5)P2 signaling zones in the adipocyte plasma membrane that in turn control regulators of endocytosis, actin dynamics, and membrane ruffling.

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Dynamic localization of yeast Fus2p to an expanding ring at the cell fusion junction during mating

The Journal of Cell Biology ◽

10.1083/jcb.200801101 ◽

2008 ◽

Vol 181 (4) ◽

pp. 697-709 ◽

Cited By ~ 22

Author(s):

Joanna Mathis Paterson ◽

Casey A. Ydenberg ◽

Mark D. Rose

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Fusion ◽

Fluorescent Protein ◽

Cell Junction ◽

Dynamic Localization ◽

Actin Organization ◽

Pheromone Signaling ◽

Fusion Junction ◽

Green Fluorescent ◽

Actin Cables

Fus2p is a pheromone-induced protein associated with the amphiphysin homologue Rvs161p, which is required for cell fusion during mating in Saccharomyces cerevisiae. We constructed a functional Fus2p–green fluorescent protein (GFP), which exhibits highly dynamic localization patterns in pheromone-responding cells (shmoos): diffuse nuclear, mobile cytoplasmic dots and stable cortical patches concentrated at the shmoo tip. In mitotic cells, Fus2p-GFP is nuclear but becomes cytoplasmic as cells form shmoos, dependent on the Fus3p protein kinase and high levels of pheromone signaling. The rapid cytoplasmic movement of Fus2p-GFP dots requires Rvs161p and polymerized actin and is aberrant in mutants with compromised actin organization, which suggests that the Fus2p dots are transported along actin cables, possibly in association with vesicles. Maintenance of Fus2p-GFP patches at the shmoo tip cortex is jointly dependent on actin and a membrane protein, Fus1p, which suggests that Fus1p is an anchor for Fus2p. In zygotes, Fus2p-GFP forms a dilating ring at the cell junction, returning to the nucleus at the completion of cell fusion.

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A PDZ-containing Scaffold Related to the Dystrophin Complex at the Basolateral Membrane of Epithelial Cells

The Journal of Cell Biology ◽

10.1083/jcb.145.2.391 ◽

1999 ◽

Vol 145 (2) ◽

pp. 391-402 ◽

Cited By ~ 78

Author(s):

Amy M. Kachinsky ◽

Stanley C. Froehner ◽

Sharon L. Milgram

Keyword(s):

Skeletal Muscle ◽

Fluorescent Protein ◽

Protein Complexes ◽

Basolateral Membrane ◽

Pdz Domain ◽

Cell Types ◽

Protein Domain ◽

Ph Domain ◽

Green Fluorescent ◽

Dystrophin Complex

Membrane scaffolding complexes are key features of many cell types, serving as specialized links between the extracellular matrix and the actin cytoskeleton. An important scaffold in skeletal muscle is the dystrophin-associated protein complex. One of the proteins bound directly to dystrophin is syntrophin, a modular protein comprised entirely of interaction motifs, including PDZ (protein domain named for PSD-95, discs large, ZO-1) and pleckstrin homology (PH) domains. In skeletal muscle, the syntrophin PDZ domain recruits sodium channels and signaling molecules, such as neuronal nitric oxide synthase, to the dystrophin complex. In epithelia, we identified a variation of the dystrophin complex, in which syntrophin, and the dystrophin homologues, utrophin and dystrobrevin, are restricted to the basolateral membrane. We used exogenously expressed green fluorescent protein (GFP)-tagged fusion proteins to determine which domains of syntrophin are responsible for its polarized localization. GFP-tagged full-length syntrophin targeted to the basolateral membrane, but individual domains remained in the cytoplasm. In contrast, the second PH domain tandemly linked to a highly conserved, COOH-terminal region was sufficient for basolateral membrane targeting and association with utrophin. The results suggest an interaction between syntrophin and utrophin that leaves the PDZ domain of syntrophin available to recruit additional proteins to the epithelial basolateral membrane. The assembly of multiprotein signaling complexes at sites of membrane specialization may be a widespread function of dystrophin-related protein complexes.

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Quantification of PtdIns(3,4,5)P3 dynamics in EGF-stimulated carcinoma cells: a comparison of PH-domain-mediated methods with immunological methods

Biochemical Journal ◽

10.1042/bj20071179 ◽

2008 ◽

Vol 411 (2) ◽

pp. 441-448 ◽

Cited By ~ 20

Author(s):

Shu-Chin Yip ◽

Robert J. Eddy ◽

Angie M. Branch ◽

Huan Pang ◽

Haiyan Wu ◽

...

Keyword(s):

Fluorescent Protein ◽

Leading Edge ◽

Carcinoma Cells ◽

Immunological Methods ◽

Ph Domain ◽

Membrane Recruitment ◽

Secondary Messenger ◽

Ph Domains ◽

Green Fluorescent ◽

Kinetics Of

Class IA PI3Ks (phosphoinositide 3-kinases) generate the secondary messenger PtdIns(3,4,5)P3, which plays an important role in many cellular responses. The accumulation of PtdIns(3,4,5)P3 in cell membranes is routinely measured using GFP (green fluorescent protein)-labelled PH (pleckstrin homology) domains. However, the kinetics of membrane PtdIns(3,4,5)P3 synthesis and turnover as detected by PH domains have not been validated using an independent method. In the present study, we measured EGF (epidermal growth factor)-stimulated membrane PtdIns(3,4,5)P3 production using a specific monoclonal anti-PtdIns(3,4,5)P3 antibody, and compared the results with those obtained using PH-domain-dependent methods. Anti-PtdIns(3,4,5)P3 staining rapidly accumulated at the leading edge of EGF-stimulated carcinoma cells. PtdIns(3,4,5)P3 levels were maximal at 1 min, and returned to basal levels by 5 min. In contrast, membrane PtdIns(3,4,5)P3 production, measured by the membrane translocation of an epitope-tagged BTKPH (PH domain of Bruton's tyrosine kinase), remained approx. 2-fold above basal level throughout 4–5 min of EGF stimulation. To determine the reason for this disparity, we measured the rate of PtdIns(3,4,5)P3 hydrolysis by measuring the decay of the PtdIns(3,4,5)P3 signal after LY294002 treatment of EGF-stimulated cells. LY294002 abolished anti-PtdIns(3,4,5)P3 membrane staining within 10 s of treatment, suggesting that PtdIns(3,4,5)P3 turnover occurs within seconds of synthesis. In contrast, BTKPH membrane recruitment, once initiated by EGF, was relatively insensitive to LY294002. These data suggest that sequestration of PtdIns(3,4,5)P3 by PH domains may affect the apparent kinetics of PtdIns(3,4,5)P3 accumulation and turnover; consistent with this hypothesis, we found that GRP-1 (general receptor for phosphoinositides 1) PH domains [which, like BTK, are specific for PtdIns(3,4,5)P3] inhibit PTEN (phosphatase and tensin homologue deleted on chromosome 10) dephosphorylation of PtdIns(3,4,5)P3in vitro. These data suggest that anti-PtdIns(3,4,5)P3 antibodies are a useful tool to detect localized PtdIns(3,4,5)P3, and illustrate the importance of using multiple approaches for the estimation of membrane phosphoinositides.

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Visualization of Phosphoinositides That Bind Pleckstrin Homology Domains: Calcium- and Agonist-induced Dynamic Changes and Relationship to Myo-[3H]inositol-labeled Phosphoinositide Pools

The Journal of Cell Biology ◽

10.1083/jcb.143.2.501 ◽

1998 ◽

Vol 143 (2) ◽

pp. 501-510 ◽

Cited By ~ 534

Author(s):

Péter Várnai ◽

Tamás Balla

Keyword(s):

Fluorescent Probe ◽

Fluorescent Protein ◽

Dynamic Imaging ◽

Ph Domain ◽

Pleckstrin Homology ◽

Protein Fusion ◽

Fusion Construct ◽

Cellular Expression ◽

Ph Domains ◽

Green Fluorescent

Phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P2) pools that bind pleckstrin homology (PH) domains were visualized by cellular expression of a phospholipase C (PLC)δ PH domain–green fluorescent protein fusion construct and analysis of confocal images in living cells. Plasma membrane localization of the fluorescent probe required the presence of three basic residues within the PLCδ PH domain known to form critical contacts with PtdIns(4,5)P2. Activation of endogenous PLCs by ionophores or by receptor stimulation produced rapid redistribution of the fluorescent signal from the membrane to cytosol, which was reversed after Ca2+ chelation. In both ionomycin- and agonist-stimulated cells, fluorescent probe distribution closely correlated with changes in absolute mass of PtdIns(4,5)P2. Inhibition of PtdIns(4,5)P2 synthesis by quercetin or phenylarsine oxide prevented the relocalization of the fluorescent probe to the membranes after Ca2+ chelation in ionomycin-treated cells or during agonist stimulation. In contrast, the synthesis of the PtdIns(4,5)P2 imaged by the PH domain was not sensitive to concentrations of wortmannin that had been found inhibitory of the synthesis of myo-[3H]inositol– labeled PtdIns(4,5)P2. Identification and dynamic imaging of phosphoinositides that interact with PH domains will further our understanding of the regulation of such proteins by inositol phospholipids.

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Localization of Phosphatidylinositol (3,4,5)-Trisphosphate to Phagosomes in Entamoeba histolytica Achieved Using Glutathione S-Transferase- and Green Fluorescent Protein-Tagged Lipid Biosensors

Infection and Immunity ◽

10.1128/iai.00719-09 ◽

2009 ◽

Vol 78 (1) ◽

pp. 125-137 ◽

Cited By ~ 20

Author(s):

Yevgeniya A. Byekova ◽

Rhonda R. Powell ◽

Brenda H. Welter ◽

Lesly A. Temesvari

Keyword(s):

Green Fluorescent Protein ◽

Entamoeba Histolytica ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Major Product ◽

Pi3 Kinase ◽

Ph Domain ◽

Intestinal Protozoan ◽

Ph Domains ◽

Green Fluorescent

ABSTRACT Entamoeba histolytica is an intestinal protozoan parasite that causes amoebic dysentery and liver abscess. Phagocytosis by the parasite is a critical virulence process, since it is a prerequisite for tissue invasion and establishment of chronic infection. While the roles of many of the proteins that regulate phagocytosis-related signaling events in E. histolytica have been characterized, the functions of lipids in this cellular process remain largely unknown in this parasite. In other systems, phosphatidylinositol (3,4,5)-trisphosphate (PIP3), a major product of phosphoinositide 3 kinase (PI3-kinase) activity, is essential for phagocytosis. Pleckstrin homology (PH) domains are protein domains that specifically bind to PIP3. In this study, we utilized glutathione S-transferase (GST)- and green fluorescent protein (GFP)-labeled PH domains as lipid biosensors to characterize the spatiotemporal aspects of PIP3 distribution during various endocytic processes in E. histolytica. PIP3-specific biosensors accumulated at extending pseudopodia and in phagosomal cups in trophozoites exposed to erythrocytes but did not localize to pinocytic compartments during the uptake of a fluid-phase marker, dextran. Our results suggest that PIP3 is involved in the early stages of phagosome formation in E. histolytica. In addition, we demonstrated that PIP3 exists at high steady-state levels in the plasma membrane of E. histolytica and that these levels, unlike those in mammalian cells, are not abolished by serum withdrawal. Finally, expression of a PH domain in trophozoites inhibited erythrophagocytosis and enhanced motility, providing genetic evidence supporting the role of PI3-kinase signaling in these processes in E. histolytica.

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Alteration of Epithelial Structure and Function Associated with PtdIns(4,5)P2 Degradation by a Bacterial Phosphatase

The Journal of General Physiology ◽

10.1085/jgp.200609656 ◽

2007 ◽

Vol 129 (4) ◽

pp. 267-283 ◽

Cited By ~ 55

Author(s):

David Mason ◽

Gustavo V. Mallo ◽

Mauricio R. Terebiznik ◽

Bernard Payrastre ◽

B. Brett Finlay ◽

...

Keyword(s):

Epithelial Cells ◽

Fluorescent Protein ◽

Fluid Loss ◽

Pleckstrin Homology Domain ◽

High Avidity ◽

Ph Domain ◽

Cytosolic Ph ◽

Functional Changes ◽

Epithelial Cultures ◽

Green Fluorescent

Elucidation of the role of PtdIns(4,5)P2 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)P2, 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)P2 in intact SigD-injected cells was verified by detachment from the membrane of the pleckstrin homology domain of phospholipase Cδ, 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)P2. Similarly, anion permeability, measured using two different halide-sensitive probes, was depressed in cells expressing SigD. Depletion of PtdIns(4,5)P2 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)P2-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 (IP5) into inositol 1,4,5,6-tetrakisphosphate (IP4), 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 IP4. In contrast, the cytoskeletal and ion transport effects were replicated by hydrolyzing PtdIns(4,5)P2 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)P2 hydrolysis combine to account, at least in part, for the fluid loss observed during Salmonella-induced diarrhea.

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