Conformation, localization, and integrin binding of talin depend on its interaction with phosphoinositides

Talin is a structural component of focal adhesion sites and is thought to be engaged in multiple protein interactions at the cytoplasmic face of cell/matrix contacts. Talin is a major link between integrin and the actin cytoskeleton and was shown to play an important role in focal adhesion assembly. Consistent with the view that talin must be activated at these sites, we found that phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)) bound to talin in cells in suspension or at early stages of adhesion, respectively. When phosphoinositides were associated with phospholipid bilayer, talin/phosphoinositide association was restricted to PI4,5P(2). This association led to a conformational change of the protein. Moreover, the interaction between integrin and talin was greatly enhanced by PI4,5P(2)-induced talin activation. Finally, sequestration of PI4,5P(2) by a specific pleckstrin homology domain confirms that PI4,5P(2) is necessary for proper membrane localization of talin and that this localization is essential for the maintenance of focal adhesions. Our results support a model in which PI4,5P(2) exposes the integrin-binding site on talin. We propose that PI4,5P(2)-dependent signaling modulates assembly of focal adhesions by regulating integrin-talin complexes. These results demonstrate that activation of the integrin-binding activity of talin requires not only integrin engagement to the extracellular matrix but also the binding of PI4,5P(2) to talin, suggesting a possible role of lipid metabolism in organizing the sequential assembly of focal adhesion components.     

PIP kinase Igamma is the major PI(4,5)P(2) synthesizing enzyme at the synapse

Disruption of the presynaptically enriched polyphosphoinositide phosphatase synaptojanin 1 leads to an increase of clathrin-coated intermediates and of polymerized actin at endocytic zones of nerve terminals. These changes correlate with elevated levels of PI(4,5)P(2) in neurons. We report that phosphatidylinositol phosphate kinase type Igamma (PIPKIgamma), a major brain PI(4)P 5-kinase, is concentrated at synapses. Synaptojanin 1 and PIPKIgamma antagonize each other in the recruitment of clathrin coats to lipid membranes. Like synaptojanin 1 and other proteins involved in endocytosis, PIPKIgamma undergoes stimulation-dependent dephosphorylation. These results implicate PIPKIgamma in the synthesis of a PI(4,5)P(2) pool that acts as a positive regulator of clathrin coat recruitment and actin function at the synapse.     

Type I gamma phosphatidylinositol phosphate kinase is required for EGF-stimulated directional cell migration

Phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)) modulates a plethora of cytoskeletal interactions that control the dynamics of actin assembly and, ultimately, cell migration. We show that the type Igamma phosphatidylinositol phosphate kinase 661 (PIPKIgamma661), an enzyme that generates PI4,5P(2), is required for growth factor but not G protein-coupled receptor-stimulated directional migration. By generating PI4,5P(2) and regulating talin assembly, PIPKIgamma661 modulates nascent adhesion formation at the leading edge to facilitate cell migration. The epidermal growth factor (EGF) receptor directly phosphorylates PIPKIgamma661 at tyrosine 634, and this event is required for EGF-induced migration. This phosphorylation regulates the interaction between PIPKIgamma661 and phospholipase Cgamma1 (PLCgamma1, an enzyme previously shown to be involved in the regulation of EGF-stimulated migration). Our results suggest that phosphorylation events regulating specific PIPKIgamma661 interactions are required for growth factor-induced migration. These interactions in turn define the spatial and temporal generation of PI4,5P(2) and derived messengers required for directional migration.     

Interaction of talin with actin: sensitive modulation of filament crosslinking activity.

Talin is an adhesion plaque protein believed important in linking actin filaments to the plasma membrane. The nature of a direct talin-actin interaction, however, is complex and has remained unclear. We have systematically characterized the effects of pH, ionic strength, temperature, and protein molar ratio on the interaction between highly purified talin and actin. The ability of talin to increase viscosity of F-actin at 25 degrees C and low ionic strength increased with decreasing pH from 7.3 to 6.4 and increasing molar ratio of talin to actin. At pH 6.4 and low ionic strength, talin could extensively crosslink actin filaments into ordered bundles as shown by negative staining and could cosediment with F-actin at molar ratios as high as one talin to two actin monomers. Talin crosslinked prepolymerized actin filaments to a similar extent as actin filaments polymerized in its presence. The 190-kDa calpain-generated proteolytic fragment of talin bound poorly to actin under conditions favorable for intact talin, but was able to crosslink actin filaments at a lower pH. Increasing the ionic strength within a relatively narrow range significantly decreased ability of talin to bind to actin, regardless of pH. The effects of pH and ionic strength on the talin-actin interaction were rapid and reversible. Low-shear-viscosity studies revealed a strong temperature dependence in the talin-actin interaction with significant crosslinking activity at physiological-like ionic conditions and temperature (37 degrees C). Our results consistently demonstrated that talin crosslinks actin filaments and that this direct interaction is highly sensitive to, and dependent upon, ionic conditions and temperature.     

SNX9 couples actin assembly to phosphoinositide signals and is required for membrane remodeling during endocytosis

Multiple modes of endocytosis require actin-dependent remodeling of the plasma membrane; however, neither the factors linking these processes nor their mechanisms of action are understood. The sorting nexin, SNX9, localizes to clathrin-coated pits where it interacts with dynamin and functions in clathrin-mediated endocytosis. Here, we demonstrate that SNX9 also localizes to actin-rich structures implicated in fluid-phase uptake, including tubular membranes containing GPI-anchored proteins and dorsal membrane ruffles. Moreover, we show that SNX9 is critical for dorsal ruffle formation and for clathrin-independent, actin-dependent fluid-phase endocytosis. In vitro, SNX9 directly associates with N-WASP, an Arp2/3 complex activator, and stimulates N-WASP/Arp2/3-mediated actin assembly. SNX9-stimulated actin polymerization is greatly enhanced by PI(4,5)P(2)-containing liposomes, due in part to PI(4,5)P(2)-induced SNX9 oligomerization. These results suggest a mechanism for the spatial and temporal regulation of N-WASP-dependent actin assembly and implicate SNX9 in directly coupling actin dynamics to membrane remodeling during multiple modes of endocytosis.     

Polyphosphoinositides inhibit the interaction of vinculin with actin filaments.

Binding of vinculin to adhesion plaque proteins is restricted by an intramolecular association of vinculin's head and tail regions. Results of previous work suggest that polyphosphoinositides disrupt this interaction and thereby promote binding of vinculin to both talin and actin. However, data presented here show that phosphatidylinositol 4,5-bisphosphate (PI4,5P2) inhibits the interaction of purified tail domain with F-actin. Upon re-examining the effect of PI4,5P2 on the actin and talin-binding activities of intact vinculin, we find that when the experimental design controls for the effect of magnesium on aggregation of PI4,5P2 micelles, polyphosphoinositides promote interactions with the talin-binding domain, but block interactions of the actin-binding domain. In contrast, if vinculin is trapped in an open confirmation by a peptide specific for the talin-binding domain of vinculin, actin binding is allowed. These results demonstrate that activation of the actin-binding activity of vinculin requires steps other than or in addition to the binding of PI4,5P2.     

Eukaryotic elongation factor 1A2 cooperates with phosphatidylinositol-4 kinase III beta to stimulate production of filopodia through increased phosphatidylinositol-4,5 bisphosphate generation

Eukaryotic elongation factor 1 alpha 2 (eEF1A2) is a transforming gene product that is highly expressed in human tumors of the ovary, lung, and breast. eEF1A2 also stimulates actin remodeling, and the expression of this factor is sufficient to induce the formation of filopodia, long cellular processes composed of bundles of parallel actin filaments. Here, we find that eEF1A2 stimulates formation of filopodia by increasing the cellular abundance of cytosolic and plasma membrane-bound phosphatidylinositol-4,5 bisphosphate [PI(4,5)P(2)]. We have previously reported that the eEF1A2 protein binds and activates phosphatidylinositol-4 kinase III beta (PI4KIIIbeta), and we find that production of eEF1A2-dependent PI(4,5)P(2) and generation of filopodia require PI4KIIIbeta. Furthermore, PI4KIIIbeta is itself capable of activating both the production of PI(4,5)P(2) and the creation of filopodia. We propose a model for extrusion of filopodia in which eEF1A2 activates PI4KIIIbeta, and activated PI4KIIIbeta stimulates production of PI(4,5)P(2) and filopodia by increasing PI4P abundance. Our work suggests an important role for both eEF1A2 and PI4KIIIbeta in the control of PI(4,5)P(2) signaling and actin remodeling.     

Role of activation of PIP5Kgamma661 by AP-2 complex in synaptic vesicle endocytosis

Synaptic vesicles (SVs) are retrieved by clathrin-mediated endocytosis at the nerve terminals. Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] drives this event by recruiting the components of the endocytic machinery. However, the molecular mechanisms that result in local generation of PI(4,5)P2 remain unclear. We demonstrate here that AP-2 complex directly interacts with phosphatidylinositol 4-phosphate 5-kinase gamma661 (PIP5Kgamma661), the major PI(4,5)P2-producing enzyme in the brain. The beta2 subunit of AP-2 was found to bind to the C-terminal tail of PIP5Kgamma661 and cause PIP5Kgamma661 activation. The interaction is regulated by PIP5Kgamma661 dephosphorylation, which is triggered by depolarization in mouse hippocampal neurons. Finally, overexpression of the PIP5Kgamma661 C-terminal region in hippocampal neurons suppresses depolarization-dependent SV endocytosis. These findings provide evidence for the molecular mechanism through which PIP5Kgamma661 locally generates PI(4,5)P2 in hippocampal neurons and suggest a model in which the interaction trigger SV endocytosis.     

Plasma Membrane Phosphatidylinositol 4,5 Bisphosphate Is Required for Internalization of Foot-and-Mouth Disease Virus and Vesicular Stomatitis Virus

Phosphatidylinositol-4,5-bisphosphate, PI(4,5)P₂, is a phospholipid which plays important roles in clathrin-mediated endocytosis. To investigate the possible role of this lipid on viral entry, two viruses important for animal health were selected: the enveloped vesicular stomatitis virus (VSV) − which uses a well characterized clathrin mediated endocytic route − and two different variants of the non-enveloped foot-and-mouth disease virus (FMDV) with distinct receptor specificities. The expression of a dominant negative dynamin, a PI(4,5)P₂ effector protein, inhibited the internalization and infection of VSV and both FMDV isolates. Depletion of PI(4,5)P₂ from plasma membrane using ionomycin or an inducible system, and inhibition of its de novo synthesis with 1-butanol revealed that VSV as well as FMDV C-S8c1, which uses integrins as receptor, displayed a high dependence on PI(4,5)P₂ for internalization. Expression of a kinase dead mutant (KD) of phosphatidylinositol-4-phosphate-5-kinase Iα (PIP5K-Iα), an enzyme responsible for PI(4,5)P₂ synthesis that regulates clathrin-dependent endocytosis, also impaired entry and infection of VSV and FMDV C-S8c1. Interestingly FMDV MARLS variant that uses receptors other than integrins for cell entry was less sensitive to PI(4,5)P₂ depletion, and was not inhibited by the expression of the KD PIP5K-Iα mutant suggesting the involvement of endocytic routes other than the clathrin-mediated on its entry. These results highlight the role of PI(4,5)P₂ and PIP5K-Iα on clathrin-mediated viral entry.     

Synaptojanin 1-mediated PI(4,5)P2 hydrolysis is modulated by membrane curvature and facilitates membrane fission

Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P?] plays a fundamental role in clathrin-mediated endocytosis. However, precisely how PI(4,5)P? metabolism is spatially and temporally regulated during membrane internalization and the functional consequences of endocytosis-coupled PI(4,5)P? dephosphorylation remain to be explored. Using cell-free assays with liposomes of varying diameters, we show that the major synaptic phosphoinositide phosphatase, synaptojanin 1 (Synj1), acts with membrane curvature generators/sensors, such as the BAR protein endophilin, to preferentially remove PI(4,5)P? from curved membranes as opposed to relatively flat ones. Moreover, in vivo recruitment of Synj1's inositol 5-phosphatase domain to endophilin-induced membrane tubules results in fragmentation and condensation of these structures largely in a dynamin-dependent fashion. Our study raises the possibility that geometry-based mechanisms may contribute to spatially restricting PI(4,5)P? elimination during membrane internalization and suggests that the PI(4,5)P?-to-PI4P conversion achieved by Synj1 at sites of high curvature may cooperate with dynamin to achieve membrane fission.     

Separate functions of gelsolin mediate sequential steps of collagen phagocytosis

Collagen phagocytosis is a critical mediator of extracellular matrix remodeling. Whereas the binding step of collagen phagocytosis is facilitated by Ca2+-dependent, gelsolin-mediated severing of actin filaments, the regulation of the collagen internalization step is not defined. We determined here whether phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] regulation of gelsolin is required for collagen internalization. In gelsolin null fibroblasts transfected with gelsolin severing mutants, actin severing and collagen binding were strongly impaired but internalization and actin monomer addition at collagen bead sites were much less affected. PI(4,5)P2 accumulated around collagen during internalization and was associated with gelsolin. Cell-permeable peptides mimicking the PI(4,5)P2 binding site of gelsolin blocked actin monomer addition, the association of gelsolin with actin at phagosomes, and collagen internalization but did not affect collagen binding. Collagen beads induced recruitment of type 1 gamma phosphatidylinositol phosphate kinase (PIPK1gamma661) to internalization sites. Dominant negative constructs and RNA interference demonstrated a requirement for catalytically active PIPK1gamma661 for collagen internalization. We conclude that separate functions of gelsolin mediate sequential stages of collagen phagocytosis: Ca2+-dependent actin severing facilitates collagen binding, whereas PI(4,5)P2-dependent regulation of gelsolin promotes the actin assembly required for internalization of collagen fibrils.     

Structural basis for the interaction between the cytoplasmic domain of the hyaluronate receptor layilin and the talin F3 subdomain

Talin is a large cytoskeletal protein that is involved in coupling the integrin family of cell adhesion molecules to the actin cytoskeleton, colocalising with the integrins in focal adhesions (FAs). However, at the leading edge of motile cells, talin colocalises with the hyaluronan receptor layilin in what are thought to be transient adhesions, some of which subsequently mature into more stable FAs. During this maturation process, layilin is replaced with integrins, which are highly clustered in FAs, where localised production of PI(4,5)P₂ by type 1 phosphatidyl inositol phosphate kinase type 1γ (PIPK1γ) is thought to play a role in FA assembly. The talin FERM F3 subdomain binds both the integrin β-subunit cytoplasmic domain and PIPK1γ, and these interactions are understood in detail at the atomic level. The talin F3 domain also binds to short sequences in the layilin cytoplasmic domain, and here we report the structure of the talin/layilin complex, which shows that talin binds integrins, PIPK1γ and layilin in similar although subtly different ways. Based on structure comparisons, we designed a set of talin F3 mutations that selectively affected the affinity of talin for its targets, as determined by stopped-flow fluorescence measurements. Such mutations will help to assess the importance of the interactions between talin and its various ligands in cell adhesion and migration.     

Phosphatidylinositol-4,5-bisphosphate hydrolysis directs actin remodeling during phagocytosis

The Rho GTPases play a critical role in initiating actin polymerization during phagocytosis. In contrast, the factors directing the disassembly of F-actin required for fission of the phagocytic vacuole are ill defined. We used fluorescent chimeric proteins to monitor the dynamics of association of actin and active Cdc42 and Rac1 with the forming phagosome. Although actin was found to disappear from the base of the forming phagosome before sealing was complete, Rac1/Cdc42 activity persisted, suggesting that termination of GTPase activity is not the main determinant of actin disassembly. Furthermore, fully internalized phagosomes engineered to associate constitutively with active Rac1 showed little associated F-actin. The disappearance of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)) from the phagosomal membrane closely paralleled the course of actin disassembly. Furthermore, inhibition of PI(4,5)P(2) hydrolysis or increased PI(4,5)P(2) generation by overexpression of phosphatidylinositol phosphate kinase I prevented the actin disassembly necessary for the completion of phagocytosis. These observations suggest that hydrolysis of PI(4,5)P(2) dictates the remodeling of actin necessary for completion of phagocytosis.     

Molecular Basis for Association of PIPKI��-p90 with Clathrin Adaptor AP-2

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) is an essential determinant in clathrin-mediated endocytosis (CME). In mammals three type I phosphatidylinositol-4-phosphate 5-kinase (PIPK) enzymes are expressed, with the Iγ-p90 isoform being highly expressed in the brain where it regulates synaptic vesicle (SV) exo-/endocytosis at nerve terminals. How precisely PI(4,5)P₂ metabolism is controlled spatially and temporally is still uncertain, but recent data indicate that direct interactions between type I PIPK and components of the endocytic machinery, in particular the AP-2 adaptor complex, are involved. Here we demonstrated that PIPKIγ-p90 associates with both the μ and β2 subunits of AP-2 via multiple sites. Crystallographic data show that a peptide derived from the splice insert of the human PIPKIγ-p90 tail binds to a cognate recognition site on the sandwich subdomain of the β2 appendage. Partly overlapping aromatic and hydrophobic residues within the same peptide also can engage the C-terminal sorting signal binding domain of AP-2μ, thereby potentially competing with the sorting of conventional YXXØ motif-containing cargo. Biochemical and structure-based mutagenesis analysis revealed that association of the tail domain of PIPKIγ-p90 with AP-2 involves both of these sites. Accordingly the ability of overexpressed PIPKIγ tail to impair endocytosis of SVs in primary neurons largely depends on its association with AP-2β and AP-2μ. Our data also suggest that interactions between AP-2 and the tail domain of PIPKIγ-p90 may serve to regulate complex formation and enzymatic activity. We postulate a model according to which multiple interactions between PIPKIγ-p90 and AP-2 lead to spatiotemporally controlled PI(4,5)P₂ synthesis during clathrin-mediated SV endocytosis.     

A phosphatidylinositol (4,5)-bisphosphate binding site within mu2-adaptin regulates clathrin-mediated endocytosis

The clathrin adaptor complex AP-2 serves to coordinate clathrin-coated pit assembly with the sorting of transmembrane cargo proteins at the plasmalemma. How precisely AP-2 assembly and cargo protein recognition at sites of endocytosis are regulated has remained unclear, but recent evidence implicates phosphoinositides, in particular phosphatidylinositol (4,5)-bisphosphate (PI[4,5]P2), in these processes. Here we have identified and functionally characterized a conserved binding site for PI(4,5)P2 within mu2-adaptin, the medium chain of the clathrin adaptor complex AP-2. Mutant mu2 lacking a cluster of conserved lysine residues fails to bind PI(4,5)P2 and to compete the recruitment of native clathrin/AP-2 to PI(4,5)P2-containing liposomes or to presynaptic membranes. Moreover, we show that expression of mutant mu2 inhibits receptor-mediated endocytosis in living cells. We suggest that PI(4,5)P2 binding to mu2-adaptin regulates clathrin-mediated endocytosis and thereby may contribute to structurally linking cargo recognition to coat formation.     

Adenovirus Endocytosis Requires Actin Cytoskeleton Reorganization Mediated by Rho Family GTPases

Adenovirus (Ad) endocytosis via α_v integrins requires activation of the lipid kinase phosphatidylinositol-3-OH kinase (PI3K). Previous studies have linked PI3K activity to both the Ras and Rho signaling cascades, each of which has the capacity to alter the host cell actin cytoskeleton. Ad interaction with cells also stimulates reorganization of cortical actin filaments and the formation of membrane ruffles (lamellipodia). We demonstrate here that members of the Rho family of small GTP binding proteins, Rac and CDC42, act downstream of PI3K to promote Ad endocytosis. Ad internalization was significantly reduced in cells treated with Clostridium difficile toxin B and in cells expressing a dominant-negative Rac or CDC42 but not a H-Ras protein. Viral endocytosis was also inhibited by cytochalasin D as well as by expression of effector domain mutants of Rac or CDC42 that impair cytoskeletal function but not JNK/MAP kinase pathway activation. Thus, Ad endocytosis requires assembly of the actin cytoskeleton, an event initiated by activation of PI3K and, subsequently, Rac and CDC42.     

The yeast synaptojanin-like proteins control the cellular distribution of phosphatidylinositol (4,5)-bisphosphate

Phosphoinositides (PI) are synthesized and turned over by specific kinases, phosphatases, and lipases that ensure the proper localization of discrete PI isoforms at distinct membranes. We analyzed the role of the yeast synaptojanin-like proteins using a strain that expressed only a temperature-conditional allele of SJL2. Our analysis demonstrated that inactivation of the yeast synaptojanins leads to increased cellular levels of phosphatidylinositol (3,5)-bisphosphate and phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P(2)), accompanied by defects in actin organization, endocytosis, and clathrin-mediated sorting between the Golgi and endosomes. The phenotypes observed in synaptojanin-deficient cells correlated with accumulation of PtdIns(4,5)P(2), because these effects were rescued by mutations in MSS4 or a mutant form of Sjl2p that harbors only PI 5-phosphatase activity. We utilized green fluorescent protein-pleckstrin homology domain chimeras (termed FLAREs for fluorescent lipid-associated reporters) with distinct PI-binding specificities to visualize pools of PtdIns(4,5)P(2) and phosphatidylinositol 4-phosphate in yeast. PtdIns(4,5)P(2) localized to the plasma membrane in a manner dependent on Mss4p activity. On inactivation of the yeast synaptojanins, PtdIns(4,5)P(2) accumulated in intracellular compartments, as well as the cell surface. In contrast, phosphatidylinositol 4-phosphate generated by Pik1p localized in intracellular compartments. Taken together, our results demonstrate that the yeast synaptojanins control the localization of PtdIns(4,5)P(2) in vivo and provide further evidence for the compartmentalization of different PI species.     

Distinct specificity in the recognition of phosphoinositides by the pleckstrin homology domains of dynamin and Bruton's tyrosine kinase.

Pleckstrin homology (PH) domains may act as membrane localization modules through specific interactions with phosphoinositide phospholipids. These interactions could represent responses to second messengers, with scope for regulation by soluble inositol polyphosphates. A biosensor-based assay was used here to probe interactions between PH domains and unilamellar liposomes containing different phospholipids and to demonstrate specificity for distinct phosphoinositides. The dynamin PH domain specifically interacted with liposomes containing phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] and, more weakly, with liposomes containing phosphatidylinositol-4-phosphate [PI(4)P]. This correlates with phosphoinositide activation of the dynamin GTPase. The functional GTPase of a dynamin mutant lacking the PH domain, however, cannot be activated by PI(4,5)P2. The phosphoinositide-PH domain interaction can be abolished selectively by point mutations in the putative binding pocket predicted by molecular modelling and NMR spectroscopy. In contrast, the Bruton's tyrosine kinase (Btk)PH domain specifically bound liposomes containing phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3]: an interaction requiring Arg28, a residue found to be mutated in some X-linked agammaglobulinaemia patients. A rational explanation for these different specificities is proposed through modelling of candidate binding pockets and is supported by NMR spectroscopy.     

The activation of exocytotic sites by the formation of phosphatidylinositol 4,5-bisphosphate microdomains at syntaxin clusters

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a minor component of the lipid bilayer but plays an important role in various cellular functions, including exocytosis and endocytosis. Recently, PI(4,5)P2 was shown to form microdomains in the plasma membrane. In this study, we investigated the relationship between the spatial organization of PI(4,5)P2 microdomains and exocytotic machineries in clonal rat pheochromocytoma PC12 cells. Both PI(4,5)P2 and syntaxin, a soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein essential for exocytosis, exhibited punctate clusters in isolated plasma membranes. The number of PI(4,5)P2 microdomains colocalizing with syntaxin clusters and large dense core vesicles (LDCVs) was decreased after catecholamine release. Alternatively, the expression of type I phosphatidylinositol-4-phosphate 5-kinase (PIP5KI) increased the number of PI(4,5)P2 microdomains at syntaxin clusters with docked LDCVs and enhanced exocytotic activity, possibly by increasing the number of release sites. About half of the PI(4,5)P2 microdomains were not colocalized with Thy-1, a specific marker of lipid rafts, and the colocalization of transfected PIP5KI with syntaxin clusters was observed. These results suggest that the formation of PI(4,5)P2 microdomains at syntaxin clusters with docked LDCVs is essential for Ca2+-dependent exocytosis.     

Intrasteric inhibition mediates the interaction of the I/LWEQ module proteins Talin1, Talin2, Hip1, and Hip12 with actin

The I/LWEQ module superfamily is a class of actin-binding proteins that contains a conserved C-terminal actin-binding element known as the I/LWEQ module. I/LWEQ module proteins include the metazoan talins, the cellular slime mold talin homologues TalA and TalB, fungal Sla2p, and the metazoan Sla2 homologues Hip1 and Hip12 (Hip1R). These proteins possess a similar modular organization that includes an I/LWEQ module at their C-termini and either a FERM domain or an ENTH domain at their N-termini. As a result of this modular organization, I/LWEQ module proteins may serve as linkers between cellular compartments, such as the plasma membrane and the endocytic machinery, and the actin cytoskeleton. Previous studies have shown that I/LWEQ module proteins bind to F-actin. In this report, we have determined the affinity of the I/LWEQ module proteins Talin1, Talin2, huntingtin interacting protein-1 (Hip1), and the Hip1-related protein (Hip1R/Hip12) for F-actin and identified a conserved structural element that interferes with the actin binding capacity of these proteins. Our data support the hypothesis that the actin-binding determinants in native talin and other I/LWEQ module proteins are cryptic and indicate that the actin binding capacities of Talin1, Talin2, Hip1, and Hip12 are regulated by intrasteric occlusion of primary actin-binding determinants within the I/LWEQ module. We have also found that the I/LWEQ module contains a dimerization motif and stabilizes actin filaments against depolymerization. This activity may contribute to the function of talin in cell adhesion and the roles of Hip1, Hip12 (Hip1R), and Sla2p in endocytosis.