The Activation of Exocytotic Sites by the Formation of Phosphatidylinositol 4,5-Bisphosphate Microdomains at Syntaxin Clusters

Aoyagi, Kyota; Sugaya, Tsukiko; Umeda, Masato; Yamamoto, Seiji; Terakawa, Susumu; Takahashi, Masami

doi:10.1074/jbc.m413307200

Cited by 164 publications

(223 citation statements)

References 52 publications

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“…With additional control parameters (i.e., osmotic pressure, temperatature, composition) a transition to a nonlamellar states is then easily induced. In this context, we also found that the lipid phosphatidylinositol-4,5-bisphosphate (PIP 2 ), which is enriched at the sites of vesicle fusion of synaptic membranes (84,85), has a very pronounced effect on the phase behavior of DOPC and promotes stalk and inverted hexagonal phase formation already at concentrations of few mol% (43). As a strongly anionic lipid with three net negative charges, PIP 2 is likely to interact with Ca 2þ , possibly again facilitating localized dehydration and hydrophobic defect formation.…”

Section: Discussionmentioning

confidence: 88%

Energetics of stalk intermediates in membrane fusion are controlled by lipid composition

Aeffner

Reusch²,

Weinhausen³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

162

246

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We have used X-ray diffraction on the rhombohedral phospholipid phase to reconstruct stalk structures in different pure lipids and lipid mixtures with unprecedented resolution, enabling a quantitative analysis of geometry, as well as curvature and hydration energies. Electron density isosurfaces are used to study shape and curvature properties of the bent lipid monolayers. We observe that the stalk structure is highly universal in different lipid systems. The associated curvatures change in a subtle, but systematic fashion upon changes in lipid composition. In addition, we have studied the hydration interaction prior to the transition from the lamellar to the stalk phase. The results indicate that facilitating dehydration is the key to promote stalk formation, which becomes favorable at an approximately constant interbilayer separation of 9.0 AE 0.5 Å for the investigated lipid compositions.curvature energy | hydration force | lipid bilayer | E xocytosis, intracellular transport, neurotransmission, fertilization, or viral entry, require that two membranes merge into one. This event, membrane fusion, involves a complex interplay of different membrane lipids, proteins, and water molecules on length scales of few nm. Following the "lipidic pore hypothesis", it is now well accepted that membrane fusion involves a sequence of lipidic nonbilayer intermediates, whose formation is catalyzed and guided by a specialized protein machinery (1, 2). A stage termed "hemifusion" in which the lipids of the outer leaflets of two membrane-enclosed compartments about to fuse can mix, whereas the inner leaflets and the enclosed content remain unaltered (3), has been confirmed by a variety of methods including conical electron tomography (4) and, more indirectly; e.g., by electron spin resonance (5) and fluorescence recovery after photobleaching (6). Studying the fusion of protein-free bilayers of well defined lipid composition can contribute useful insights into the physical principles governing the merger of their more complex biological counterparts and clarify the effect of individual lipid species.The first connection between two lipid bilayers is the so-called stalk sketched in Fig. 1A (7). The proximal lipid monolayers have merged into one strongly curved monolayer, whereas the distal ones are still separated and intact. A persistent problem in membrane biophysics is to determine the precise structure of stalks and the free energy barrier for stalk formation. In a long series of papers (8-16), this determination has been attempted within the framework of the continuum theory of membrane elasticity (17, 18). More recently, with the advent of sufficient computational power, simulations including molecular details have become feasible [e.g. (19, 20) and references therein]. While stalk formation between lipid bilayers in close contact is generally accepted as the initial step in possibly all membrane fusion reactions (7), the subsequent stages from stalk to complete fusion are still debated and different pathways may exist (21)....

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Section: Discussionmentioning

confidence: 88%

Energetics of stalk intermediates in membrane fusion are controlled by lipid composition

Aeffner

Reusch²,

Weinhausen³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

162

246

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“…Recent studies have shown that vertebrate CAPS-1 activity and binding to the plasma membrane of PC12 cells depend on the lipid PIP 2 (Grishanin et al 2004), which is concentrated in submicrometer-size microdomains that correlate with sites of dense-core vesicle exocytosis in PC12 and chromaffin cells (Aoyagi et al 2005;Milosevic et al 2005). Further studies will be necessary to determine whether or not UNC-31's recruitment to high focal concentrations at active zones is PIP 2 dependent.…”

Section: Discussionmentioning

confidence: 99%

Presynaptic UNC-31 (CAPS) Is Required to Activate the Gαs Pathway of the Caenorhabditis elegans Synaptic Signaling Network

et al. 2006

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C. elegans mutants lacking the dense-core vesicle priming protein UNC-31 (CAPS) share highly similar phenotypes with mutants lacking a neuronal Ga s pathway, including strong paralysis despite exhibiting near normal levels of steady-state acetylcholine release as indicated by drug sensitivity assays. Our genetic analysis shows that UNC-31 and neuronal Ga s are different parts of the same pathway and that the UNC-31/Ga s pathway is functionally distinct from the presynaptic Ga q pathway with which it interacts. UNC-31 acts upstream of Ga s because mutations that activate the Ga s pathway confer similar levels of strongly hyperactive, coordinated locomotion in both unc-31 null and (1) backgrounds. Using cell-specific promoters, we show that both UNC-31 and the Ga s pathway function in cholinergic motor neurons to regulate locomotion rate. Using immunostaining we show that UNC-31 is often concentrated at or near active zones of cholinergic motor neuron synapses. Our data suggest that presynaptic UNC-31 activity, likely acting via dense-core vesicle exocytosis, is required to locally activate the neuronal Ga s pathway near synaptic active zones.A T least two distinct vesicle systems, synaptic vesicles and dense core vesicles, mediate the presynaptic release of neurotransmitters. The synaptic vesicle system mediates the highly localized, rapid release of classical neurotransmitters from active zones. Presynaptic dense core vesicle release does not occur at morphologically defined active zones and is generally associated with neuropeptide release. Little is known about whether and how these two vesicle systems interact at the synapse during the execution of behaviors. Both vesicle systems release their contents in response to electrically induced calcium influx (Martin 2003), and each system has a mechanism for priming vesicles, which is the process by which vesicles become competent to fuse in response to the calcium signal. Synaptic vesicles use an UNC-13-based system (Aravamudan et al. 1999;Augustin et al. 1999;Richmond et al. 1999Richmond et al. , 2001. Dense core vesicles use a CAPS-based system (Grishanin et al. 2004). UNC-13 and CAPS are distantly related as indicated by a shared UNC-13 homology domain (Grishanin et al. 2002).In either system, if vesicles are not primed, they will not release neurotransmitter in response to stimuli, even if calcium enters the synapse or cell (Hay and Martin 1992;Aravamudan et al. 1999;Augustin et al. 1999;Richmond et al. 1999). Although the pathways regulating dense core vesicle priming are poorly understood, previous studies have shown that diacylglycerol (DAG) and cAMP can regulate the readily releasable (primed) pool of synaptic vesicles (Chen and Regehr 1997;Stevens and Sullivan 1998;Waters and Smith 2000;Kidokoro et al. 2004;Virmani et al. 2005). Genetic studies in the model organism Caenorhabditis elegans have identified a network of pathways that appears to regulate priming in the synaptic vesicle system. These studies have suggested a model, the synaptic signal...

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“…One interesting possibility is the PIP2-mediated regulation of Wiskott-Aldrich syndrome protein (WASP)-Arp2/3 pathway, which is required for actin nucleation 55 and for the formation of an organized cytolytic synapse 56,57 ; another possibility is the control of vesicle fusion step via PIP2 interaction with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) factor syntaxin, as reported in other secretory systems. 58 Notably, WASP and syntaxin 11 mutations are responsible for Wiskott-Aldrich syndrome 56,57 and familiar hemophagocytic lymphohistiocytosis 4 immunodeficiencies, 59 respectively, both characterized by a defective cytotoxic function. Experiments are ongoing to elucidate whether PIP2 pools are actually implicated in the regulation of actin-and SNAREdependent events in cytotoxicity.…”

Section: Org Frommentioning

confidence: 99%

PI5KI-dependent signals are critical regulators of the cytolytic secretory pathway

Micucci

Capuano

Marchetti

et al. 2008

Blood

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Although membrane phospholipid phosphatidylinositol-4,5bisphosphate (PIP2) plays a key role as signaling intermediate and coordinator of actin dynamics and vesicle trafficking, it remains completely unknown its involvement in the activation of cytolytic machinery. By live confocal imaging of primary human natural killer (NK) cells expressing the chimeric protein GFP-PH, we observed, during effector-target cell interaction, the consumption of a preexisting PIP2 pool, which is critically required for the activation of cytolytic machinery. We identified type I phosphatidylinositol-4-phosphate-5-kinase (PI5KI) ␣ and ␥ isoforms as the enzymes responsible for PIP2 synthesis in NK cells. By hRNA-driven gene silencing, we observed that both enzymes are required for the proper activation of NK cytotoxicity and for inositol-1,4,5-trisphosphate (IP3) generation on receptor stimulation. In an attempt to elucidate the specific step controlled by PI5KIs, we found that lytic granule secretion but not polarization resulted in impaired PI5KI␣-and PI5KI␥-silenced cells. Our findings delineate a novel mechanism implicating PI5KI␣ and PI5KI␥ isoforms in the synthesis of PIP2 pools critically required for IP3-dependent Ca 2؉ IntroductionNatural killer (NK) cells and cytotoxic T lymphocytes are critical effectors in the defense against tumor and viral infections 1 ; they exert cytotoxic function through the polarized secretion of granules containing proteolytic molecules, such as perforin and granzymes. This process involves several steps, including the formation of a cytolytic synapse between cytolytic effector and target cell, the rapid reorientation of the microtubule-organizing center along with lytic granules toward the target contact area followed by granule docking and fusion at specialized secretory domains within the cytolytic synapse. 2,3 Several structurally distinct receptors have been implicated in the activation of NK-cell cytolytic machinery: when cross-linked by the corresponding ligands on target cell, they trigger multiple and intersecting signaling pathways responsible for functional activation. 4 A vast array of activating NK receptors belonging to different families are coupled to the lipid modifying enzymes phosphatidylinositol3-kinase (PI3K) and phospholipase C␥ (PLC␥), which provide signals critically required for the activation of the cytolytic machinery [5][6][7][8] ; notably, both enzymes use the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PIP2) as common substrate. 9 Besides its role as signaling intermediate, PIP2 also acts as critical regulator of various cellular processes, including actin remodeling, membrane and vesicle trafficking, adhesion, and ion transport. 10,11 Surprisingly, the role of PIP2 and its regulatory mechanisms in lymphocyte-mediated cytotoxicity remain completely undefined.The main cellular source of PIP2 are type I phosphatidylinositol-4-phosphate-5-kinase (PI5KI) family members which phosphorylate PI4P on the D5 position of the inositol ring. Three major PI5KI is...

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The Activation of Exocytotic Sites by the Formation of Phosphatidylinositol 4,5-Bisphosphate Microdomains at Syntaxin Clusters

Cited by 164 publications

References 52 publications

Energetics of stalk intermediates in membrane fusion are controlled by lipid composition

Energetics of stalk intermediates in membrane fusion are controlled by lipid composition

Presynaptic UNC-31 (CAPS) Is Required to Activate the Gαs Pathway of the Caenorhabditis elegans Synaptic Signaling Network

PI5KI-dependent signals are critical regulators of the cytolytic secretory pathway

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