Ca2+-regulated Pool of Phosphatidylinositol-3-phosphate Produced by Phosphatidylinositol 3-Kinase C2 on Neurosecretory Vesicles

Molecular Dynamics of Synaptic Function Laboratory, Queensland Brain Institute and School of Biomedical Sciences, The University of Queensland, St. Lucia, 4072 Queensland, Australia.
Molecular biology of the cell (Impact Factor: 4.47). 11/2008; 19(12):5593-603. DOI: 10.1091/mbc.E08-06-0595
Source: PubMed


Phosphatidylinositol-3-phosphate [PtdIns(3)P] is a key player in early endosomal trafficking and is mainly produced by class III phosphatidylinositol 3-kinase (PI3K). In neurosecretory cells, class II PI3K-C2alpha and its lipid product PtdIns(3)P have recently been shown to play a critical role during neuroexocytosis, suggesting that two distinct pools of PtdIns(3)P might coexist in these cells. However, the precise characterization of this additional pool of PtdIns(3)P remains to be established. Using a selective PtdIns(3)P probe, we have identified a novel PtdIns(3)P-positive pool localized on secretory vesicles, sensitive to PI3K-C2alpha knockdown and relatively resistant to wortmannin treatment. In neurosecretory cells, stimulation of exocytosis promoted a transient albeit large increase in PtdIns(3)P production localized on secretory vesicles sensitive to PI3K-C2alpha knockdown and expression of PI3K-C2alpha catalytically inactive mutant. Using purified chromaffin granules, we found that PtdIns(3)P production is controlled by Ca(2+). We confirmed that PtdIns(3)P production from recombinantly expressed PI3K-C2alpha is indeed regulated by Ca(2+). We provide evidence that a dynamic pool of PtdIns(3)P synthesized by PI3K-C2alpha occurs on secretory vesicles in neurosecretory cells, demonstrating that the activity of a member of the PI3K family is regulated by Ca(2+) in vitro and in living neurosecretory cells.

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Available from: Frederic A Meunier, Sep 29, 2015
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    • "Using the phosphatidylinositol 3-kinase inhibitor LY294002, a correlation between the level of the plasma membrane PtdIns(4,5)P2 and the size of the primed vesicle pool was found (19, 20). Wen et al. (21) further demonstrated that selective inhibition of phosphatidylinositol 3-kinase delta isoform was responsible for this effect. Importantly, such an inhibition promotes a transient rise in PtdIns(4,5)P2 that was sufficient to mobilize secretory vesicles to the plasma membrane via activation of the small GTPase Cdc42 and actin polymerization. "
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    ABSTRACT: The regulated secretory pathway in neuroendocrine cells ends with the release of hormones and neurotransmitters following a rise in cytosolic calcium. This process known as regulated exocytosis involves the assembly of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, the synaptic vesicle VAMP (synaptobrevin), and the plasma membrane proteins syntaxin and SNAP-25. Although there is much evidence suggesting that SNARE proteins play a key role in the fusion machinery, other cellular elements regulating the kinetics, the extent of fusion, and the preparation of vesicle for release have received less attention. Among those factors, lipids have also been proposed to play important functions both at the level of secretory vesicle recruitment and late membrane fusion steps. Here, we will review the latest evidence supporting the concept of the fusogenic activity of lipids, and also discuss how this may be achieved. These possibilities include the recruitment and sequestration of the components of the exocytotic machinery, regulation of protein function, and direct effects on membrane topology.
    Frontiers in Endocrinology 09/2013; 4:125. DOI:10.3389/fendo.2013.00125
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    • "Cells were fixed in 4% paraformaldehyde in phosphate-buffered saline and processed for immunocytochemistry as described previously [24], [25]. Permeabilisation was performed using 0.1% saponin (for EEA1) or 0.05% Triton X-100 (other antibodies). "
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    ABSTRACT: The lipid phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P 2), synthesised by PIKfyve, regulates a number of intracellular membrane trafficking pathways. Genetic alteration of the PIKfyve complex, leading to even a mild reduction in PtdIns(3,5)P 2, results in marked neurodegeneration via an uncharacterised mechanism. In the present study we have shown that selectively inhibiting PIKfyve activity, using YM-201636, significantly reduces the survival of primary mouse hippocampal neurons in culture. YM-201636 treatment promoted vacuolation of endolysosomal membranes followed by apoptosis-independent cell death. Many vacuoles contained intravacuolar membranes and inclusions reminiscent of autolysosomes. Accordingly, YM-201636 treatment increased the level of the autophagosomal marker protein LC3-II, an effect that was potentiated by inhibition of lysosomal proteases, suggesting that alterations in autophagy could be a contributing factor to neuronal cell death.
    PLoS ONE 03/2013; 8(3):e60152. DOI:10.1371/journal.pone.0060152 · 3.23 Impact Factor
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    • "Interestingly, the ATP-dependent priming stage of exocytosis was originally found to be regulated by Ca 2+ [86]. In this view it is interesting to note that the kinetics of the PtdIns3P increase detected on secretory vesicles closely followed that of the Ca 2+ signal in PC12 cells [85]. Although the functional significance of this PtdIns3P increase is still unknown, it is tempting to speculate that the Ca 2+ signal elicited during the stimulation not only triggers the fusion of LDCVs but also prepares the next round of fusion by promoting the priming of new LDCVs through this and perhaps other pathways. "
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    ABSTRACT: Phosphoinositides (PIs) are a family of phospholipids derived from phosphatidylinositol (PtdIns), whose location, synthesis, and degradation depend on specific PI kinases and phosphatases. PIs have emerged as fundamental regulators of secretory processes, such as neurotransmitter release, hormone secretion, and histamine release in allergic responses. In neurons and neuroendocrine cells, regulated secretion requires the calcium-dependent fusion of transmitter-containing vesicles with the plasma membrane. The role played by PIs in exocytosis is best exemplified by the Ca(2+)-dependent binding of vesicular Synaptotagmin1 to the plasma membrane PtdIns(4,5)P(2), and the recently demonstrated role of PtdIns(4,5)P(2) in the mobilization of secretory vesicles to the plasma membrane. New evidence has also recently emerged of an alternative PI pathway that can control exocytosis positively (via PtdIn3P) or negatively (via PtdIns(3,5)P(2)). However, the positive or negative effectors for these pathways remain to be established. Reducing PtdIns(3,5)P(2) potentiates neuroexocytosis but leads to neuronal degeneration and has been linked to certain forms of Charcot-Marie-Tooth disease and amyotrophic lateral sclerosis. The goal of this review is to describe the role of PIs in neuroexocytosis and explore the current hypotheses linking these effects to human diseases.
    Current topics in microbiology and immunology 10/2012; 362:87-98. DOI:10.1007/978-94-007-5025-8_4 · 4.10 Impact Factor
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