Vesicle Docking Is a Key Target of Local PI(4,5)P2 Metabolism in the Secretory Pathway of INS-1 Cells

Abstract

SUMMARY Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) signaling is transient and spatially confined in live cells. How this pattern of signaling regulates transmitter release and hormone secretion has not been addressed. We devised an optogenetic approach to control PI(4,5)P2 levels in time and space in insulin-secreting cells. Combining this approach with total internal reflection fluorescence microscopy, we examined individual vesicle-trafficking steps. Unlike long-term PI(4,5)P2 perturbations, rapid and cell-wide PI(4,5)P2 reduction in the plasma membrane (PM) strongly inhibits secretion and intracellular Ca2+ concentration ([Ca2+]i) responses, but not sytaxin1a clustering. Interestingly, local PI(4,5)P2 reduction selectively at vesicle docking sites causes remarkable vesicle undocking from the PM without affecting [Ca2+]i. These results highlight a key role of local PI(4,5)P2 in vesicle tethering and docking, coordinated with its role in priming and fusion. Thus, different spatiotemporal PI(4,5)P2 signaling regulates distinct steps of vesicle trafficking, and vesicle docking may be a key target of local PI(4,5)P2 signaling in vivo.

PtdIns(4,5)P2 is not required for secretory granule docking

“…This discrepancy to our live cell work indicates that intact cellular membranes are different with regards to PtdIns aggregation, either because of unknown cellular mechanisms that prevent clustering of PtdIns, or because disruption of cellular membranes promotes PtdIns aggregation. We demonstrate here that permeabilization of the cells with α‐toxin causes slow clustering of PtdIns in the plasma membrane, reminiscent of previous reports . It can be envisioned that such treatment relieves physiological membrane tension, and that this in turn leads to membrane ruffling and clustering of PtdIns, syntaxin‐1 and other proteins.…”

“…We show here that acute depletion of PtdIns(4,5)P 2 in the plasma membrane strongly inhibits exocytosis, which confirms earlier reports . However, PtdIns(4,5)P 2 depletion did not cause changes in granule docking, and the density of docked granules was unaffected.…”

Cdk5‐mediated phosphorylation regulates phosphatidylinositol 4‐phosphate 5‐kinase type I γ 90 activity and cell invasion

“…Moreover, inhibition of PIPKIg90 activity with UNC3230 also inhibited fibronectin secretion. These data suggest that PIPKIg90 activity is essential for fibronectin secretion, which is consistent with the role of PIP 2 in vesicle docking (7,8,54).…”

“…Phosphatidylinositol 4 phosphate 5-kinase type I g (PIPKIg90) catalyzes ATP-dependent phosphorylation of phosphatidylinositol 4-phosphate to generate phosphatidylinositol 4,5-bisphosphate (PIP 2 ), which functions as a vesicle docking site, thus regulating vesicle exocytosis (7,8). It is also a precursor of several lipid second messengers, such as phosphatidylinositol 3,4,5-triphosphate, inositol 1,4,5-triphosphate, and diacylglycerol.…”

The fusion pore, 60 years after the first cartoon

“…In contrast, rapid PIP 2 reduction in the plasma membrane of INS1 cells strongly inhibited secretion but not sytaxin1a clustering. However, selective local PIP 2 reduction at vesicle docking sites caused vesicle undocking from the plasma membrane at these sites [166]. Another very recent study in live INS1 cells revealed also no evidence for PI(3,4,5)P3 or PIP 2 association with syntaxin clusters and actually no evidence for PIP 2 clustering at all [167].…”