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

Abstract

Phosphoinositides (PtdIns) play important roles in exocytosis and are thought to regulate secretory granule docking by co-clustering with the SNARE protein syntaxin to form a docking receptor in the plasma membrane. Here we tested this idea by high-resolution total internal reflection imaging of EGFP-labeled PtdIns markers or syntaxin-1 at secretory granule release sites in live insulin-secreting cells. In intact cells, PtdIns markers distributed evenly across the plasma membrane with no preference for granule docking sites. In contrast, syntaxin-1 was found clustered in the plasma membrane, mostly beneath docked granules. We also observed rapid accumulation of syntaxin-1 at sites where granules arrived to dock. Acute depletion of plasma membrane phosphatidylinositol (4,5) bisphosphate (PtdIns(4,5)P ) by recruitment of a 5'-phosphatase strongly inhibited Ca -dependent exocytosis, but had no effect on docked granules or the distribution and clustering of syntaxin-1. Cell permeabilization by α-toxin or formaldehyde-fixation caused PtdIns marker to slowly cluster, in part near docked granules. In summary, our data indicate that PtdIns(4,5)P accelerates granule priming, but challenge a role of PtdIns in secretory granule docking or clustering of syntaxin-1 at the release site.

Phosphatidylinositol(4,5)bisphosphate: diverse functions at the plasma membrane

“…There can be preferential trapping of PI(4,5)P 2 in lipid rafts, binding proteins such as MARCKS (myristoylated alanine-rich C-kinase substrate), syntaxin-1 and K-Ras that sequester PI(4,5)P 2 , or localised recruitment of PIP5K to generate PI(4,5)P 2 . Although there is strong evidence to support segregation of PI(4,5)P 2 , as discussed above, there remains many caveats due to technical limitations [132,133].…”

Insulin granule biogenesis and exocytosis

“…Indeed, both syntaxin-1 and PI(4,5)P 2 have been shown to form microdomains in cell-free membranes and fixated cells that at least partially colocalize with docked granules [123][124][125]. However, their presence in the plasma membrane of living cells is controversial and may depend on cell type and experimental methods [126][127][128]. However, selective removal of PI(4,5)P 2 at docking sites affects tethering and docking [129].…”

Local PI(4,5)P2 generation controls fusion pore expansion during exocytosis

“…Acute depletion of PI(4,5)P 2 strongly inhibits regulated exocytosis (Milosevic et al, 2005; Omar-Hmeadi et al, 2018), suggesting an important role of this phospholipid during late stages of exocytosis. To understand this role, we studied PI(4,5)P 2 dynamics during stimulated exocytosis.…”