Nanoscale clustering and dynamics of phosphatidylinositol 4,5-bisphosphate in an immune cell model

Mast cells mediate their immuno- and neuro-modulatory effects by releasing granules containing bioactive substances. Phosphatidylinositol 4,5-bisphosphate (PIP₂), enriched at the plasma membrane, is a key signaling lipid involved in numerous physiological functions including the calcium entry needed for antigen-stimulated mast cell degranulation. However, functional nanoscale PIP₂ clustering and dynamics have not been previously investigated in immune cells. Using the pleckstrin homology domain from PLCδ (PH) tagged with photoswitchable fluorescent protein Dendra2, clustering was revealed in the mast cell model RBL-2H3, both fixed and live. We also discovered that live RBL-2H3 cells have PH clusters that evolve over timescales of ∼100 s. Additionally, the distribution of PIP₂, and specifically PIP₂ clusters themselves, are disrupted upon addition of the cationic, lipidic drug cetylpyridinium chloride (CPC). CPC led to smaller, less dense, and more circular clusters. Furthermore, PH molecular mobility increased after the addition of CPC, suggesting interference of this drug with PH binding to PIP₂. In addition to this pharmacological relevance, the physiology of PIP₂ clusters during functional stimulation by antigen was investigated. Antigen stimulation led to increased cluster size, which was counteracted by CPC. In live cells, PH density outside clusters was altered by CPC but not by antigen. CPC increased the proportion of regions of high-density PH compared to all other regions of the plasma membrane. Although PH diffusion was, interestingly, not affected by antigen, it was increased by CPC, particularly in lower-density regions. Under all live-cell dynamics observed, PH demonstrated confinement that was consistent with simulated diffusion within potential wells with an elliptical shape. These findings illuminate the nanoscale behavior of PIP₂ in immune cells and the correlation of that behavior with cell function.