Immunostaining evidence for PI(4,5)P2 localization at the leading edge of chemoattractant-stimulated HL-60 cells

It is well known that in fMLP-stimulated neutrophils, phosphatidyl inositol 3,4,5-trisphosphate [PI(3,4,5)P-3] localizes at the leading edge of the cells. However, no effort has been made to study the PI 4,5-bisphosphate [PI(4,5)P-2] distribution in these cells. In fact, it has been suggested that PI(4,5)P-2 is unlikely to localize, as its basal level is orders of magnitude higher than that of PI(3,4,5)P-3. We developed an optimized immunostaining protocol for studying the endogenous distribution of PI(4,5)P-2 in neutrophil-like HL-60 cells. We show that PI(4,5)P-2 localizes sharply at the leading edge with an intensity gradient similar to that for PI(3,4,5)P-3. The enzymes for the production of PI(4,5)P-2, namely, PI5KI alpha and PI5KI gamma, were also found to localize at the leading edge, further supporting our finding that PI(4,5)P-2 localizes at the leading edge. These results imply that complementary regulation of PI3K and phosphate and tensin homolog (PTEN) is not the sole or dominant mechanism of PI(3,4,5)P-3 polarization in HL-60 cells.