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)P3] localizes at the leading edge of the cells. However, no effort has been made to study the PI 4,5-bisphosphate [PI(4,5)P2] distribution in these cells. In fact, it has been suggested that PI(4,5)P2 is unlikely to localize, as its basal level is orders of magnitude higher than that of PI(3,4,5)P3. We developed an optimized immunostaining protocol for studying the endogenous distribution of PI(4,5)P2 in neutrophil-like HL-60 cells. We show that PI(4,5)P2 localizes sharply at the leading edge with an intensity gradient similar to that for PI(3,4,5)P3. The enzymes for the production of PI(4,5)P2, namely, PI5KIalpha and PI5KIgamma, were also found to localize at the leading edge, further supporting our finding that PI(4,5)P2 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)P3 polarization in HL-60 cells.
Sharma
Ved P
VP
Department of Chemical Engineering, University of Florida, Gainesville, FL 32611, USA.
DesMarais
Vera
V
Sumners
Colin
C
Shaw
Gerry
G
Narang
Atul
A
D015415:Biomarkers / Q000032:analysis; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002462:Cell Membrane / Q000648:ultrastructure; D004797:Enzyme-Linked Immunosorbent Assay; D018922:HL-60 Cells / Q000166:cytology*; D006801:Humans; D007150:Immunohistochemistry; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism
D015415:Biomarkers; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; C060974:phosphatidylinositol 3,4,5-triphosphate