TY - JOUR
T1 - Immunostaining evidence for PI(4,5)P2 localization at the leading edge of chemoattractant-stimulated HL-60 cells
AU - Sharma, Ved P.
AU - DesMarais, Vera
AU - Sumners, Colin
AU - Shaw, Gerry
AU - Narang, Atul
PY - 2008/8/1
Y1 - 2008/8/1
N2 - 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)P 2 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, PI5KIα and PI5KIγ, 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)P3 polarization in HL-60 cells.
AB - 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)P 2 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, PI5KIα and PI5KIγ, 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)P3 polarization in HL-60 cells.
KW - Chemotaxis
KW - Gradient sensing
KW - Phosphoinositides
UR - http://www.scopus.com/inward/record.url?scp=48649090400&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=48649090400&partnerID=8YFLogxK
U2 - 10.1189/jlb.0907636
DO - 10.1189/jlb.0907636
M3 - Article
C2 - 18477691
AN - SCOPUS:48649090400
SN - 0741-5400
VL - 84
SP - 440
EP - 447
JO - Journal of Leukocyte Biology
JF - Journal of Leukocyte Biology
IS - 2
ER -