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Published online before print May 13, 2008

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(Journal of Leukocyte Biology. 2008;84:440-447.)
© 2008 by Society for Leukocyte Biology

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

Ved P. Sharma^*,, Vera DesMarais, Colin Sumners, Gerry Shaw and Atul Narang^*,1

^* Department of Chemical Engineering and Departments of
Physiology and Functional Genomics and
Neuroscience, McKnight Brain Institute, University of Florida, Gainesville, Florida, USA; and
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York, USA

¹ Correspondence: Department of Chemical Engineering, University of Florida, Gainesville, FL 32611, USA. E-mail: narang{at}che.ufl.edu

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, PI5KI and PI5KI, 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.

Key Words: chemotaxis • gradient sensing • phosphoinositides