Expression and translocation of fluorescent-tagged p21-activated kinase-binding domain and PH domain of protein kinase B during murine neutrophil chemotaxis.
Neutrophils are key cells of the innate immune system; they are terminally differentiated and therefore difficult to genetically manipulate and study in vitro. In the present study, we describe a protocol to transiently express two fluorescent markers, the PH domain of protein kinase B fused to red fluorescent protein and the p21-activated kinase-binding domain fused to a yellow fluorescent protein, in primary neutrophils. Using this approach, we are able to achieve a transfection efficiency of approximately 30%. The expression of the transfected probes occurred within 2 h and allowed for real-time monitoring of intermediates in key neutrophil activation pathways at the leading edge of migrating cells. We describe here a transfection protocol for primary neutrophils, which preserves fMLP-mediated cell polarization and cytoskeleton reorganization with simultaneous accumulation of PI-3K products and active Rac at the leading edge. The visualization and analysis of transfected fluorescent markers in primary neutrophils are a powerful technique to monitor chemotaxis signaling pathways in real time.
Magalhães
Marco A O
MA
CIHR Group in Matrix Dynamics and Dental Research Institute, Faculty of Dentistry, University of Toronto, 241 Fitzgerald Building, 150 College Street, Toronto, Canada M5S 3E2.
Zhu
Fei
F
Sarantis
Helen
H
Gray-Owen
Scott D
SD
Ellen
Richard P
RP
Glogauer
Michael
M
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002634:Chemotaxis, Leukocyte; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D009504:Neutrophils / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D044042:Receptors, Formyl Peptide / Q000235:genetics / Q000378:metabolism; D054462:p21-Activated Kinases; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000097573:Red Fluorescent Protein
D008164:Luminescent Proteins; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D044042:Receptors, Formyl Peptide; D054462:p21-Activated Kinases; D020744:rac GTP-Binding Proteins; D000097573:Red Fluorescent Protein