Quantitative Monitoring Spatiotemporal Activation of Ras and PKD1 Using Confocal Fluorescent Microscopy.
Receptor activation upon ligand binding induces activation of multiple signaling pathways. To fully understand how these signaling pathways coordinate, it is essential to determine the dynamic nature of the spatiotemporal activation profile of signaling components at the level of single living cells. Here, we outline a detailed methodology for visualizing and quantitatively measuring the spatiotemporal activation of Ras and PKD1 by applying advanced fluorescence imaging techniques, including multichannel, simultaneous imaging and Förster resonance energy transfer (FRET).
Chemotaxis Signaling Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, NIH, 5601 Fishers Lane, MSC 9806, Rockville, MD, USA. xxu@niaid.nih.gov.
Xuehua
X
Xu
Chemotaxis Signaling Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, NIH, 5601 Fishers Lane, MSC 9806, Rockville, MD, USA.
Michelle
M
Yun
Chemotaxis Signaling Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, NIH, 5601 Fishers Lane, MSC 9806, Rockville, MD, USA.
Xi
X
Wen
Imaging Core Facility, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, NIH, Rockville, MD, USA.
Joseph
J
Brzostowski
Chemotaxis Signaling Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, NIH, 5601 Fishers Lane, MSC 9806, Rockville, MD, USA.
Wei
W
Quan
Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Q Jane
QJ
Wang
Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, NIH, 5601 Fishers Lane, MSC 9806, Rockville, MD, USA.
Tian
T
Jin
D045744:Cell Line, Tumor; D002630:Chemotactic Factors / Q000378:metabolism / Q000494:pharmacology; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer; D015870:Gene Expression; D017930:Genes, Reporter; D018922:HL-60 Cells; D006801:Humans; D018613:Microscopy, Confocal / Q000379:methods; D057054:Molecular Imaging; D010766:Phosphorylation; D050396:TRPP Cation Channels / Q000235:genetics / Q000378:metabolism*; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
D002630:Chemotactic Factors; D050396:TRPP Cation Channels; D018631:ras Proteins
Confocal fluorescence microscopy; Förster resonance energy transfer (FRET); Phosphorylation of PKD1; Protein kinase D 1 (PKD1); Ras activation