Microspectroscopic imaging tracks the intracellular processing of a signal transduction protein: fluorescent-labeled protein kinase C beta I.
We have devised a microspectroscopic strategy for assessing the intracellular (re)distribution and the integrity of the primary structure of proteins involved in signal transduction. The purified proteins are fluorescent-labeled in vitro and reintroduced into the living cell. The localization and molecular state of fluorescent-labeled protein kinase C beta I isozyme were assessed by a combination of quantitative confocal laser scanning microscopy, fluorescence lifetime imaging microscopy, and novel determinations of fluorescence resonance energy transfer based on photobleaching digital imaging microscopy. The intensity and fluorescence resonance energy transfer efficiency images demonstrate the rapid nuclear translocation and ensuing fragmentation of protein kinase C beta I in BALB/c3T3 fibroblasts upon phorbol ester stimulation, and suggest distinct, compartmentalized roles for the regulatory and catalytic fragments.
Bastiaens
P I
PI
Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Jovin
T M
TM
D016475:3T3 Cells; D000818:Animals; D002451:Cell Compartmentation / Q000187:drug effects; D002462:Cell Membrane / Q000201:enzymology; D002467:Cell Nucleus / Q000201:enzymology; D005456:Fluorescent Dyes; D007091:Image Processing, Computer-Assisted; D007527:Isoenzymes / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D011493:Protein Kinase C / Q000378:metabolism*; D064546:Protein Kinase C beta; D011994:Recombinant Proteins; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors
D005456:Fluorescent Dyes; D007527:Isoenzymes; D011994:Recombinant Proteins; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D013755:Tetradecanoylphorbol Acetate