Fluorescence imaging of mobility shifts: an expression cloning method for identification of cell signaling targets.
There is a need for a simple global approach to identify signaling targets that are posttranslationally modified in response to physiologic or pathologic stimuli within living cells. Reported here is a simple method, fluorescence imaging of mobility shifts (FIMS), which relies on in-gel detection of cell-expressed green fluorescent protein fusion proteins undergoing electrophoretic mobility shifts. This detection method is applied to a small pool cDNA library screening protocol. The readout is essentially a differential display of posttranslational modifications. Unlike biochemical approaches to identifying signaling targets, the screen is performed in living cells using standard methods for transient transfection. This enables detection of intracellular targets modified in response to either molecularly defined stimuli, such as growth factors or drugs, or complex pathologic stimuli, such as oxidative stress or hypoglycemia. FIMS is rapid, sensitive, inexpensive, and nonradioactive and easily adapted to automated high throughput methods, including capillary electrophoresis. The technique is sufficiently sensitive to easily detect fluorescent proteins expressed in a single well in 384-well format. FIMS is applicable to traditional cDNA library screening, but the method will be especially attractive for screening preselected collections of autofluorescent fusion proteins. A bonus of the technique is that examination of transfected cells by fluorescence microscopy provides immediate information about intracellular localization and stimulus-induced translocation of putative targets. We illustrate the utility of the technique with pilot screens for apoptotic and mitogenic targets modified by staurosporine and serum stimulation, respectively.
Mandell
James W
JW
Department of Pathology, University of Virginia, Charlottesville, Virginia 22908, USA.
Manabe
Ri-Ichiroh
R
Horwitz
Alan F
AF
Baumgart
Joel P
JP
D000818:Animals; D019556:COS Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents / Q000032:analysis / Q000378:metabolism; D008164:Luminescent Proteins / Q000032:analysis / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals; D012334:RNA, Neoplasm / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D021382:Protein Sorting Signals; D012334:RNA, Neoplasm; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D019311:Staurosporine