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GFP-Tagged Protein Domains as Tools to Study Localized Second Messenger Function in Living Vells

Published online by Cambridge University Press:  02 July 2020

T. Meyer ,
E. Oancea ,
T. Stauffer  and
M. N. Teruel
T. Meyer
Affiliation:
Department of Cell Biology, Department of Pharmacology and Cancer Biology, Box 3709, Duke University Medical Center, Durham, NC27710
E. Oancea
Affiliation:
Department of Cell Biology, Department of Pharmacology and Cancer Biology, Box 3709, Duke University Medical Center, Durham, NC27710
T. Stauffer
Affiliation:
Department of Cell Biology, Department of Pharmacology and Cancer Biology, Box 3709, Duke University Medical Center, Durham, NC27710
M. N. Teruel
Affiliation:
Department of Cell Biology, Department of Pharmacology and Cancer Biology, Box 3709, Duke University Medical Center, Durham, NC27710
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Extract

We have investigated the translocation of different GFP-tagged protein domains in response to various receptor stimuli. Confocal fluorescent imaging was used to study the localization of the GFP-tagged protein domain before, during and after the receptor stimuli was applied. A minimal cysteine-rich domain from protein kinase C was shown to be a useful tool to study localized diacylglycerol changes. Furthermore, the calcium-induced plasma membrane translocation of the GFP-tagged C2-domain from PKC could be used to study receptor-mediated changes in near plasma membrane calcium concentration. In addition, different pleckstrin homology domains (PH-domains) could be employed to measure changes in the concentration of phosphatidylinositol 4,5-bisphosphate (PI4.5P2) as well as of P13,4P2and PO,4,5P3. In combination, these fluorescent translocation probes provide a new means to investigate the local and temporal control of cell signaling processes in individual living cells.

Type
Detection and Application of Green (and other Colored) Fluorescent Proteins
Information
Microscopy and Microanalysis , Volume 4 , Issue S2: Proceedings: Microscopy & Microanalysis '98, Microscopy Society of America 56th Annual Meeting, Microbeam Analysis Society 32nd Annual Meeting, Atlanta, Georgia July 12-16, 1998 , July 1998 , pp. 1014 - 1015
Copyright
Copyright © Microscopy Society of America

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References

1.Oancea, E., Teruel, M. N., Quest, A.., and Meyer, T. (1998) GFP-tagged cysteine-rich domains from protein kinase C as fluorescent indicators for diacylglycerol signaling in living cells.J. Cell Biol, (in press)Google Scholar
2.Teruel, M. N., and Meyer, T. (1997) Electroporation Induced Formation of Calcium Entry Sites in Cell Body and Processes of Adherent Cells. Biophys. J. 73, 1785.CrossRefGoogle ScholarPubMed
3.Yokoe, H., H. and Meyer, T. (1996) Spatial Dynamics of GFP-Tagged Proteins Investigated by Local Fluorescence Enhancement. Nature Biotechnol. 14,1252.CrossRefGoogle ScholarPubMed
4. This work was supported by National Insitute of Health grants GM-48113 and GM-51457 and Proctor and gamble grant SRA 1617.Google Scholar