Proc. SPIE. 4967, Genetically Engineered and Optical Probes for Biomedical Applications
KEYWORDS: Proteins, Statistical analysis, Green fluorescent protein, Microscopy, Luminescence, Molecules, Control systems, Chemical analysis, Biological research, Fluorescence resonance energy transfer
Adopted for the live cell, the methods of fluorescence resonance energy transfer (FRET) together with GFP-technology have taken us beyond the limits of optical resolution and allow to resolve proteinÐprotein interactions in 3-7 nm proximity in real time in a live cell. To reliably study cellular proteins involved in trafficking of cholera toxin we first analysed the maturation time of each chimeric protein. Cytosolic partners of proteins responsible for transport were fused to cyan fluorescent protein (CFP) and co-expressed with yellow fluorescent protein (YFP)-fused transmembrane Golgi proteins. Transiently transfected cells (4 - 10 h after transfection) were used in experiments since expression from inducible systems gave high background fluorescence of residual GFPs depending on different turnover of the investigated proteins. Cholera toxin induced interactions were visualised upon binding of cytosolic partners-CFP to the transmembrane proteins by an increase in sensitised emission of the acceptor (e.g. KDEL-receptor-YFP) and were confirmed by acceptor bleach.
Recently we have combined two cellular systems: retrograde transport (PM -Golgi-ER, using cholera toxin as a cargo) and anterograde transport - (trafficking of newly expressed connexins from the ER-via Golgi-to the PM) and analysed them simultaneously in the same cell. Four-colour resolution of GFP, YFP, Cy3, and Cy5 was achieved by a combination of multiphoton and single photon laser techniques and by selective filter sets. The data obtained reveal a limited capacity of the Golgi to perform both types of transport, i.e. retrograde and anterograde, simultaneously.