Green fluorescent protein: Use of GFP-chimeras in the analysis of microtubule-associated protein 4 domains and microtubule dynamics

Abstract

Green Fluorescent Protein (GFP) is an endogenous 27 kDa fluorophore of the jellyfish, Aequorea victoria. Chalfie et al., first described the exogenous expression of this molecule in bacteria, and its utility as a reporter in higher eukaryotes. Potential applications of GFP have been expanded through the construction of variants with enhanced brightness and/or different spectral properties.We have explored using GFP for the analysis of the real-time behaviors of microtubules and their associated proteins. Constructs of microtubule-associated protein 4 (MAP 4) or β-tubulin were generated in pRC/CMV vectors and used in either transient or stable transfection assays in a variety of cultured cell lines (3T3, PtKl, BHK, CHO, Cos). The GFP-chimeras were visualized using conventional fluorescence microscopy and confocal laser scanning microscopy. Unusual features of the GFP reporter are that fluorescence intensity increased 2-10 fold upon illumination, and that phototoxicity was low.