Analysis of microtubule organization and dynamics in living cells using green fluorescent protein-microtubule-associated protein 4 chimeras.

Publisher Summary The inherent dynamic instability of microtubules is essential for a variety of cellular processes ranging from cell division and cell motility to establishment of cell shape and polarity. Even subtle perturbation of microtubule dynamics during mitosis can result in mitotic defects and cell death. Evidence has established a link between regulation of cellular kinases, microtubule dynamics, and cell cycle progression. Thus, detailed in vivo analysis of the cellular proteins and events involved in regulation of microtubule dynamics is necessary for a better understanding of the cell biology involved in processes such as cellular morphogenesis, mitosis, progression through the cell cycle, and apoptosis. Prior studies of microtubule dynamics in living cells have utilized either video-enhanced differential interference contrast (DIC) microscopy or microinjection of purified tubulin subunits that have been covalently modified with fluorescent molecules. This chapter describes an alternative method for in vivo analysis of microtubule dynamics following transfection of mammalian expression constructs encoding fusion proteins of the green fluorescent protein (GFP) and MAP4, a ubiquitously expressed cellular protein that localizes to the microtubule cytoskeleton. This chapter describes the methods and presents data illustrating the utility of three GFP-MAP4 fusion proteins for in vivo analysis of microtubule organization and dynamics. One chimera is generated using the full-length MAP4 protein to which GFP is fused at the amino terminus. Amino-terminal fusions of GFP and the P4-3 spectral mutant of GFP are also engineered using a fragment of MAP4 comprising the complete basic microtubule-binding domain of MAP4 (BD). These fusion proteins are referred to as GFP-BD and GFPP43-BD chimeras, respectively.