UC Irvine

The use of focused high-intensity light sources for ablative perturbation has been an important technique for cell biological and developmental studies. In targeting subcellular structures many studies have to deal with the inability to target, with certainty, an organelle or large macromolecular complex. Here we demonstrate the ability to selectively target microtubule-based structures with a laser microbeam through the use of enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP) variants of green fluorescent protein fusions of tubule. Potorous tridactylus (PTK2) cell lines were generated that stably express EYFP and ECFP tagged to the alpha-subunit of tubulin. Using microtubule fluorescence as a guide, cells were irradiated with picosecond laser pulses at discrete microtubule sites in the cytoplasm and the mitotic spindle. Correlative thin-section transmission electron micrographs of cells fixed one second after irradiation demonstrated that the nature of the ultrastructural damage appeared to be different between the EYFP and the ECFP constructs suggesting different photon interaction mechanisms. We conclude that focal disruption of single cytoplasmic and spindle microtubules can be precisely controlled by combining laser microbeam irradiation with different fluorescent fusion constructs. The possible photon interaction mechanisms are discussed in detail.