Controlled ablation of microtubules using a picosecond laser

Abstract

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.

FIGURE 1

The laser “scissors” system. The second harmonic (λ = 532 nm) laser line from a mode-locked Nd-YAG laser emitting 80-ps pulses at 76 MHz was used for laser ablation. The beam was sampled by a photodiode after partial reflection off a glass coverslip and attenuation by a neutral density filter (N.D.). A shutter provided 3-ms exposures, or macropulses, comprised of 228,000 individual pulses, or micropulses. A flip-up mirror mount and externalized epifluorescence optics allowed both the laser and arc lamp light into the back of the microscope. The filter cube slider holds cubes for EYFP and ECFP imaging, and for reflecting the laser light into the microscope objective. A high-sensitivity camera recorded fluorescence and phase contrast images before and after each irradiation.

FIGURE 2

Individual cytoplasmic microtubule ablation. Two individual ECFP-labeled microtubules in interphase cells irradiated at supra-threshold irradiances. Arrows indicate irradiation target. The newly formed microtubule fragments can be seen depolymerizing. (Top) 8 s between frames; (bottom) 4 s between frames. Scale bar = 10 μm.

FIGURE 3

Spindle cutting with EYFP probe. (A) Combined phase contrast and EYFP fluorescence of metaphase cell; (B) fluorescent image before irradiation; (C) postirradiation to microtubule bundle (arrow); 0.5 nJ/micropulse; total 6.74 × 10⁴ J/cm² per macropulse; (D) low magnification TEM of cell fixed <5 s postirradiation; (E) medium magnification TEM illustrating cut zone, pole, and chromosome (arrows); (F) high magnification TEM illustrating cleanly cut microtubules in the cut zone. (A–C) Scale bar = 10 μm. (D–F) Scale bar = 2, 0.5, 0.2 μm, respectively.

FIGURE 4

Supra-threshold spindle cutting with ECFP probe. (A) Combined phase contrast and ECFP fluorescence of metaphase cell; (B) fluorescent image before irradiation; (C) postirradiation to microtubule bundle (arrow); two consecutive macropulses; 0.92 nJ/micropulse, 1.24 × 10⁵ J/cm² per macropulse; (D) low magnification TEM of cell fixed <5 s postirradiation; note vacuole (arrow); (E) medium magnification TEM illustrating ends of cut microtubule bundle (arrows); (F) high magnification of ablation zone; note microtubules truncated at 0.2-μm diameter vacuole. (A–C) Scale bar = 10 μm. (D–F) Scale bar = 2, 0.5, 0.2 μm, respectively.

FIGURE 5

Centriole cutting with EYFP probe. (A) Combined phase contrast and fluorescence of metaphase cell expressing EYFP-tubulin at both poles; (B) fluorescent image ∼2 s postirradiation; 0.66 nJ/micropulse, total 8.90 × 10⁴ J/cm² per macropulse; (C) recovery of fluorescence 15 min postirradiation; (D) low-magnification TEM of cell fixed >15 min postirradiation ; (E) TEM of unexposed centrosome illustrating tubular structure of centrioles; (F) irradiated centrosome illustrating amorphous altered structure of centrioles; Both poles have normal appearing pericentriolar material and microtubule nucleation. (A–C) Scale bar = 10 μm. (D–F) Scale bar = 2, 0.5, 0.2 μm, respectively.