Continuation of mitosis after selective laser microbeam destruction of the centriolar region.

Abstract

The centriole regions of prophase PTK2 cells were irradiated with a laser microbeam. Cells continued through mitosis normally. Ultrastructural analysis revealed either an absence of centrioles or severely damaged centrioles at the irradiated poles. Microtubules appeared to focus into pericentriolar cloud material. KEY WORDS laser 9 microbeam centrioles mitosisIn a previous study (2), it was demonstrated that damage produced in the pericentriolar material of prophase cells of rat kangaroo (Potorous tridactylis) by argon ion laser microbeam irradiation was effective in blocking the anaphase movements of chromosomes. However, these cells did appear to contain microtubules emanating from kinetochores, and they did undergo cytokinesis. The centrioles in these cells appeared normal ultrastructurally. In another study (3), cells undergoing an unusual meiotic-like reduction division were examined, and centrioles were found to be lacking from otherwise normal spindle poles. In these cells, microtubules appeared to be associated with electron-dense pericentriolar cloud material even though no centrioles could be found by careful examination of serial thin sections.In the present study, we have examined the question of centriole function by producing selective laser microbeam damage in the centriole proper while apparently leaving the pericentriolar material unaffected. The cells were irradiated in prophase and subsequently went through normal anaphase movements and cytokinesis. MATERIALS AND METHODSCells of the PTK2 (P. tridactylis) rat kangaroo were grown in Rose culture chambers after the procedures described previously (2, 3). Immediately before microbeam irradiation, the cells were exposed to the vital dye acridine orange (AO) in a concentration of 0.25 /zg/ml culture medium for 5 min and then placed in normal medium for irradiation. Cells in early prophase were selected for irradiation. Only cells in which the centriolar duplexes were clearly visible in the perinuclear clear zone (4) were irradiated. After irradiation, the cells were followed by time-lapse videotape and fLxed for electron microscope analysis at varying time periods. All ultrastructural procedures were identical to those described in our earlier studies (2, 4).The laser microbeam employed the second harmonic blue wavelength, 473 nm, of a neodymium-YAG laser. These were the first experiments in which this particular wavelength has been used. Laser pulse duration was 100 ns, and peak output was 2-3 kW. Approx. 3% of this power was contained within the 0.5-/~m focal spot of the microbeam. The basic configuration of the laser microbeam was the same as described in earlier studies (1). RESULTSLaser microbeam irradiation of both centriolar duplex regions in early prophase did not affect the ability of the cell to undergo mitosis. Two spindle poles were established; chromosomes aligned on a metaphase plate and underwent subsequent anaphase movements. These results were observed in seven repeats of the experiment.Light and electron micrographs of a ty...

Laser Microsurgery in Cell and Developmental Biology

“…Recent ribonuclease digestion studies support this finding (24). A final series of laser microbeam studies on the centriolar region involved selective destruction of the centriole proper without damage to the pericentriolar cloud (25). In these studies, the blue second harmonic wavelength of the Y AG laser was used with acridine orange.…”

Aster-free spindle poles in insect spermatocytes: evidence for chromosome-induced spindle formation?

“…Electron microscopical investigations show that normal centrosomes are compound structures composed of a centriole pair, a cloud of darkly staining pericentriolar material (PCM),' and microtubules radiating from it ("aster" microtubules). It became evident that the capacity of the centrosome to nucleate microtubules is due to the PCM (l 5,30), and the PCM is regarded as the important component for spindle pole formation, while the centrioles do not seem to play a critical role during this process (3,5,6,17). The presence of PCM could recently even be verified at the spindle poles in plant cells, a well known example of acentric spindle formation (8,32).…”

Microtubule Dynamics may Embody a Stationary Bipolarity Forming Mechanism Related to the Prokaryotic Division Site Mechanism (Pole-to-Pole Oscillations)

“…Thus in experiments with laser destruction of one or both centrosomes and subsequent cold treatment destruction of polymerized tubulin, bipolar spindles devoid of centrosomes reformed. Anaphase and cytokinesis appeared normal [1,37]. The possibility exists that the ability to form a bipolar spindle without centrosomes is present throughout eukaryotes [32].…”