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Electron-Microscopic and Immunochemical Analysis of Kinetochore Microtubules After Ultraviolet Microbeam Irradiation of Kinetochores

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Electron-Microscopic and Immunochemical Analysis of Kinetochore Microtubules After Ultraviolet Microbeam Irradiation of Kinetochores Electron-microscopic and immunochemical analysis of kinetochore microtubules after ultraviolet microbeam irradiation of kinetochores JULIA A. M. SWEDAK, CYNTHIA LEGGIADRO and ARTHUR FORER Biology Department, York University, North York, Ontario, Canada M3J 1P3 Summary We used an ultraviolet microbeam to irradiate kinetochore microtubules are in smaller numbers in kinetochores of chromosomes in crane-fly spermato- the irradiated half-spindle than in the non-irradiated cytes. We used one of two doses, low (0.106 erg fim~2) half-spindle or in non-irradiated cells. Since ir- or high (0.301 erg /«n~2), and then studied the micro- radiation with low doses alters interchromosomal tubules in those spindles using electron microscopy 'signals', but microtubules remain attached to the or immunofluorescence microscopy. After irrad- kinetochore, we argue that low doses of ultraviolet iation with low doses microtubules are present as light damage a signal-related function of kineto- usual, with normal fluorescence and in normal chores without altering the ability of the kineto- numbers. After irradiation with high doses micro- chores to bind microtubules. tubules are no longer associated with the irradiated kinetochore. After irradiation with either dose, non- Key words: kinetochores, ultraviolet microbeam, spindles. Introduction Louis, MO). The halocarbon oil was replaced with insect Ringer's solution prior to irradiation (Czaban and Forer, 1985). Spermato- After kinetochores in anaphase cells were irradiated with cytes were placed on a 0.35 mm thick quartz coverslip (ESCO low doses of ultraviolet light (UV), using an UV Products Inc., Oak Ridge, NJ), and after irradiation cells were processed for immunofluorescence or electron microscopy as microbeam, there was no loss in birefringence of the described below. spindle fibre associated with the irradiated kinetochore, but all six half-bivalents temporarily stopped moving Microbeam irradiations (Swedak and Forer, 1991). After kinetochores were Ultraviolet microbeam irradiation was as described previously irradiated with high doses of UV the birefringence of the (Swedak and Forer, 1991). Briefly, all irradiations were with a chromosomal spindle fibre disappeared, and all six half- wavelength of 280 nm, with a half-band-width of 3.6 nm. The bivalents permanently stopped moving. We argued that a irradiating spot on the specimen was 1.2 /un in diameter. The 'signalling' function of kinetochores is damaged by the low total energy of UV incident on the specimen, controlled by doses of ultraviolet light, without loss of ability to bind varying the irradiation time (between 10 and 90s), was microtubules. In making this argument we assumed that 0.106erg^m~2±0.05erg;nn~2 (standard deviation) in low-dose 2 2 the presence of spindle fibre birefringence indicates the irradiations (rc=21) and 0.301 erg/<m~ ±0.034 erg/im~ in high- presence of kinetochore microtubules; it is important to dose irradiations (rc=24). our argument to test this assumption using other For microbeam experiments images of cells were relayed to a techniques. In this paper we report on the effects on monitor via a Panasonic camera (WV1550) and videotaped with a Panasonic video cassette recorder (model 6300). Photographs spindle microtubules of high- and low-dose irradiation of were taken from the video monitor using a Nikon F3 35 mm kinetochores as seen using immunofluorescence mi- camera and a 1 s exposure while the video cassette recorder was croscopy and electron microscopy. These data confirm our playing at normal speed. original supposition, that kinetochore microtubules are still present after irradiation of kinetochores with low Immunofluorescence doses. At various times after irradiation cells were lysed for 10 min (lysis medium: 100 mM Pipes, 10 HIM EGTA, 5 HIM MgSO4, 0.1% NP40, 5.0% dimethyl sulphoxide (DMSO), final pH6.9; a medium developed by B.B. Czaban), after which they were fixed in cold Materials and methods methanol (-20°C) or 0.5% glutaraldehyde in PBS for 6min. (Pipes, EGTA and MgSO4 were from Sigma Chemical Company Animal preparations (St. Louis, MO), DMSO from Anachemia (Mississauga, Ontario, Spermatocytes from fourth instar larvae were obtained from Canada) and NP40 from BDH Chemicals (Toronto, Ontario).) The Nephrotoma suturalis (Loew) or Nephrotoma abbreviata (Loew), lysis and fixation was done in the well formed by coverslip and using rearing methods and preparation of spermatocytes that slide. The coverslip was then removed from the slide and have been described by Forer (1982). Briefly, spermatocyte smears immersed in phosphate-buffered saline (PBS: 0.12 M NaCl, 6mM under halocarbon oil (series 27) were held in place by a fibrin clot phosphate buffer, pH 6.9) in a Petri dish for 2 min. If the cell was (Fibrinogen: Calbiochem, CA/ Thrombin: Sigma Chem. Co., St fixed with glutaraldehyde the coverslip was removed from the Journal of Cell Science 100, 269-277 (1991) Printed in Great Britain © The Company of Biologists Limited 1991 269 x slide and placed in sodium borohydride (lmgml ) for 20min Results before immersing the coverslip in PBS for 2min. After a quick rinse with Triton X-100 (Sigma Chemical Co.) (0.1 % Triton X-100 We have shown previously (Swedak and Forer, 1991) that in PBS) cells were then processed for indirect immunofluor- birefringent fibres remain after low-dose irradiation of escence with rat monoclonal antibody against tubulin (YL 1/2; Cedarlane, Hornby, Ontario) diluted 1:4000 in PBS; the antibody kinetochores, that spindle fibre birefringence disappears was added for 20min, and after two 5-min rinses in PBS, after high-dose irradiation of kinetochores, and that flourescein-conjugated anti-rat antibodies (dilution, 1:10; Ceder- interchromosomal 'signals' are affected in both cases. lane Labs Ltd., Hornby, Ontario) were added for 20min. The coverslip was then placed in a mixture of mowiol (Polysciences, Analysis using immunofluorescence Inc.) and 0.02 g per 100 ml p-phenylenediamine (PPD; Sigma Chemical Co., St Louis, MO) and the slides were stored at 4°C in We determined whether immunofluorescent staining (of the dark until studied by fluorescence microscopy. tubulin) was altered by irradiating kinetochores of Irradiated cells were relocated in the following way. Prior to autosomes in either metaphase or early anaphase. Cells making preparations, a grid was scratched on the coverslip with a were fixed either immediately following irradiation diamond scribe, and markings were placed on the grid. During the (n=10, low dose; n=ll, high dose) or 5min after lOmin lysis period the cell was located relative to the fixed irradiation (?i=9, low dose; re=ll, high dose). By 'immedi- markings and a map of the area was drawn. After staining, cells ately' we mean less than 2min after irradiation, as were located with respect to the map. determined by analysis of videotapes taken during the Stained cells were viewed with a Nikon inverted Diaphot-TMD course of fixation. The results for cells fixed immediately microscope equipped with epifluorescence (Nikon, 100 x phase- contrast fluor-DL objective lens, oil immersion, NA=1.3). were the same as for cells fixed 5 min after irradiation. Immunofluorescent images from a RCA SIT camera were recorded In cells in which the kinetochore had been irradiated on videotape using an Electrohome HO2 videocasette recorder. with a low dose, the spindle fibre associated with the Photographs were taken as described above. irradiated kinetochore stained with what appeared to be normal intensity (Fig. 1). Thus, these irradiations did not cause the loss of microtubules, though from these data we Electron microscopy cannot rule out that some kinetochore microtubules were Cells to be studied using electron microscopy (EM) were fixed at lost. various times after irradiation using 2 % agar-treated glutaralde- hyde (Nicklas et al. 1982) in the insect Ringer's solution described In cells in which the kinetochore had been irradiated by Czaban and Forer (1985). Coverslips were prepared for electron with a high dose the spindle fibre associated with the microscopy and serially sectioned as described by Hughes et al. irradiated kinetochore did not stain with tubulin anti- (1988). (The irradiated cells were located on coverslips as bodies (Fig. 2), though spindle fibres associated with non- described above.) Sections were observed with a Jeol CX100 irradiated kinetochores stained with normal intensity. electron microscope operated at 120 kV, and micrographs were Thus, irradiation of kinetochores with high doses causes taken on 8 cm x 10 cm film. Electron micrograph serial reconstruc- loss of all microtubules in the associated spindle fibre. tion, as described by Wilson and Forer (1988), involved tracing the chromosomes and spindle microtubules onto acetate sheets from micrographs of each section and stacking the acetates on top of Electron microscopic analysis each other to obtain a representation of the spindle. Irradiation with low doses. We studied two cells Fig. 1. Low-dose irradiation of a kinetochore in N. suturalis spermatocyte. (A) Cell viewed with polarizing optics prior to irradiation. Site of irradiation is indicated by an arrow. (B) During the irradiation. (C) Following the irradiation. x800. (D) Cell in C viewed with fluorescence optics to reveal anti-tubulin staining. The irradiated area appears normal. X750. Fig. 2. High-dose irradiation of a kinetochore in a N. suturalis spermatocyte. (A and B) Polarization microscopy. (A) Site of irradiation is indicated by an arrow. (B) Following the irradiation. X200. (C) Cell in B viewed with fluorescence optics to reveal anti-tubulin staining. There is little or no staining of the chromosomal spindle fibres for those 2 chromosomal kinetochores that were irradiated. (In this cell, 2 chromosome kinetochores were irradiated, because they were on top of each other during UV irradiation). X400. 270 J.A.M. Swedak et al. electron microscopically, both of which were irradiated with low doses and then fixed immediately. (We estimate from the videotape that fixation occurred less than 3 min after the end of the irradiation.) A representative section of cell 1 is illustrated in Fig.
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