Journal of Photochemistry and Photobiology В: Biology 29 (1995) 163-170
Role of the centrosome in mitosis: UV micro-irradiation study
R.E. Uzbekov, M.S. Votchal, I.A. Vorobjev *
Division of Electron Microscopy, A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119899 Moscow, Russia
Abstract
Ultraviolet micro-irradiation (UV-MI) of the PK (pig kidney embryo) cell centrosome (Amax = 280 nm, spot diameter 1.6 mm, exposure time 515 s) at metaphase and anaphase resulted in functional damage of the centrosome. After UV-MI of the centrosome at early metaphase,
chromosomes quickly (in 1-3 min) moved away from the irradiated pole and then encircled the non-irradiated pole. Within 10 min after UV-MI
the spindle disassembled and chromosomes remained unseparated. The minimal dose inducing this effect in 90% of cells was accumulated in 5 s.
After the same UV-MI at late metaphase, chromosomes shifted towards the non-irradiated pole; however, anaphase started and
chromosome motion towards the non-irradiated pole continued normally.
UV-MI of the centrosome at early anaphase for 5-15 s slowed down and then stopped chromosome motion towards the irradiated pole. This
was a result of rapid (within 2-3 min) disorganization of the half-spindle. Chromosomes continued to move towards the opposite pole normally,
while cytokinesis was significantly retarded.
No visible lesion was revealed by electron microscopy after 5 s UV-MI, while 15s irradiation resulted in the truncation of the microtubule
bundles 1.5-2 µm from the centrosome.
We concluded that UV-MI inactivates the centrosome and induces disaggregation of microtubule initiation sites. The critical point
(checkpoint) in mitosis up to which this damage induces mitotic arrest is mid-metaphase.
Keywords: Centrosome; Mitosis; Micro-irradiation; Microtubules
1
. Introduction
In animal cells the mitotic spindle is organized by the
centrosome containing a pair of centrioles. The centrioles
duplicate at S phase and at the onset of mitosis two centrosomes serving as mitotic poles initiate microtubule assembly
and form the spindle where chromosomes are captured,
aligned and then separated via kinetochore-microtubule
interaction [ 1 ]. However, despite numerous studies, the role
of the centrosome in the control of mitosis remains far from
being understood. In some cells mitosis occurs without a
prominent centrosome. Centrosome core structures — centrioles — are absent in higher plants, diatoms [2] and even
in one Drosophila cell line [ 3 ]. To gain insight into the role
of the centrosome in mitosis, direct experiments are essential.
Such experiments have been performed using the micro-irradiation technique. Two main set-ups, namely UV microbeam
and laser systems, were used.
Experiments on UV micro-irradiation of the centrosome
(spindle pole) performed in the 1960s gave some controversial results. Irradiation of the mitotic pole in telophase sper* Corresponding author.
matocytes of Bombyx mori resulted in a rapid shift of
chromosomes from the irradiated pole f 4]. However, Zirkle
found the mitotic blockage induced by UV irradiation in newt
pneumocytes to be independent of the presence or absence
of the centrosome in the irradiated area [5].
In a series of studies, Berns and coworkers used an argon
laser microbeam for centrosome irradiation [6-9]. They
found irradiation of the mitotic pole in prophase after acridine
orange treatment to inhibit anaphase onset [7], but in some
cases mitosis was completed despite the damage to the centrosome and even in the absence of centrioles in the mitotic
poles [6]. More recently, re-evaluating these studies, Vorobjev et al. showed that the cell behaviour after centrosome
micro-irradiation with an argon laser microbeam (with and
without acridine orange pretreatment) depended strongly on
the stage of mitosis at which irradiation had been performed
[ 10]. Irradiation of the centrosome at prophase and early
prometaphase always blocked mitosis at metaphase; when
performed at metaphase, the irradiation had no effect on the
course of mitotis. In all cases no lesion was observed by
electron microscopy [ 10].
To gain a better insight into the role of the centrosome in
mitosis, we have undertaken a study of mitosis in PK (pig
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R.E. Uzbekov et al. /Journal of Photochemistry and Photobiology B: Biology 29 (1995) 163-170
kidney embryo) cells using a UV microbeam. A UV microbeam was chosen for several reasons. First, a UVC microbeam reversibly severs microtubules in a living spindle [11],
so we were able to compare the effect of centrosome damage
with spindle destruction. Second, UV micro-irradiation gives
rapid visible alterations of the living spindle, being much
more convenient than laser microbeam irradiation. Third, a
UV micro-irradiation device based on the high pressure mercury lamp is compact and more easily adjustable as compared
with an argon laser micro-irradiation system.
In this paper we report the effect of centrosome damage
induced by UV micro-irradiation at various stages of cell
division. Our initial premise was that the centrosome is an
essential part of the spindle at the beginning of cell division
but becomes a passive ''passenger'' at the later stages. Therefore we made efforts to get direct evidence for the centrosome-dependent critical point(s) in mitosis. Some evidence
for this supposition comes from a previous laser microbeam
study [10]. To tackle the problem, we performed UV irradiation of the centrosome at various stages of mitosis and
followed the fate of the cells. Observations of living cells
were supplemented with immunofluorescent staining of
microtubules and with ultrastructural study. Some similarities
of our results to those of previous laser microbeam studies
[6,9,10,12] as well as some striking differences in cell behaviour immediately after irradiation were revealed.
2. Materials and methods
2.1. Cell culture
PK cells were used. They were grown on 199 tissue culture
medium (Biomedical Drugs and Chemicals Factory, Moscow) supplemented with 10% bovine serum (Medical Drugs
and Chemicals Factory, Moscow) and antibiotics (penicillin
and streptomycin, 100 units ml–1 each). The cells were subcultured on to round quartz coverslips (d = 25 µm, 0.17 µm
thickness) 48-72 h before use. For the experiments the coverslips were mounted in modified Dvorak-Stottler chambers,
where the cells continued dividing for 6-8 h without change
of the medium.
2.2. Microscope and micro-irradiation instruments
A modified transmission light microscope was used (Fig.
1). It was supplemented with a UV epi-illumination device.
The quartz objective used for irradiation was a Zeiss Ultrafluar 100X/1.25 (glycerol immersion). The incident light
was collected from an HBO-100 lamp through a high numerical aperture (NA) quartz collector lens, passed through a
UV wide bandpass filter, then through a UV bandpass interference filter (Amax = 280 nm, 50% transmission, bandwidth
+10 nm) and focused by a quartz condenser lens in the rear
focal plane of the objective. A field diaphragm was placed in
the front focal plane of the condenser lens to cut a small area
Fig. 1. Micro-irradiation instrumentation: 1, photographic or video camera 2,
UV dichroic mirror; 3, Ultrafluar objective 100 X /1.25; 4, cells in chamber;
5, pinhole diaphragm; 6, set of UV filters; 7, quartz collector lenses; HBO100 mercury lamp; 9, halogen lamp.
in the objective focal plane. To measure the diameter of the
beam, a uranium slide was put on the microscope stage and
the diameter of the fluorescent spot was measured either using a
video camera or from a photograph. The diameter of tl
beam in the front focal plane of the objective lens was
1.6 µm.
The average power of the UV light directed on to the
specimen in all experiments was approximately 0.04 erg
µm-2 s-1 (as measured under the objective). The do
depended on the exposure time (see Section 3). Cells we
observed using eyepieces with a graticule to focus the micro
beam. After irradiation, cells were either observed with
Plan-neofluar 40 X/0.9 phase-contrast objective lens
(glycerol immersion) using a charge-coupled device (CCD)
video camera (400 TV lines) or photographed using a
mm single-lens reflex (SLR) camera.
2.3. Light microscopy: immunofluorescence
Cells were briefly rinsed in warm phosphate-buffered
saline (PBS), then lysed on the coverslips with microtubule
stabilizing buffer containing 50 mM imidazole (pH 6.8), 5
mM MgCl2,1 mM EGTA, 0.1 mM EDTA, 35% glycerol and
0.5% Nonidet P-40 (Sigma) and fixed with 0.5% glutaral
dehyde in PBS for 30 min-2 h. The coverslips were rinsed in
PBS and placed in 2% sodium borohydride for 20 min, then
washed twice in PBS and incubated with mouse monoclonal
anti-a-tubulin antibodies (clone DM-1A, Sigma) for 30 min
37 °C. The antibodies were rinsed off with three changes of
PBS and the coverslips were incubated in secondary FITClabeled rabbit-antimouse IgG (Sigma) for 30 min at 37o C.
The stained specimens were mounted in Elvanol (Sigma) or
Moviol (Calbiochem). The cells were examined in a Zeiss
Photomicroscope 3 equipped for phase-contrast and epiflu
R.E. Uzbekov et al. /Journal of Photochemistry and Photoblology B: Biology 29 (1995) 163-170
Table 1
Results of micro-irradiation of centrosome at early metaphase (exposure time 5s)
Site of
Number
Normal
Delayed
Scattering of
Abnormal
Anaphase
irradiation
of cells
irradiated
75
17
7
division
division
chromosomes
cytokinesis
blockage
0
10
0
15
7
6
74
0
1
15
0
0
60
0
0
Centrosome
Cytoplasm
Chromosome
Note. Several alterations of mitosis were sometimes observed in one and the same cell after irradiation. Thus the sum of the last five numbers in each line can
exceed the total number of irradiated cells.
orescence optics using a 63 X /1.4 Phanapochromate phasecontrast objective. Photomicrographs were taken on RF-3
film (1500 ASA).
2.4. Electron microscopy
Cells were fixed with 2.5% glutaraldehyde (Merck) on
0.1 M K-Na phosphate buffer (pH7.2) for 1-2 h, then rinsed
in PBS, postfixed with osmium tetroxide, stained with uranyl
acetate, dehydrated and embedded in Epon 812 mixture.
Serial ultrathin (70 nm) sections were obtained parallel to
the substrate plane using an LKB-III ultramicrotome (LKB,
Sweden) and mounted on single-slot grids. Sections were
examined and photographed in HU-11В and HU-12 electron
microscopes (Hitachi, Japan) operating at 75 kV.
3. Results
Initial experiments were conducted to determine the minimal dose of irradiation of the pole sufficient for a prominent
change in mitosis. We found that upon irradiation of the
centrosome at metaphase the minimum time it took for mitotic
blockade accompanied by gradual disassembly of the spindle
to be established in more than 90% of the cells was 5 s (Table
1). After 15 s irradiation the effect was identical. However, 3
s irradiation of the pole blocked anaphase onset in roughly
50% of cells treated. It was concluded that 5 s irradiation
(dosage 0.2 X 10~7 J) sufficed to damage the centrosome at
metaphase.
Irradiation of the pole for 5 s (low dose) or even 15 s
(high dose) at early anaphase did not stop mitotic progression
but caused some changes in chromosome movement (see
below). Irradiation for 30 s or more inflicted gross damage
on the cell. In the subsequent experiments the anaphase cells
were irradiated for 15 s as a maximum dose giving specific
effects.
3.1. Control irradiations
Different regions of mitotic cells were irradiated at metaphase and anaphase to determine whether the effects of microirradiation of the pole were specific. Cells were irradiated in
the cytoplasm outside the spindle and in the spindle area
between chromosomes and one pole. Chromosomes were
also irradiated.
Irradiation of the cytoplasm at metaphase and anaphase for
as long as 30 s had little effect on mitotic progression. The
spindle appeared normal and the chromosome plate was not
displaced. In some instances anaphase onset and cytokinesis
were retarded for 10-15 min (Table 1), but when started they
ran normally. The two daughter cells were of the same shape
and had equivalent nuclei and a normal microtubule system
(data not shown).
Chromosomes are far more sensitive to UVC light than are
cytoplasmic organelles [13]. Thus the most effective control
of non-specific damage was irradiation of chromosomes in
the metaphase plate. Indeed, irradiation of chromosomes for
15 s resulted in visible lesions; however, this only postponed
anaphase onset but did not inhibit mitotic progression and
cytokinesis (Table 1).
Irradiation of the spindle between chromosomes and the
pole was described in detail elsewhere [11,14-16]. Our
results are consistent with the previous data. Irradiation of
this area locally destroyed the spindle for some minutes (data
not shown) and anaphase progression slowed down. After
recovery the cell behaviour returned to normal mode.
The filter set we used transmitted about 7% of UV light at
wavelengths above 300 nm. To make sure that such UV
irradiation of the centrosome was without effect, we repeated
the experiment with cells grown on regular (glass) coverslips. The glass coverslips used transmitted more than 90%
of light with A>310 nm (UVB and UVC) but were not
transparent to light of wavelength below 295 nm. In these
cells, mitosis was not affected by 1 min irradiation of the
spindle pole.
3.2. Cell behaviour after irradiation of the centrosome at
metaphase
During the first 10 min after irradiation the metaphase plate
drifted towards the non-irradiated pole. Chromosomes near
the spindle axis moved slower than those on the edges and
after several minutes all chromosomes surrounded the nonirradiated pole in a semicircle or almost a complete circle.
The average velocity (20 cells measured) of central chromosomes was 0.095 + 0.026 µm min-1, while on the sides it
was 0.73 + 0.14 µm min-1 at the beginning and declined
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R.E. Uzbekov et al. /Journal of Photochemistry and Photobiology B: Biology 29 (1995) 163-170
enter anaphase. Anaphase was observed in 15 out of 25 cells
irradiated 35 min after prometaphase onset, while in the other
10 it was blocked. All 25 cells with the pole irradiated 40 min
after the beginning of prometaphase (i.e. 5 min prior to normal anaphase onset) and all 50 cells irradiated in early anaphase divided into two daughter cells.
3.3. Behaviour of the spindle after centrosome irradiation
at metaphase
Within 1 min after irradiation of the pole the half-spindle
at the opposite pole shortened; 2-3 min after irradiation the
half-spindle at the irradiated pole disorganized (Fig. 3); 5-8
min after irradiation the whole spindle was disrupted and
several microtubule convergence centres appeared. The metaphase plate dissolved within 10-20 min after irradiation of
the pole. At that time no spindle was visible. The number of
microtubule convergence centres increased with time and
ranged up to 10 or more. They were located on or very close
to chromosomes. One hour after irradiation the overall picture
did not change (Fig. 4). Sometimes the spindle partially
rehabilitated — two microtubule convergence centres dominated over the others, but the metaphase plate was not reestablished.
3.4. Cell behaviour after irradiation of the centrosome at
anaphase
Micro-irradiation of the spindle pole was performed
within 50-60 s after chromosome splitting. During this
time chromosomes had separated by a distance of 1.96 +
Fig. 2. Chromosome shift after irradiation at metaphase: (a), before irradiation; (b), 10 min after irradiation; (c), 40 min after irradiation. Arrow
indicates irradiated pole (bar, 5 µm).
toO.27 + 0.21 µm min-1 (10 min after irradiation). On average the total shift of the metaphase plate 10 min after irradiation of the centrosome was 0.95 + 0.26 µm in the centre
and 5.38 + 0.21 µm on the sides. Later on, different
chromosomes came out of the plate and were randomly
distributed in the central part of the cell (Fig. 2).
As mentioned above, initial experiments showed that the
cell behaviour after micro-irradiation of the pole depended
strongly on the moment irradiation had been performed. We
constructed a time scale taking as zero point the visible dissolution of the nuclear envelope at the end of prophase. In
PK cells the period between the beginning of prometaphase
and anaphase onset lasted for 45 + 3 min. Prometaphase continued for approximately 10 min and metaphase took the rest
of the time (about 35 min). Irradiation was performed at
various times after the beginning of prometaphase. Fifty cells
were irradiated in the first 10 min of metaphase (with the low
dose). All of them were blocked in metaphase and did not
Fig. 3. Immunofluorescent staining of cell 3 min after irradiation of pole at
metaphase. Arrow indicates irradiated pole (bar, 5 µm).
0.09 µm.
R.E. Uzbekov et al /Journal of Photochemistry and Photobiology B: Biology 29 (1995) 163-170
Fig. 4. Immunofluorescent staining of cell 73 min after irradiation of pole at
metaphase (bar, 5 µm).
Immediately after irradiation the chromosome movement
towards the irradiated pole slowed down while that towards
the opposite pole continued normally (Fig. 5). At telophase
the distance between two groups of chromosomes was 30%
less than in control cells (Fig. 5C).
Immediately after irradiation of the centrosome no difference between the two half-spindles was observed; however,
within 3 min after irradiation a distinction became evident.
On the site of the irradiated pole the spindle disassembled
and only short bundles of kinetochore microtubules remained
in a non-convergent array (Fig. 6). Within 8-15 min after
irradiation numerous centres of microtubule convergence
appeared on this site, while the opposite half-spindle was still
arranged in normal fashion.
In cells with the irradiated pole, cytokinesis was postponed
for 10-15 min. Sometimes lobes of the cytoplasm were separated near the cleavage furrow by additional constrictions
(Table 2).
3.5. Fine structure of the centrosome after irradiation with
low (5 s) and high (15 s) doses
Irradiation with the low dose leaves the centrosome
unchanged within the first 5 min. The two centrioles remained
perpendicular to each other. Numerous microtubules emanated from the mitotic halo. The irradiated pole looked
exactly as the opposite one did (data not shown).
Irradiation with the high dose resulted in severing of spindle microtubules around the centrosome. With 1-3 min after
irradiation, bundles of kinetochore microtubules terminated
at a distance of 1.5-2 µm from the centrosome (Fig. 7, arrow-
Fig. 5. Chromosome behaviour after irradiation of pole at anaphase (phase
contrast): (a), 2 min before irradiation; (b), 1 min after irradiation; (c), 17
min after irradiation; (d), 45 min after irradiation. Arrow indicates irradiated
pole (bar, 5 µm).
heads). The distance between chromosomes and the irradiated pole exceeded that between chromosomes and the
opposite pole (Fig. 7). Only a few short, randomly oriented
microtubules remained around the centrioles (Fig. 7, inset).
Sometimes centriole lesions were also observed — occasionally triplets of microtubules appeared destroyed (data not
shown).
4. Discussion
In the micro-irradiation studies the mechanism of damage
and the diameter of lesions depend on the wavelength of the
light used, the intensity of the incident light and the diameter
of the focused beam.
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R.E. Uzbekov et al. /Journal of Photochemistry and Photobiology B: Biology 29 (1995) 163-170
When using visible light (400-700 nm), one needs to
employ extremely high power densities or to sensitize cells
with dyes [9]. Using an argon laser, Berns and coworkers
obtained certain effects on mitosis with a power density of
1012 W m -2 (107 erg µm-2 s-1) in the focal plane of the
objective lens. With acridine orange or psoralens they managed to slightly reduce the power used, but it still was very
high [7,8].
UV light at wavelengths of 260-280 nm is strongly
absorbed by cells and a power density of only 102-104 Wm-2
(10-3-10-1 erg µ-2 s-1) is sufficient to produce specific
lesions [11,13]]. The molecular targets for UVC irradiation
are proteins and nucleic acids. Photochemical damage of
proteins occurs via a variety of mechanisms as a result of high
absorption by tryptophan and tyrosine at 280 nm [ 17]. This
gives strong evidence for a local and direct effect of UVC
micro-irradiation. Another line of evidence for determination of
lesion area comes from disruption of micro-tubules. They are
truncated by a UV microbeam, with a mercury lamp as light
source, exactly in the irradiated region [11,18,19]. It is
worth noting that a pulsed UV laser (266 nm) produces in
interphase cells a lesion area much larger than the beam spot
[20] and has little effect on mitotic progression even at higher
power [2].
In 1965 Forer described the formation of an area of reduced
birefringence (ARB) by UV micro-irradiation of the spindle [
14]. Subsequently it was shown that formation of the ARB
corresponds to local depolymerization of microtubules
[19,21,22]. Recently Spurck et al., using a high pressure
mercury lamp as UV light source, made it clear that the area of
microtubule depolymerization is strictly identical with the
irradiated spot [11]. Quantitative data provided by various
authors show that microtubules are destroyed by irradiation
doses of 0.2—0.6 erg µm-2, which are achieved by 3 s [ 11 ] to
90 s [15] exposure. Microtubules in the irradiated area
rehabilitate within a few minutes and the ARB disappears [
11,18,23]. The action spectrum of ARB formation in living
cells and that of microtubule truncation in vitro are nearly
identical [19].
In our micro-irradiation experiments the beam was applied to
the centrosome area, thus affecting the centrosome as well as
microtubules (MTs) emanating from it. The influence of
centrosome irradiation on mitosis observed in our experiments is much stronger than that described for irradiation of
Fig. 6. Immunofluorescent staining of cell 3 min after irradiation of pi
anaphase. Arrow indicates irradiated pole (bar, 5 µm).
spindle MTs [16,18,19,23-25] and is irreversible when per
formed at metaphase. It is worth noting that the dose
of irradiation required for centrosome damage was three
times less than that necessary for truncating
microtubules. This means that specific targets located in
the centrosome essential for its function in mitosis are more
sensitive to UV damage than are MTs.
4.1. Centrosome-dependent mitotic checkpoint is in midmetaphase
The marked difference in cell behaviour following
irradiation of the pole in early (first 10 min) and late (last
10min) metaphase clearly demonstrates that during midmetaphase a cell overcomes a certain checkpoint. At
present it may be assumed that a centrosome-dependent
mechanism essential for anaphase onset and subsequent
cytokinesis occurs in PK cells in mid-metaphase.
Micro-irradiation of the mitotic pole with an argon la
revealed a centrosome-dependent checkpoint in PtK cells
Table 2
Results of micro-irradiation at anaphase (exposure time 15 s)
Site of
Number
Normal
Altered
Delay of
Incomplete
irradiation
of cells
irradiated
63
10
20
division
chromosome
separation
60
0
12
normal
cytokinesis
58
1
18
cytokinesis
Centrosome
Cytoplasm
Chromosome
3
9
2
2
0
0
Note, beveral alterations of mitosis were sometimes observed in one and the same cell after irradiation. Thus the sum of the last four numbers in each 1 can
exceed the total number of irradiated cells.
R.E. Uzbekov etal. /Journal of Photochemistry and Photobiology B: Biology 29 (1995) 163-170
Fig. 7. Electron microscopy of anaphase cell fixed 5 min after irradiation of centrosome. Inset: higher magnification of irradiated centrosome (subsequent
section). IP, irradiated pole; NP, opposite (non-irradiated) pole. Arrows indicate bundles of microtubules (bar, 0.5 µm).
Prometaphase or prometaphase-metaphase transition
[9,10]. In PtK cells, prominent metaphase last for about 10
min, i.e. it is very short compared with metaphase in PK cells.
Therefore, taken together, experiments on centrosome
(mitotic pole) irradiation suggest that the centrosome is
essential to mitosis until the last 10 min before chromosome
separation. Following Picket-Heaps' [26] terminology, it
may be inferred that 10 min prior to anaphase onset the
centrosome becomes a "passenger" and plays no further
part in mitotic progression.
4.2. UV micro-irradiation at anaphase results in
asymmetric destruction of the spindle
As discussed above, irradiation of one pole at metaphase
leads to a rapid shift of all chromosomes towards the opposite
pole and a coordinated disassembly of the entire spindle. If
the structure of the spindle is assumed to be the same at
anaphase, then after irradiation of the pole at anaphase all
chromosomes will be expected to shift and chromosomes
moving towards the non-irradiated pole will speed up.
Instead, no increase in the average speed of chromatids moving
to the opposite pole was observed. Chromosomes continued
moving to the irradiated pole but at reduced speed. This
suggests that the two half-spindles at anaphase become independent of each other. Our data are contradictory to earlier
observations [5,15] but consistent with the modern view on
mechanisms guiding chromosome separation [27].
Comparing the data presented here with previous microirradiation experiments, two points should be emphasized.
(i) After laser irradiation of mitotic poles at anaphase no
alterations in chromosome motions were described even
when some pole structures were destroyed [7,12]. We are
apt to explain this fact by only partial destruction of the
essential centrosome structures by an extremely sharp microbeam about 0.25 yum in diameter [28].
(ii) In early experiments on UV irradiation of the pole at
early telophase all chromosomes shifted towards the opposite
pole and cytokinesis was abnormal [4]. Taking into account
that the cells used were rather thick, the absorption of UV
light by structures lying above the centrosome was high [ 13 ].
This result might thus be explained as non-specific damage
of the whole cell.
Therefore we suppose that our instrumentation causes specific damage to the centrosome at mitosis. Our approach for
studying cells lacking normal centrosomes offers a number
of advances over other methods proposed by Karsenti et al.
[29] and Maniotis and Schliwa [30]. For the method of
Karsenti etal. [29] only cytoplasts without centrosomes were
available. The cytoplasts had a short lifespan and could not
be used for long-term experiments. Maniotis and Schliwa
[30] could obtain only a restricted number of cultured cells,
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R.E. Uzbekov et al. /Journal of Photochemistry and Photobiology B: Biology 29 (1995) 163-170
since many of them did not survive after microsurgical dissection into two parts [ 30]. Our instrumentation may be used
with any cultured cells that remain flat during mitosis, e.g.
PtK, newt lung epithelial cells, etc. Furthermore, in our
method the sister cell with the normal centrosome serves as
a natural control to the treated one.
Acknowledgments
This work was supported by Russian Fundamental
Research Foundation Grant 93-04-6523 to I.A.V. and in part
by International Science Foundation Grant MRJOOO to I.A.V.
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