1 University of Cambridge, Department of Genetics, Downing Street, Cambridge CB2
3EH, UK
2 European Molecular Biology Laboratory, Cell Biology and Biophysics Programme,
Meyerhofstrasse 1, 69117 Heidelberg, Germany
* Present address: NYU School of Medicine, Skirball Institute of Biomolecular
Medicine, Developmental Genetics Program, 450 First Avenue, New York, NY
10016, USA
Author for correspondence (e-mail:
dmg25{at}mole.bio.cam.ac.uk)
Accepted 28 November 2002
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Summary |
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Key words: Cell division, Mitotic spindle apparatus, Centrosome, Microtubules, Tubulin
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Introduction |
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Drosophila has two genes for -tubulin one is
located at salivary gland chromosome region 37C and is restricted in its
expression to ovaries and precellularized embryos; the other is located at 23C
and is expressed in somatic tissues and testes
(Sunkel et al., 1995
;
Tavosanis et al., 1997
;
Wilson and Borisy, 1998
). The
brains of
-tub23C Drosophila have highly condensed
chromosomes, and spindles with defective poles
(Sunkel et al., 1995
). The
gene encoding Dgrip91 is known as discs-degenerate 4 (dd4)
and has a similar phenotype, with highly condensed chromosomes, but shows a
higher mitotic index as if arrested at the spindle integrity checkpoint
(Barbosa et al., 2000
). The
poles of mitotically arrested dd4 spindles contain some centrosomal
antigens, whereas others may be dispersed rather than present in a single
centrosomal body. Moreover, centrioles were found to be missing from one of
the two spindle poles in a high proportion of mutant dd4 cells. In
many respects, the dd4 spindles resemble those from polo
neuroblasts, reflecting the requirement for polo to recruit
-tubulin to the centrosomes and to phosphorylate and activate Asp
(Avides et al., 2001
;
Donaldson et al., 2001
). The
disorganized dd4 centrosomes are associated with fewer spindle
microtubules, but nevertheless, stable bipolar spindles are formed and
maintained in the mutant cells (Barbosa et
al., 2000
).
It has been particularly informative to study the phenotypes of
Drosophila cell division cycle mutants to examine not only somatic
cells, but also spermatocytes undergoing male meiosis. This is because the
spindle assembly checkpoint is not effective at blocking spermatocyte division
(Lin and Church, 1982;
Miyazaki and Orr-Weaver, 1994
;
Rebollo and Gonzalez, 2000
;
Savoian et al., 2000
) but only
briefly delays meiotic progression and allows spermatocytes to complete
division. Thus, examination of the mutant phenotypes of spermatocytes
revealed, for example, that Polo kinase and Asp, known to function early in M
phase to organize the spindle poles, also have additional functions in
cytokinesis (Carmena et al.,
1998
; Riparbelli et al.,
2002
; Wakefield et al.,
2001
). A recent study of the meiotic phenotype exhibited in
-tub23CPI males also suggested novel aspects of
function of the
TuRC, as centrosomes were still capable of nucleating
microtubules but conical structures formed in place of bipolar spindles
(Sampaio et al., 2001
). Such
cones had centrosomes at their base and Klp3A and Polo kinase at their pointed
ends, which, in some cases, could be the site of highly asymmetric
cytokinesis.
In this paper we cast further light on the functions of the TuRC
through observations of meiosis in males in which
-tubulin has been
lost from the centrosome as a result of hypomorphic mutations in the gene for
Dgrip91. We show that the nucleation of astral microtubules still occurs in
such dd4 males, and conical spindle structures are formed that
strongly resemble those described in
-tub23C testes
(Sampaio et al., 2001
). These
dd4 alleles have allowed us to identify a novel role for the
TuRC in maintaining the separation of spindle poles around the time of
metaphase. Bipolar spindles that form in these cells collapse at this time and
their poles re-associate. In some cells, spindle poles either never separate
or collapse very early, and these nucleate robust asters of microtubules. If
the chromosomes associated with such hemi-spindles organize bipolar arrays of
microtubules, then contractile cytokinetic rings are able to form. We discuss
the role of the
TuRC in the nucleation of asters, maintaining the
separation of centrosomes and in the formation of the central spindle.
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Materials and Methods |
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Preparation of live testes for phase-contrast microscopy
Testes were dissected from young dark pupae in Testis Buffer (183 mM KCl,
47 mM NaCl, 10 mM Tris-HCl, pH 6.8, 1 mM EDTA) containing 1 µM PDMF
protease inhibitor (Sigma), following the method described by Gonzalez and
Glover (Gonzalez and Glover,
1993). The testes were transferred to a drop of the same buffer
placed on a clean slide and cut with tungsten needles near the apical tip to
release the cysts through the testes wall. The testes were gently squashed by
placing a siliconized coverslip over them on the slide, and then applying a
small piece of blotting paper to one of the edges of the coverslip. The
process of squashing was monitored using a 40x phase-contrast objective.
When the appropriate degree of squashing was attained, the blotting paper was
removed. The criteria used to assess the degree of squashing were the
integrity of the cysts outside the testes and the contrast of the phase-dense
material within each cell. Specimens were screened for intact cysts of primary
spermatocytes in phase-contrast with a Nikon Microphot-FX microscope at low
magnification (25x). The morphology and number of cells in those cysts
were analysed in photographs taken by a Nikon Coolpix 990 digital camera at
higher magnification (60x). Onion-stage cysts were also photographed
with the 60x objective but only isolated spermatids were counted on
photographs at the same magnification. The nuclear diameter of the early
spermatid cysts with one nucleus and one Nebenkern was measured
(Gonzalez et al., 1988
) on the
digital photographs using OpenLab software, the ruler of which was calibrated
with a scale that had minimum divisions of 1 µm. Ten dd43,
dd4S, and Oregon R (OrR) male pupae were dissected for this
experiment.
Spermatocyte culture and time-lapse microscopy
Spermatocytes were cultured as described by Church and Lin
(Church and Lin, 1985) and the
phase-contrast observations were made as in Rebollo and Gonzalez
(Rebollo and Gonzalez, 2000
),
with a Leica DM IRB/E microscope equipped with a 63x/1.32 objective.
Time-lapse images were captured with a Cohu camera at a rate of 20
frames/minute. Cultured dd4S spermatocytes from a
dd4S/FM6 | Y were obtained from dark pupae stages
and OrR spermatocytes from males of the same age were used as controls.
Indirect immunolocalization of centrosomal and central spindle
proteins in testes
Testes were dissected from young OrR and dd4S dark
pupae and squashed as for phase-contrast observations, except that five pairs
of testes were placed in one slide and up to three slides of each genotype
were prepared per experiment. After squashing, the slide was immersed in
liquid nitrogen and the coverslip flicked off with a scalpel blade. The
preparation was fixed with methanol and acetone as described by Gonzalez and
Glover (Gonzalez and Glover,
1993) and then `blocked' by pre-incubation in 10% fetal calf serum
in 1x phosphate buffered saline (PBS). Primary antibody incubations were
done overnight at 4°C in 1x PBS containing 10% fetal calf serum.
Samples were washed in PBT (1x PBS, 0.1% Tween 20) before secondary
antibodies diluted in PBT, 10% fetal calf serum were added. The secondary
incubation was for at least 1 hour at room temperature. The preparation was
then washed in PBT, rinsed in PBS and incubated for 15 minutes in PBS
containing the DNA dye TOTO-3 (Molecular Probes) before being mounted. We used
the monoclonal anti-body YL1/2 (Kilmartin
et al., 1982
) 10% diluted and anti-rat FITC-conjugated
immunoglobulin G (Jackson Immunochemicals) to detect microtubules.
-Tubulin was localized by using the monoclonal antibody from clone
GTU88 (Sigma) diluted 1:50. Anti-Asp was a polyclonal rabbit serum Rb3133
(Saunders et al., 1997
) and
anti-Pav-KLP (Pavarotti) was Rb3301 (Adams
et al., 1998
) diluted 1:25. Centrosomin (CNN) was revealed with a
rabbit CNN-specific R19 antibody (Heuer, 1995) at a dilution of 1:100, kindly
provided by Thomas Kaufman (Indiana University, Bloomington, IN). Peanut was
detected with MAb4C9 (Neufeld and Rubin,
1994
) diluted 1:5. The secondary antibodies used to detect all
antigens, with the exception of tubulin and F-actin, were conjugate with
TexasRed, obtained from Jackson Immunochemicals, and were used according to
the supplier's instructions. To visualize the distribution of F-Actin, fixed
testes were incubated in a 1:200 dilution of rhodamine-conjugated phalloidin
(Molecular Probes) in 1x PBS for 20 minutes. After washing with PBS,
testes were mounted as usual. Preparations were visualized using a Bio-Rad
1024 confocal scanning head coupled to a Nikon Optiphot microscope. The
brightness and contrast of images collected for the figures were adjusted with
Adobe Photoshop 5.5 software.
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Results |
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Analysis of squashed preparations of testes by phase-contrast microscopy
revealed two main types of cytological abnormality. The first was in the
appearance of cysts of primary spermatocytes. In wild type, a primary
spermatogonial cell undergoes four mitotic divisions with incomplete
cytokinesis to produce a cyst of 16 primary spermatocytes connected at ring
canals through cytoplasmic bridges
(Fuller, 1993). In both
dd4 alleles, several such cysts contained fewer than 16 cells
(Table 1;
Fig. 1D). The second type of
abnormality was in the morphology of the meiotic spindles. In wild-type
meiosis, a system of parafusorial membranes and mitochondria line up along the
nuclear membranes and appear in the phase-contrast microscope as dark bands
outlining the equatorial region of the spindle
(Fig. 1B). In both
dd4S and dd43, phase-dense membranous
material similar to the wild type accumulated unevenly around the nuclear
region (Fig. 1E, arrowhead).
Very frequently, the dark material was organized in cone-like shapes with less
dense material, probably corresponding to chromatin, sitting on the base of
the cone (Fig. 1E, arrows). In
some cases, biconical figures sharing the same base were found
(Fig. 1E, arrowhead). Intact
cysts of spermatocytes having the characteristics of meiosis II were not found
in any sample taken from dd4 males, suggesting that cells progress
through the second cycle without dividing.
|
|
In wild type, the meiotic divisions normally result in a syncytium of 64
spermatids, each of which contains a single haploid (N) nucleus and a
phase-dense spherical mitochondial aggregate, the Nebenkern
(Fig. 1C). The morphology,
size, and number of Nebenkerns per nucleus within a mutant spermatid indicate
defects in organization of mitochondria on the spindle or failure in
cytokinesis, or both. Similarly, the number and size of spermatid nuclei at
this stage reflects the fidelity of chromosome segregation and karyokinesis
(Cross and Shellenbarger,
1979; Gonzalez et al.,
1988
; Sunkel and Glover,
1988
; Gonzalez et al.,
1989
). Cysts of early spermatids in both dd4 mutants
displayed fewer than 32 Nebenkerns with abnormal morphology and variable size
(compare dark inclusions in Fig.
1C with those in Fig.
1F). Nuclei in the cysts were also variable in number and size
(Table 1;
Fig. 1F). More rarely, the
abnormal meiotic divisions resulted in onion stages with only 16 cells
(Table 1). 16-cell cysts at
onion-stage with a 1:1 ratio of nuclei to Nebenkerns were found more
frequently in dd43 than in dd4S males
(Table 1). Such cells had
nuclei of a size compatible with a tetraploid content of chromosomes
(Gonzalez et al., 1989
). In
general, dd43 showed a stronger hypomorphic phenotype than
dd4S mainly reflected in the relative viability of adult
males, the number of early spermatid cysts with 4N nuclei, and the presence of
sperm tails inside the testes (Fig.
1F, cyst on the right).
To clarify the nature of the spindle defects observed by phase-contrast
microscopy, we carried out immunostaining to localize spindle microtubules.
Morphologically, dd4S primary spermatocytes were
indistinguishable from wild type except in the number of cells in some cysts
(Table 1). Masses of
microtubules were observed around presumably bivalent chromosomes at the onset
of meiosis in dd4 (Fig.
1J, arrows). Spindle-like structures then appeared to develop that
fell into three main categories on the basis of their morphology. One category
included conical structures with a radial and slightly concave microtubule
array at the wider basal region and bundles of microtubules converging to an
apex (Fig. 1K, arrow). Conical
spindles usually displayed bundles of microtubules that extended distally from
their apexes (Fig. 1K-L,
arrows). In those instances a constriction around the apex together with a
dark band interrupting the continuity of the microtubule `tracks' suggested a
rudimentary central spindle structure
(Cenci et al., 1994;
Gatti et al., 2000
). The
second category of abnormal spindle appeared as umbrella cup-like structures
in which the only visible microtubules irradiated from the centre
(Fig. 1K, arrowhead). These
latter figures resembled astral arrays of microtubules or halves of normal
spindles at early anaphase I in which the inter-polar microtubules were not
yet visible (Fig. 1K,
arrowhead). More rarely, `biconical' figures such as those observed with the
light microscope (Fig. 1E, arrowhead) were also found by indirect immunofluorescence of microtubules
(Fig. 5D, arrows). We refer to
these three types of spindles as `cones', `hemi-spindles', and `biconical
figures', respectively.
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It was also evident that chromatin had a scattered distribution in most of the mutant meiotic figures (Fig. 1J-L). DNA was often found at two sides of the cones namely, at the base and beyond the constriction, suggesting chromosome segregation on a bipolar structure. Generally, hemi-spindles contained chromatin in contact with the astral microtubules varying, however, in its position relative to the main microtubule organizing centre (Fig. 1K).
We also stained such preparations to reveal -tubulin. In wild type,
-tubulin concentrates in the duplicated centrosomes as the nucleus
enters meiosis (Fig. 1G) and
remains associated with centrosomes throughout meiosis
(Fig. 1H,I). In late anaphase,
the
-tubulin-containing body at the spindle pole splits and the central
spindle matures (Fig. 1I). In
dd4S testes, however, we were unable to detect
-tubulin in any particular structure of the meiotic apparatus of
dividing spermatocytes (Fig.
1J-L).
Defects in pole segregation of dd4S
meiotic spindles are inferred from CNN localization
Two explanations may be offered to account for the abnormal meiotic
spindles in dd4S spermatocytes. First, as with
dd4 neuroblasts, the microtubule organizing centres (MTOCs) are
defective in duplication or segregation, or both. In male meiosis, where the
spindle assembly checkpoint is less stringent, such a defect would be expected
to have visible consequences for the later stages of the division cycle
(Gonzalez et al., 1988;
Sunkel and Glover, 1988
).
Second, the bipolar spindle in meiosis I first forms and then collapses around
an abnormal central spindle, reuniting both poles and leaving an aneuploid
complement of DNA per cyst cell similar to the effect proposed in
Tub23C mutants (Sampaio et
al., 2001
).
In order to understand the behaviour of the spindle poles in the abnormal
meiotic spindles of dd4S males, we studied the
localization of centrosomin (CNN) in fixed preparations of
dd4S testes. In wild-type meiosis, CNN appears in
discrete bodies at the poles of the spindle that correlate with the expected
number of centrosomes (Fig.
2A-C). As the chromosomes segregate and microtubules accumulate in
the central spindle to organize cytokinesis, the CNN-containing bodies split
in two, reflecting the distribution of this pericentriolar material (PCM)
component around the centrioles (Li et
al., 1998). In dividing dd4S
spermatocytes, CNN-containing bodies accumulated together in a cluster
(Fig. 2D-F, arrows) or showed
an abnormal position relative to each other
(Fig. 2D, arrow). These
CNN-containing bodies were generally present in variable numbers (ranging from
three to five) at the centre of the asters in both spindle types. However,
independent CNN-containing bodies were, in some, cases found embedded in the
microtubule network, distal from the rest of the CNN-staining fragments
(Fig. 2D, arrowhead). Cones
always contained CNN accumulated in the centre of their base
(Fig. 2F). Hemi-spindles showed
several CNN-stained bodies comparable to those in the bases of cones
(Fig. 2E). The finding of some
examples of both cones and hemi-spindles with greater than four CNN fragments
per pole, in cysts undergoing meiosis (Fig.
2E,F), suggests that failure of centrosome segregation can occur
from the gonial divisions onwards.
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Localization of Asp suggests the nature of hemi-spindles and reveals
central spindle abnormalities in cones
As Asp participates in the organization of spindle poles
(Avides and Glover, 1999) and
in the subsequent stabilization of the central spindle
(Wakefield et al., 2001
;
Riparbelli et al., 2002
), we
wished to examine its localization in the abnormal spindles in
dd4S spermatocytes. Asp associates with the
putative minus ends of microtubules and thus in wild-type meiosis, it is seen
as a hemispherical cup-like structure overlying the spindle-facing side of the
centrosomes of the meiotic spindle in late anaphase/telophase
(Fig. 3B, arrowhead). It also
localizes to the edges of the central spindle
(Riparbelli et al., 2002
)
(Fig. 3B, arrows). In
dd4S spermatocytes, Asp was localized in an
irregular manner at the core of the astral poles of the nascent spindles
(Fig. 3D, arrowheads).
Curiously, it was also possible to see Asp extending along fibres emanating
from the asters in both cones and in hemi-spindles
(Fig. 3E, inset;
Fig. 3F, arrowheads). Although
the fragmented distribution of Asp appeared restricted to the centre of the
asters, in some cones the Asp-containing fibrous material extending from the
asters seemed to reach the apex (Fig.
3F, inset). So, if Asp is marking the putative minus ends of
microtubules it would appear that many have been released from the poles as
seems to occur when the central spindle forms in wild type
(Riparbelli et al., 2002
).
However, in this case discrete staining at the edges of the central
spindle-like structure was not observed. This suggests that the central
spindle-like structures associated with cones are not correctly organized and
that the correct interaction of Asp with the putative minus ends of
microtubules may require
TuRC function. In the hemi-spindle structures
spreading of Asp from the organizing centre was restricted and it was never
found around the periphery of these astral structures
(Fig. 3E, arrowheads).
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Time-lapse studies indicate that bipolar spindles can form and
subsequently collapse
To gain insight into the process that leads to formation of the abnormal
spindle structures in dd4S spermatocytes, we
followed meiosis in these cells by time-lapse microscopy. In wild-type meiosis
(Fig. 4A-E), centrosome
separation occurs around an intact nucleus
(Fig. 4A). Subsequently, the
spindle elongates and chromosomes align during prometaphase
(Fig. 4B). Homologues separate
in anaphase (Fig. 4C, arrows,
and Fig. 4D) before the
reformation of nuclei at telophase (Fig.
4E). In dd4S mutant spermatocytes we
observed two main types of event: in one case
(Fig. 4F-J) bipolar spindles
assembled (Fig. 4F,G) and
bioriented bivalents localized in the central region of the spindle
(Fig. 4G). However, instead of
further elongating, such spindles collapsed and their poles approached one
another (Fig. 4H,I). After the
collapse, microtubules continued to grow as if attempting to undertake
anaphase B but with no evidence of segregation of homologues
(Fig. 4H). These collapsed
spindles thus produced biconical figures with a common basal region containing
the asters (Fig. 4I). Finally,
the spindle appeared to disassemble and nuclear-like vesicles formed
(Fig. 4J). In the other type of
event (Fig. 4K-O), asters
remained very close to each other throughout. Phase dense membranous material
(pdm) that would probably correspond to the parafusorial membranes accumulated
along these apparently monopolar spindles. This material then became
concentrated at the distal part (Fig.
4L,M), which developed an increasingly conical shape
(Fig. 4N). In this process
chromosomes were seen in rapid movements towards the astral pole
(Fig. 4L, arrow) and sporadic
slow movements away from it. In later stages most of the chromosomes seemed to
localize in the vicinity of the pole (Fig.
4M, arrows). Hemi-spindles thus seem to be formed either as a
result of a failure of centrosome separation or through a collapse of a
bipolar spindle shortly after centrosome separation. Such hemispindles
invariably developed into cones when observed by time lapse microscopy.
|
Pav-KLP localizes to the putative plus ends of hemispindles and the
apexes of cones
As our time-lapse observations indicated that the two major spindle defects
arose at or shortly after metaphase I, we were curious about the extent to
which events associated with cytokinesis occurred in dd4S
males. To verify whether the formation of the contractile ring was compromised
by the disorganization of the dd4S spindle, we first
investigated the localization of Pav-KLP (Pav-KLP), a kinesin-like protein
related to the mammalian MKLP-1 and essential for cytokinesis in mitosis
(Fig. 5)
(Adams et al., 1998). In
Drosophila spermatogenesis, Pav-KLP localizes to the ring canals,
remnants of the contractile rings from earlier divisions
(Fig. 5A, arrow). It localizes
to the central spindle in anaphase and concentrates in the mid-zone in
telophase (Fig. 5B, arrow)
(Carmena et al., 1998
;
Adams et al., 1998
). In
dd4S males, the localization of Pav-LKP in ring canals
derived from premeiotic divisions (Fig.
5C, large arrow) did not seem to be affected. In the hemi-spindles
of dd4S spermatocytes Pav-KLP was seen to localize at the
putative plus ends of the microtubules
(Fig. 5C, arrowheads). In cones
with a morphology consistent with their derivation from hemispindles, Pav-LKP
was associated with microtubules at the point of constriction
(Fig. 5C, small arrow;
Fig. 5D, arrowheads). Biconical
figures, which are probably derived from the collapse of bipolar spindles,
showed Pav-KLP at the vertex of each cone
(Fig. 5D, arrows). These
observations support the hypothesis that hemi-spindles are initially monopolar
structures. However, Pav-KLP never appeared in a ring shape in cones but
rather as a `knot' at their apexes. This could reflect abnormalities in the
structure of the central spindle.
Peanut fails to localize on hemi-spindles and forms rings around the
rudimentary central spindles in cones
The Drosophila septin Peanut
(Neufeld and Rubin, 1994) is a
component of both mitotic and meiotic ring canals
(Fig. 6A, inset)
(Hime et al., 1996
).
Interestingly, Peanut was localized to the apex of a subset of `cones' in
dd4S spermatocytes, forming circular structures which
usually ringed the very mid-zone of the putative central spindle structure
whenever such a structure could be discerned
(Fig. 6A, arrows). The rings of
Peanut staining in these cones are morphologically similar to the
Peanut-decorated contractile rings around the mid-zone of wild-type meiotic
spindles during cytokinesis (Fig.
6A, inset). Peanut rings displayed a variable range of diameters
correlated with the degree of constriction of microtubules (compare split
channels in Fig. 6A). The
pattern of distribution of Peanut in such cones suggests that it is part of a
dynamic structure able to constrict around the abnormal central spindles
leading to asymmetrical cytokinesis. A second set of cones that appeared to
have no extension of microtubules showed only slight or no Peanut staining at
their vertexes (Fig. 6B,
arrow), whereas equivalent structures had Pav-KLP present in the same region
(Fig. 5D, arrowheads). These
observations suggest either that Peanut was lost or never associated with such
structures. Hemi-spindles did not appear to contain any sort of Peanut
staining in their microtubule arrays (Fig.
6B, arrowhead), suggesting that contractile rings cannot be
properly assembled at the dispersed putative plus ends of their
microtubules.
|
Actin filaments are part of the major contractile structure during
cytokinesis (Hime et al.,
1996). We stained both dd4S and wild-type
testes with phalloidin to reveal actin and found a similar distribution to
that of Peanut with respect to the contractile rings (data not shown). Thus,
the central spindle would appear in some cells to be formed when microtubules
were organized by a single pole and focused on a centrosomal aggregate on the
one hand and by chromosomes on the other. In some cases sufficient of a
central spindle was generated that enabled ring-like structures of Peanut and
F-actin to form.
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Discussion |
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Aster formation
The spindle poles of dd4 primary spermatocytes usually have the
expected number of centrioles by the criteria of discrete bodies of CNN, a
component of the PCM that has been described to closely surround the
centrioles in such cells. However, the finding of some spermatocytes with more
than four such bodies suggests that there can be failure in centriole
separation in the pre-meiotic divisions as has been described in mutant
dd4 neuroblast divisions (Barbosa
et al., 2000). The CNN-containing bodies in dd4
spermatocytes appear either to have never fully separated or have become
reunited after spindle collapse and so the four such bodies are usually at the
focus of the astral poles. In common with dd4 mutant neuroblasts,
these pole bodies lack the
TuRC but are associated with Asp. The
ability of these poles to nucleate asters thus goes against the accepted dogma
that the proper localization of
-tubulin and centrosomal integrity is
absolutely required for the function of a polar MTOC to direct the formation
of asters (Bonaccorsi et al.,
1998
; Khodjakov and Rieder,
2001
). At present we can only speculate why astral microtubule
arrays are not seen in dd4 neuroblasts
(Barbosa et al., 2000
) and yet
appear robust in dd4S spermatocytes. On the one hand it
could reflect a general deterioration of the spindle throughout a prolonged
period of metaphase delay due to the more robust spindle integrity checkpoint
in neuroblasts. On the other it could reflect underlying differences in
spindle structure and function between these cell types
(Casal et al., 1990
). It is
possible, for example, that Asp in the focus of asters in
dd4S spindles may play more of a role in maintaining
astral microtubules in spermatocytes than it does in neuroblasts. This would
be consistent with the known function of Asp in the reorganization of radial
arrays of microtubules around isolated Drosophila centrosomes
(Avides and Glover, 1999
).
Moreover, meiotic spindles in asp spermatocytes are abnormal in
shape, and the morphology of their asters is considerably affected
(Gonzalez et al., 1990
;
Wakefield et al., 2001
).
However, it would seem that Asp may not be as efficient at stabilizing asters
in the dd4 larval CNS as in dd4 spermatocytes.
Many of the astral structures revealed by the immunostaining of dd4 testes appeared sufficiently asymmetric to have the appearance of hemi-spindles. These were truly monopolar by the criteria of having Asp at the focused putative minus ends of microtubules and with Pav-KLP located at their periphery, the putative plus ends. Such hemi-spindles are quite different structures from the asymmetric spindles sometimes observed in dd4 mutant neuroblasts in which one Asp containing pole can be focused and the other comprised of scattered bundles of microtubules whose putative minus ends are associated with Asp. However, real-time imaging suggests the hemi-spindles seen in dd4 meiocytes are an intermediary in the development of cones. In this process it seems that bipolarity is developed by the chromatin apparently acting to stabilize the diverging microtubules. Such spindles have one pole with multiple centrioles and the other with none.
Spindle-pole separation
The difficulties in either establishing or maintaining the separation of
spindle poles in male meiosis in dd4 mutants point towards a novel
role for the TuRC in maintaining the function of spindle microtubules
per se. It is possible that there could be two stages to this process
that differ in their sensitivity to the compromised function of the
TuRC. This is suggested by the finding that in some cells, bipolar
spindles either never form or collapse early (to form initially a
hemi-spindle). Thus, the first crucial requirement of
-tubulin function
may be to nucleate a subset of spindle microtubules that maintain bipolarity.
If a bipolar and bi-astral spindle does form then it seems to undergo a crisis
around metaphase when it appears to collapse. The collapsing spindles do
elongate however, suggesting that collapse may in part be driven by anaphase
events. In some ways the spindle collapse is reminiscent of the consequences
of inactivating
-Tub function by RNAi in Caenorhabditis
elegans embryos that result in separated asters re-approaching each other
at late prophase (Strome et al.,
2001
). Moreover, conical spindles in
Tub23CPI spermatocytes seem to appear from a
collapse of bipolar spindles around prophase and elongate in a timeframe
comparable to the assembly of the central spindle in wild type
(Sampaio et al., 2001
). It is
possible that a second, stabilizing effect of the
TuRC at the minus
ends of the microtubules (Wiese and Zheng,
2000
) is specially required before metaphase in meiosis I. In
vertebrate cells, low doses of taxol have shown to preferentially stabilize
kinetochore microtubules plus ends leading to a slight collapse of the spindle
around the time of metaphase (Waters,
1997
; Compton,
2000
). Perhaps the reduction of centrosomal
TuRCs in
Tub23CPI and dd4 cells is reproducing this
effect by destabilizing the minus ends of microtubules.
Central spindle formation
The normal origin of the central spindle microtubules in wild-type cells is
obscure. Treatment of cells with microtubule destabilizing agents after the
onset of anaphase suggests that the central spindle may be assembled from
newly nucleated microtubules and not from remains of the mitotic spindle
material left in the cell equator (Canman
et al., 2000; Gorbsky et al.,
1998
; Mastronarde et al.,
1993
; Shelden and Wadsworth,
1990
). However, although the localization of
-tubulin in
the central spindle of mammalian dividing cells has been reported by several
groups (Julian et al., 1993
;
Shu et al., 1995
), the
presence of
-tubulin in Drosophila central spindle is still a
matter of debate (Callaini, 1997; Carmena
et al., 1998
; Raff et al.,
1993
; Wakefield et al.,
2000
). The spindle collapse that occurs in dd4 meiocytes
could be related to the onset of reorganization of the spindle that occurs at
the metaphase-anaphase transition when some microtubules appear to detach from
the centrosomes as the central spindle structure begins to form. In wild-type
meiocytes this is seen by the generation of a new set of central spindle
microtubules with Asp at their putative minus ends
(Fig. 3B,C)
(Riparbelli et al., 2002
).
Central spindle microtubules never become fully organized in the dd4
spermatocytes although this seems to progress further in cones. Consistently,
Asp never undertakes its normal redistribution but rather adopts a fibrous
pattern of organization extending from the spindle poles. If as it has been
suggested, Asp works as an anchor to the putative minus ends of microtubules,
it is possible that microtubules are released from the spindle poles and
rather dispersed throughout the conical microtubule structure in dd4
meiocytes. But the lack of Asp capped microtubules of central spindle-like
structures in these cells suggests some degree of co-operation with the
-TuRC is necessary to correctly co-ordinate this transition in spindle
structure.
Despite the absence of clearly organized central spindle microtubules, the
mutant cells do show several features typical of post-metaphase stages of
meiosis that differ in two pathways of spindle development. The hemi-spindles
that give rise to cones harbour homologues that are initially mono-oriented as
they move towards and away from the asters without evidence of segregation. As
cones develop from the hemi-spindles, bipolarity appears to arise from some
ability of chromosomes to stabilize microtubules as discussed above. At this
time, microtubules stabilized by distal chromatin in some hemi-spindles would
appear to interdigitate with microtubules from the astral pole in an
anti-parallel manner to form cones with the motor protein Pav-KLP then
becoming associated with a `knot-like' structure at the centre of the spindle
but never forming a ring. Rings of septin and actin can then form around
structures equivalent to those where the Pav-KLP `knots' appear. Sometimes
these enable cytokinesis to be achieved. In the pathway in which bipolar
spindles collapse there is an elongation of spindle microtubules analogous to
the lengthening that takes place in anaphase B. Such spindles have no
arrangement of microtubules that resembles a central spindle. They lack the
bipolarity usually associated with central spindle formation and unlike the
hemi-spindles they appear to lack the ability for regenerating such a bipolar
structure. The presence of Pav-KLP at the apexes of the biconical figures
(Fig. 5D) suggests that
although Pav-KLP is a known prerequisite for central spindle formation
(Adams et al., 1998), this
localization is in itself insufficient for this process. Thus, central
spindle-like structures do not form in the biconical figures possibly
reflecting the absence of interdigitating microtubules inherent in a bipolar
structure and this in turn leads to a failure in formation of rings of septin
and actin. Thus as previously observed in
Tub23CPI
spermatocytes there seems to be some limited ability to organize some of the
components required for cytokinesis when
TuRC function is compromised,
the extent of which appears to reflect the ability to reorganize central
spindle microtubules.
In summary our observations indicate that the TuRC may provide
several functions to the spindle. It is not absolutely essential for
microtubule nucleation to form asters in all cell types. Rather, it may be
required for the specific function of subsets of spindle microtubules that
maintain pole separation. It appears to co-operate with other proteins
associated with the minus ends of microtubules, notably Asp in
Drosophila cells, and this appears to be important in the
reorganization of the spindle that occurs following the metaphase-anaphase
transition. Further work will be required to determine the extent to which
defects in the reorganization of the central spindle at this stage reflect a
direct requirement for the
TuRC or are a consequence of earlier defects
in spindle organization.
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Acknowledgments |
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Footnotes |
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References |
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