Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Genetica e Biologia Molecolare, Universita' di Roma La Sapienza, 00185 Rome, Italy
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Abstract |
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While Drosophila female meiosis is anastral,
both meiotic divisions in Drosophila males exhibit
prominent asters. We have identified a gene we call
asterless (asl) that is required for aster formation during
male meiosis. Ultrastructural analysis showed that asl
mutants have morphologically normal centrioles. However, immunostaining with antibodies directed either to
tubulin or centrosomin revealed that these proteins
do not accumulate in the centrosomes, as occurs in
wild-type. Thus, asl appears to specify a function required for the assembly of centrosomal material around
the centrioles.
Despite the absence of asters, meiotic cells of asl mutants manage to develop an anastral spindle. Microtubules grow from multiple sites around the chromosomes, and then focus into a peculiar bipolar spindle that mediates chromosome segregation, although in a highly irregular way.
Surprisingly, asl spermatocytes eventually form a morphologically normal ana-telophase central spindle that has full ability to stimulate cytokinesis. These findings challenge the classical view on central spindle assembly, arguing for a self-organization of this structure from either preexisting or newly formed microtubules. In addition, these findings strongly suggest that the asters are not required for signaling cytokinesis.
Key words: centrosome; spindle assembly; cytokinesis; male meiosis; Drosophila ![]() |
Introduction |
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CHROMOSOME segregation during both mitosis and
meiosis is mediated by the spindle, a complex bipolar structure consisting of microtubules and associated proteins. Although the basic structure of the spindle is similar in all cell types of all higher eukaryotes, the
routes through which the spindle assembles can be substantially different (reviewed by Rieder et al., 1993; Merdes and Cleveland, 1997
; Waters and Salmon, 1997
).
In animal mitotic cells, spindle formation is mediated by the centrosomes. During prophase, duplicated centrosomes, while moving to the opposite poles of the cell, nucleate radial arrays of microtubules called the asters. After the breakdown of the nuclear envelope, the plus ends of astral microtubules are captured and stabilized by the kinetochores, allowing the formation of a bipolar spindle.
In contrast, higher plant cells and female meiotic cells of
several animal species such as Caenorhabditis, Drosophila,
or Xenopus, do not contain centrosomes (Smirnova and
Bajer, 1992; Albertson and Thompson, 1993; Theurkauf
and Hawley, 1992
; Gard, 1992
). In these systems, microtubules grow from multiple sites around the chromosomes
and progressively self-organize into a bipolar spindle.
Studies on Drosophila female meiosis and in vitro spindle
assembly from Xenopus egg extracts have shown that microtubule focusing into spindle poles is mediated by minus-end-directed motor proteins. In Drosophila, the assembly and maintenance of a bipolar meiotic spindle
requires the action of Ncd, a minus-end directed kinesin
motor protein (Hatsumi and Endow, 1992
; Matthies et al.,
1996
; Endow and Komma, 1997
). Similarly, in Xenopus
egg extracts spindle pole formation is mediated by cytoplasmic dynein, another minus-end-directed motor protein (Heald et al., 1996
; Heald et al., 1997
). Dynein forms a
complex with NuMA (nuclear/mitotic apparatus protein)
and dynactin, both of which are also necessary for proper
microtubule focusing at the spindle poles (Merdes et al.,
1996
; Merdes and Cleveland, 1997
). Thus, in acentrosomal spindles the minus-ends of the microtubules that grow
around the chromatin move and converge towards the
poles through the action of minus-end microtubule-based
motors and their associated proteins.
Recent studies have shown that cells without centrosomes and cells with centrosomes share common mechanisms of spindle pole assembly (reviewed by Merdes and
Cleveland, 1997). Inhibition of cytoplasmic dynein by a
dynein-specific antibody disrupts spindle pole formation
in both centrosome-free and centrosome-containing spindles (Gaglio et al., 1997
; Heald et al., 1997
). Furthermore, in the latter systems dynein depletion results in the detachment of centrosomes from the spindle poles (Gaglio et al.,
1995
; Echeverri et al., 1996
). These observations, and the
finding that most interpolar microtubules are not connected to the centrosomes (Mastronarde et al., 1993
), suggested a model for pole formation in centrosome-containing spindles (Gaglio et al., 1997
; Heald et al., 1997
). It has
been proposed that a substantial fraction of the microtubules nucleated by the centrosomes is released from these
structures during the prometaphase search and capture
process (Kirschner and Mitchison, 1986
). The free minus-ends of these microtubules are then focused at the spindle
poles through the action of the same structural and motor
proteins that mediate pole formation in acentrosomal systems. The translocation of the microtubule minus-ends towards the spindle poles, coupled with microtubule elongation at the plus-ends and microtubule shortening at the
minus-ends, would then create a poleward microtubule
flux that exerts force through the spindle (Waters et al.,
1996
; Waters and Salmon, 1997
). In addition, lateral interactions between the astral microtubules and the free minus ends of poleward-migrating microtubules would tether
the centrosomes to the spindle poles, restoring the connection between these organelles and the rest of the spindle.
If the poles are assembled with similar mechanisms in
both acentrosomal and centrosomal systems, centrosome-containing cells should be able to assemble a spindle even
in the absence of centrosomes. In most cell types, however, this is not the case. For example, micromanipulation
experiments carried out in echinoderm embryonic cells,
vertebrate somatic cells, and grasshopper spermatocytes have clearly shown that removal of centrosomes from
prophase cells prevents spindle formation (Sluder and
Rieder, 1985; Sluder et al., 1986
; Rieder and Alexander,
1990
; Rieder et al., 1993
; Zhang and Nicklas, 1995
). However, if centrosomes are removed or lost during anaphase,
the spindle poles remain focused, and chromosome segregation is not affected (for review see Waters and Salmon, 1997
). On the other hand, experiments on spermatocytes
of the crane fly Pales ferruginea indicate that in these cells
spindle pole assembly is independent of the presence of
centrosomes (Steffen et al., 1986
). The reason why Pales
spermatocytes can assemble spindle poles in the absence
of centrosomes whereas the other systems cannot, is not
understood. An intriguing possibility is that the requirement of centrosomes for spindle assembly simply reflects
the fact that in some cell types these organelles are the
only source of microtubule nucleation. Thus, in the absence of centrosomes, there would not be enough microtubules to be focused at the spindle poles, and spindle assembly would be prevented (Waters and Salmon, 1997
).
In this paper we describe another centrosome-containing system that does not require centrosomes for spindle
formation. While Drosophila female meiosis is anastral
(Theurkauf and Hawley, 1992), both meiotic divisions in
Drosophila males exhibit prominent asters (Cenci et al.,
1994
; see Fig. 2). We have genetically micromanipulated Drosophila male meiosis by means of mutations in asterless (asl), a gene required for centrosome assembly and aster formation. In asl spermatocytes, despite the absence of
functional centrosomes, microtubules grow from multiple
sites around the chromosomes, and self-organize into peculiar anastral spindles. These spindles manage to mediate
chromosome segregation, although in a very irregular way.
Surprisingly, asl spermatocytes develop a morphologically normal ana-telophase central spindle. The finding that asl
mutants are completely devoid of asters and have normal
central spindles gave us the opportunity to test the relative
role of these structures in signaling cytokinesis. Our results
show that central spindles are fully able to induce cytokinesis, indicating that asters are not required for the cytokinetic signal.
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Materials and Methods |
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Drosophila Stocks and Mutagenesis
To isolate the asl2 and asl3 mutant alleles we mutagenized es ca males with
a 25-mM ethyl methane sulfonate (EMS)1 solution (Lewis and Bacher,
1968) and mated them with Oregon-R virgin females. The F1 es ca/++
males were crossed individually to asl1 es/TM6C, Sb e Tb ca females, and
their es ca/asl1 es male progeny were tested for fertility. The es ca/TM6C
brothers of the sterile males were then mated to apXa/TM6C females to
balance the putative asl alleles. The asl mutations (asl1, asl2, and asl3) were
kept over the TM6C balancer that carries the body-shape marker Tubby
(Tb), allowing identification of homozygous asl larvae and pupae. All the
balancers and markers used for mutagenesis and mapping are described in
Lindsley and Zimm (1992)
. The flies were reared on standard Drosophila
medium at 25 ± 1°C; dissections were performed at room temperature.
Immunofluorescence Microscopy
Cytological preparations were made with testes from third instar larvae
or from young pupae. For tubulin immunostaining, KLP3A plus tubulin
immunostaining, and anillin plus tubulin immunostaining, testes were
fixed as described previously (Cenci et al., 1994; Williams et al., 1995
). For
phalloidin staining and tubulin immunostaining, testes were fixed according to Gunsalus et al., 1995
. For
tubulin plus tubulin immunostaining or
centrosomin plus tubulin immunostaining, testes were dissected and frozen in liquid nitrogen as described (Cenci et al., 1994
). Preparations were
then fixed in cold methanol for 15 min and acetone for 30 s, and were then
immersed for 10 min in PBS containing 0.1% Tween 20 and 0.1% acetic
acid. Before incubation with antibodies, slides were rinsed several times in PBS containing 0.1% Tween 20.
Tubulin immunostaining and phalloidin staining plus tubulin immunostaining have been described previously (Cenci et al., 1994; Gunsalus et
al., 1995
). For double immunostainings, testes were first incubated overnight at 4°C with any of the following rabbit primary antibodies diluted in
PBT (PBS containing 0.1% Triton X-100) containing 1% BSA: anti-
tubulin (1:200; Callaini et al., 1997
); anti-centrosomin (1:1,000; Li and Kaufman, 1996
); anti-KLP3A (1:500; Williams et al., 1995
); or anti-anillin
raised against amino acids 1-371 (1:300; Field and Alberts, 1995
). These
primary antibodies were detected by 2-h incubation at room temperature with TRITC-conjugated anti-rabbit IgG (Cappel Laboratories, Malvern, PA) diluted 1:100 in PBT. Slides were then incubated with a monoclonal anti-
tubulin antibody (Pharmacia Biotech, Inc., Piscataway, NJ) diluted
1:50 in PBS, which was detected by FLUOS-conjugated sheep anti-mouse
IgG (Boehringer Mannheim, Mannheim, Germany) diluted 1:10 in PBS.
After these immunostainings, testis preparations were air-dried and
stained with Hoechst 33258 as described (Cenci et al., 1994
).
All preparations were examined with an Axioplan (Carl Zeiss,
Oberkochen, Germany) microscope equipped with an HBO 50W mercury lamp for epifluorescence, and with a cooled charge-coupled device (CCD;
Photometrics Inc., Woburn, MA). Hoechst 33258, FLUOS, and TRITC
fluorescence were detected as described (Gunsalus et al., 1995). Gray-scale digital images were collected separately using the IP Lab Spectrum
software. Images were then converted to Photoshop 2.5 format (Adobe
System, Inc., Mountain View, CA), pseudocoloured, and merged.
Electron Microscopy
Testes dissected from asl1 adult males were fixed in 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) at room temperature for 1 h, washed four times in phosphate buffer (5 min each), and postfixed in 1% OsO4 in the same buffer for 1 h. After four washes in phosphate buffer (5 min each), testes were dehydrated with ethanol (30, 50, and 70% 3×, 5 min each at 4°C; and 95 and 100%, 3×, 10 min each at room temperature). Testes were embedded in Epon and, after sectioning, were stained with 3% uranyl acetate and lead citrate.
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Results |
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Isolation and Characterization of asterless Mutations
The first asl mutant allele (asl1) was isolated in the course
of a cytological screen of a collection of 16 EMS-induced
male sterile mutations kindly provided by Barbara Wakimoto (University of Washington, Seattle, WA). Living
preparations of mutant testes were examined by phase
contrast microscopy for defects in the onion stage spermatids. In wild-type, each spermatid contains one phase-light
nucleus and one phase-dense mitochondrial derivative called the Nebenkern (reviewed by Fuller, 1993). At the
onion stage of spermatid development, the nuclei and
Nebenkern have spherical shapes and very similar sizes
(Fig. 1). The regular size of both nuclei and Nebenkern depends on the correct execution of the meiotic process; abnormal-sized nuclei and Nebenkern are diagnostic of errors in chromosome segregation and in the partition of
mitochondria, respectively (Gonzalez et al., 1989
; Fuller, 1993
). As shown in Fig. 1, asl1 spermatids are composed of
nuclei and Nebenkern of very different sizes, suggesing
that asl mutations disrupt both meiotic chromosome segregation and the correct distribution of mitochondria between the daughter cells.
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asl1 is perfectly viable but sterile in both sexes. The phenotypes of male sterility, female sterility, and aberrant spermatids associated with asl1 were mapped using a ri Ki pp chromosome by examining 44 recombinants between ri and pp (these markers define an interval of 1 cM). This analysis showed that these three phenotypes comap just to the left of Kinked (Ki), from which they are separated by only one recombination event.
To determine whether the asl1 phenotype is specifically elicited by this mutation or is a general characteristic of lesions in the asl locus, we isolated two additional mutant alleles. We treated 2,360 chromosomes with EMS and tested them for allelism with asl1. This screen yielded two new mutations, asl2 and asl3, that are viable over asl1 and fail to complement asl1 for male and female sterility as well as for the aberrant spermatid phenotype. However, asl2/asl2, asl2/ asl3, and asl3/asl3 individuals are lethal; asl2/asl2 and asl2/ asl3 larvae die at the larval pupal boundary, whereas asl3/ asl3 individuals have an earlier lethal phase. In addition, recombination experiments failed to separate the late lethal phenotype from asl2 and the earlier lethal phenotype from asl3. Thus, we conclude that asl is an essential locus required for viability. At present, however, we do not know whether asl3 is a null mutation. The fact that asl maps very close to Triplolethal (Tpl) prevented examination of the phenotype of asl3 over deficiency and its comparison with that of asl3/asl3 individuals. In this context, it is of interest that the pattern and frequency of abnormal spermatids is very similar in all mutant combinations (asl1/ asl1, asl1/asl2, asl2/asl2, asl1/asl3, and asl2/asl3), indicating that the three mutant alleles cause similar disruptions of the asl+ function during male meiosis.
asl Spermatocytes are Devoid of Asters and have Defective Centrosomes
To define the primary lesion leading to the formation of
aberrant spermatids in asl mutants, we analyzed cytologically the meiotic division. Testis preparations were stained
with anti-tubulin antibodies and Hoechst 33258 for simultaneous visualization of both microtubules and chromatin.
Examination of male meiosis in asl1/asl1, asl1/asl2, asl2/asl2,
asl1/asl3, and asl2/asl3 animals revealed that all these mutant combinations cause a common cytological phenotype.
Whereas wild-type spermatocytes exhibit prominent asters
throughout meiotic cell division (Fig. 2; see Cenci et al.
1994 for a detailed description of male meiosis), asl spermatocytes are completely devoid of asters (Fig. 3).
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To determine whether the absence of asters in asl mutants was the consequence of a primary defect in centrosome structure, we immunostained mutant testes with
antibodies directed to either tubulin or centrosomin, two
components of Drosophila centrosomes (Zheng et al.,
1991
; Li and Kaufman, 1996
). In wild-type testes, anti-
tubulin antibodies immunostain the centrosomes in premeiotic primary spermatocytes and throughout meiosis (Fig.
4). In mature primary spermatocytes, the centrosomes are
located near the plasma membrane (not shown). Before
the first meiotic division they migrate to the periphery of
the nuclear envelope where they nucleate prominent asters that move to the opposite poles of the cell (Fig. 4 A'). In anaphase and early telophase I there is a single centrosome at each spindle pole, which in late telophase I
splits into two centrosomes that start migrating to the
poles of secondary spermatocytes while nucleating new asters (Fig. 4, B' and C'). These centrosomes remain at the
spindle poles throughout the second meiotic division and
do not split into two separate entities in late telophase II
so that each spermatid receives a single centrosome. In
contrast, in asl mutants
tubulin is not concentrated in the
centrosomes during any phase of primary spermatocyte
growth and meiotic cell division (Fig. 4, D-F'). Instead, it
is dispersed in multiple small aggregates that do not appear to have the ability to nucleate microtubules.
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Similar but not identical results were obtained with centrosomin. In wild-type, centrosomin accumulates in the
centrosomes of both premeiotic primary spermatocytes
and meiotic cells, just as tubulin (Fig. 5). In asl mutants,
antibodies directed to centrosomin fail to detect discrete
centrosomal entities in most mature primary spermatocytes at the S5 stage (Fig. 5 F). However, in late prophase/
prometaphase primary spermatocytes at the M1 stage, anti-centrosomin antibodies immunostain two structures located near the nuclear envelope. These structures are consistently paired, as are the wild-type centrosomes before
their migration to the cell poles, but are much less fluorescent than regular centrosomes and fail to nucleate astral
microtubules (Fig. 5, G and G'). During ana-telophase I,
the centrosomin-enriched bodies are always detected at
only one of the cell poles, whereas the other pole is consistently devoid of them (Fig. 5, H-I'). In addition, although
they usually appear as a pair of fluorescent spots (Fig. 5
H), they are occasionally resolved into four entities (Fig. 5
I). At telophase I, the centrosomin-positive bodies are
transmitted to only one of the daughter cells, and are
therefore inherited by only one half of the secondary spermatocytes. These bodies tend to remain associated either
as doublets or quartets during the second meiotic division,
and are usually transmitted together to one-fourth of the
spermatids (Fig. 5 J). These observations strongly suggest
that each element of the fluorescent doublets corresponds
to a pair of centrioles, and that each element of the quartets consists of a single centriole. However, neither the
doublets nor the quartets have nucleating ability, as they are never associated with astral arrays of microtubules.
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To ascertain the presence of centrioles in asl spermatocytes and spermatids, we examined thin sections of testes by EM. This analysis revealed that asl spermatocytes have morphologically normal centrioles (Fig. 6). However, centriole separation is abnormal in that we observed that in some spermatids, Nebenkern are associated with two centrioles instead of a single one, as occurs in the wild-type (Fig. 6). Moreover, the two centrioles of the spermatid shown in Fig. 6, C and D, are lying parallel to each other instead of at a right angle, as do the parent and its daughter centriole in the wild-type. This spermatid may therefore contain four centrioles, with only two of them in the plane of the section.
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asl Mutants Organize Anastral Spindles that Mediate Chromosome Segregation
Despite the absence of asters, asl primary spermatocytes develop a peculiar anastral spindle. After the breakdown of the nuclear envelope, microtubules grow from multiple sites near the chromosomes, and form radial arrays extending from each bivalent (Fig. 3 B). These microtubules then organize into bipolar bundles, creating minispindles associated with individual bivalents (Fig. 3 C). However, in many asl spermatocytes, not all the bivalents within the same cell develop clear minispindles; some bivalents remain associated with a nonpolarized or poorly polarized network of microtubules (Fig. 3 C). In addition, the bundles of microtubules associated with the bivalents are often oriented in different directions (Fig. 3, C-F; Fig. 4 E'). As a consequence, the bivalents never congregate into a metaphase I plate during male meiosis in asl mutants.
Interestingly, the network of microtubules associated
with the tiny fourth chromosomes is always much smaller
than that associated with the larger bivalents, indicating
that microtubule growth around the chromosomes is promoted by the whole chromatin, and not by the kinetochores alone. It is worth noting that in most metaphase-like figures the fourth chromosomes exhibit precocious segregation (Fig. 3 C), as occurs in wild-type metaphase I
female meiosis (McKim and Hawley, 1995).
Despite these problems in congression, asl meiotic chromosomes progress into a highly irregular anaphase A (Fig.
3, D-F; Fig. 4 E'). The homologs manage to segregate, but
their separation is often asynchronous (Fig. 3 D; Fig. 4 E').
Moreover, in ~35% of anaphase I-like figures, the sister
chromatids of one or more half bivalents split and separate
from each other (Fig. 3, E and F). These peculiar ana-
phases are genuine anaphase I figures, and are not cells
undergoing anaphase II. This conclusion is suggested by
the finding that in telophase I figures we never observed
abnormal segregations with all the chromosomes migrating to a single pole (see below). Thus, most if not all the dividing cells with a 2N complement are likely to be primary
spermatocytes undergoing meiosis I, and not diploid secondary spermatocytes in meiosis II. The phenomenon of
precocious sister chromatid separation observed in asl
anaphase I figures is probably due to the structure of the
kinetochore of their half bivalents. During wild-type
prometaphase, each half bivalent has a single hemispherical kinetochore that differentiates into two planar kinetochores between late prometaphase I and early anaphase I
(Goldstein, 1981). In asl mutants where spindle formation
is likely to be delayed with respect to the wild-type (see
below), kinetochore duplication may occur before the onset of anaphase I. As a consequence, some half bivalents may become connected to both poles through their duplicated kinetochores, leading to separation of their component sister chromatids.
Regardless of the type of segregation they exhibit, anaphase I chromosomes of asl mutants are never organized into two discrete sets, but are instead scattered throughout the cell (compare Fig. 2 C with Fig. 3, D-F and Fig. 4 E'). Most likely this irregular anaphase chromosome arrangement reflects both the poor polarization of the spindles and the asynchrony in chromosome segregation.
After anaphase A, asl primary spermatocytes undergo anaphase B. Despite the aberrant configuration of anaphase A, ~85% of these cells develop a central spindle, which is indistinguishable from its wild-type counterpart (compare Figs. 2 D and 4 C' with Figs. 3 G and 4 F'; Table I). The remaining 15% of ana-telophases form tripartite or multipartite central spindles (Fig. 3 H; Table I). Moreover, central spindles elongate normally and are pinched in the middle during cytokinesis (Figs 3 G, 4 F', and 5 H', I'; see below). However, in about 80% of the ana-telophases with morphologically normal central spindles, these structures are asymmetrically located with respect to the cell poles, so that cytokinesis would produce two daughter cells of different size (Fig. 7; Table I). In addition, in most cells (60%) chromosomes also remain scattered during anaphase B and do not congregate into two daughter nuclei as in wild type (compare Figs. 2 D and 4 C' with Figs. 3 G, 4 F' and 5 H', I'; Table I).
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About 70% of telophase I figures exhibit unequal chromosome segregation (Fig. 4 F'; Fig. 7 C; Table I). In cells showing both an asymmetrically located central spindle and unequal chromosome segregation, there is no correlation between the size of the daughter cells and their chromosomal content, indicating that central spindle positioning and chromosome segregation are independent events.
As a consequence of the abnormal first meiotic division, asl secondary spermatocytes receive variable numbers of chromosomes. These cells undergo an anastral second meiotic division that has the same features reported above for mutant first meiotic divisions. Mutant secondary spermatocytes form an irregular apolar spindle that mediates chromosome segregation, and eventually assemble an apparently normal central spindle (data not shown).
To obtain insight into the dynamics and timing of the meiotic process in asl mutants, we determined the frequencies of the various meiotic figures in asl testes, and compared them with those observed in wild-type controls (Table II). An inspection of Table II reveals that the frequencies of late prophase/early prometaphase I and anaphase/telophase I figures found in asl testes are only slightly higher than those observed in controls. In contrast, the frequencies of prometaphase/metaphase I and early anaphase I figures are much higher in asl than in controls. Because the frequency of each meiotic stage should be proportional to its duration in vivo, these findings suggest that the duration of asl prophase/prometaphase I is only slightly increased with respect to the control. However, prometaphase I, metaphase I, and especially early anaphase I appear to last much longer in asl mutants than in the wild-type. A likely explanation of this observation is that the process of spindle organization in asl mutants lasts longer than it does in wild-type because of the absence of astral microtubules nucleated by the centrosomes. The fact that asl mutants and wild-type controls exhibit similar frequencies of ana-telophases with a well-formed central spindle strongly suggests that most if not all the asl cells that enter meiosis I progress until ana-telophase I.
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The frequencies of meiosis II figures are substantially
lower in asl than in the control, but the ratios between interphase-early anaphase II cells and anaphase/telophase II
figures are similar in both mutants and control. Because
there is not reason to postulate that the second meiotic division is more rapid in asl than in the wild-type, the most
straightforward explanation for these results is that only a
fraction of asl secondary spermatocytes has the ability to
organize an anastral spindle. However, once this anastral
spindle is assembled, the cells can progress to anatelophase and complete the second meiotic division.
The Central Spindle has the Ability to Stimulate Cytokinesis
An open question about cell cleavage in animal cells is the
source of signals that stimulate contractile ring formation
and cytokinesis. At present it is unclear whether these signals emanate from the asters or from the central spindle
(reviewed by Fishkind and Wang, 1995; Glotzer, 1997
;
Goldberg et al., 1997). The fact that asl mutants form a
central spindle in the absence of asters provided us with a
unique opportunity to discriminate between these alternatives. We stained asl testes with rhodamine-phalloidin, which detects the actomyosin contractile ring during male
meiotic cytokinesis (Gunsalus et al., 1995
). In addition, we
immunostained asl testes for KLP3A (Williams et al.,
1995
) and anillin (Field and Alberts, 1995
), two proteins
that concentrate in the cleavage furrow during wild-type
meiotic cytokinesis (Williams et al., 1995
; Hime et al.,
1996
). As shown in Fig. 7, both symmetrically and asymmetrically located central spindles exhibit a regular actin-based contractile ring and normal accumulations of both KLP3A and anillin. Regardless, the positioning of the central spindle within the cell, actin, anillin, and KLP3A are
always localized in the middle of this structure, as occurs in
the wild-type. In addition, in correspondence with the localization of these proteins, the central spindle is pinched,
suggesting regular execution of cytokinesis.
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Discussion |
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asl Mutants are Defective in Centrosome Assembly
We have identified a gene we call asterless (asl), that specifies a function necessary for aster formation during Drosophila male meiosis. In interphase primary spermatocytes and
meiotic cells of wild-type males, centrosomes are enriched
in tubulin. In contrast, in the same cell types of asl mutants this protein does not accumulate in the centrosomes
but remains dispersed in multiple cytoplasmic aggregates
that do not have microtubule-nucleating ability. Most
likely, this primary defect in centrosome assembly prevents aster formation throughout meiotic cell division in
asl mutants.
A similar but not identical situation has been observed
in the acentriolar Drosophila cell line 1182-4, established
from aploid embryos produced by the female sterile mutant mh 1182 (Gans et al., 1975; Debec, 1978
; Debec et al.,
1995
). In control embryonic cell lines,
tubulin accumulates in both interphase and mitotic centrosomes. In 1182-4 acentriolar cells
tubulin fails to associate with the interphase centrosomes, but it concentrates in the spindle poles
where it exhibits different patterns of accumulation (Debec et al., 1995
). However, the
tubulin polar spots seen
in the acentriolar cells are not true centrosomes in that
they readily disappear upon microtubule disassembly with
either cold or colchicine treatment (Debec et al., 1995
).
Based on these results, Debec et al. (1995)
suggested that
centrioles play an important role in the assembly of centrosomal material.
We have shown that in wild-type testes, antibodies directed to centrosomin immunostain the centrosomes in mature primary spermatoytes and throughout meiosis. In asl mutants these antibodies detect either doublets or quartets of discrete structures that are present in all late prophase/prometaphase primary spermatocytes, but are transmitted to only one half of the secondary spermatocytes and to one fourth of the spermatids. The behavior of these centrosomin-enriched bodies seen in asl mutants can be easily explained if one assumes that they correspond to the centrioles.
In wild-type, each mature primary spermatocyte contains two pairs of duplicated centrioles, with the daughter
centriole lying at a right angle with respect to its parent. In
preparation of meiosis I, both pairs of centrioles migrate
together from the plasma membrane to the nuclear envelope, become associated with centrosomal material, and
move to the cell poles while nucleating astral microtubules. Thus, during meiosis I each centrosome contains a
pair of duplicated centrioles. However, there is not centriole duplication before the second meiotic division; in secondary spermatocytes each pair of centrioles splits into
two single centrioles that migrate to the opposite cell
poles. Therefore, each spermatid inherits a single centriole
that becomes the basal body of the elongating axoneme
(reviewed by Fuller, 1993).
Based on centriole behavior in the wild-type, we propose that the centrosomin-enriched doublets seen in asl
primary spermatocytes correspond to the centrioles. The
fact that these doublets are occasionally resolved into four
entities further suggests that each element of the doublets
does in fact consist of a pair of centrioles. In addition, we
propose that the two pairs of centrioles, due to the absence
of astral microtubules (Waters and Salmon, 1997), fail to
separate and migrate to the cell poles during both meiotic
divisions of asl mutants. Thus, during each meiotic division they are transmitted together to only one of the two
daughter cells. This model for centriole behavior in asl
mutants is supported by the results obtained by EM. EM
analysis has shown that asl cells contain morphologically
normal centrioles that in several cases fail to separate
properly. We have observed several Nebenkern associated
with two instead of a single centriole. Moreover, in some
asl spermatids, these two centrioles are lying parallel to each other instead of at a right angle, as do the parent and
its daughter centriole in wild-type. This parallel centriole
arrangement is consistent with the possibility that the two
centrioles in the plane of the section belong to different
pairs of centrioles that have been transmitted together to
the sectioned spermatid.
Centrosomin immunostaining and EM analysis clearly
indicate that asl meiotic cells contain centrioles of regular
morphology that duplicate normally. Thus, the asl1 function does not appear to be required for either centriole fine structure or duplication. However, the observation
that asl centrioles are never associated with tubulin and
accumulate much less centrosomin than their wild-type
counterparts, strongly suggests that asl specifies a function
required for the assembly of centrosomal material around
the centrioles. The identification of such a function must
await the molecular analysis of asl, which, however, may
turn out to be particularly difficult. We have not succeeded in isolating asl alleles by P-mutagenesis, and molecular cloning of asl by chromosome walking is hampered
by its vicinity to the Tpl locus.
Spindle Assembly in asl Mutants
We have shown that despite the absence of asters, asl mutants assemble a peculiar anastral spindle. Meiotic chromosomes appear to play an important role in this process,
acting as microtubule-organizing centers and promoting
formation of bipolar minispindles. This finding was anticipated by micromanipulation experiments showing that
Drosophila male bivalents detached from the spindle can
trigger the formation of minispindles (Church et al., 1986).
The aberrant meiosis observed in asl males has many
similarities with naturally occurring anastral divisions,
such as those accompanying female meiosis in mice, Caenorhabditis, Xenopus, and Drosophila (reviewed in
McKim and Hawley, 1995). The asl spindle formation
pathway is also reminiscent of the in vitro spindle assembly induced by DNA-coated beads in Xenopus egg extracts (Heald et al., 1996
; Heald et al., 1997
). In all these
systems, chromatin can induce microtubule nucleation and
stabilization. These microtubules are initially randomly
oriented; their minus-ends then focus at the spindle poles
through the action of minus-end-directed motors and their
associated proteins (Hatsumi and Endow, 1992
; Heald et al.,
1996
; Matthies et al., 1996
; Merdes et al., 1996
; Heald et al.,
1997
). However, the minispindles associated with the asl bivalents are not always clearly organized into a bipolar
array. Moreover, when they do exhibit a bipolar configuration, the poles are broad and are never as focused as
those observed in Drosophila female meiosis or in the Xenopus in vitro systems. This result suggests that Drosophila spermatocytes do not have sufficient minus-end motor
activity to complete spindle polarization in the absence of
centrosomes.
Our results on asl mutants indicate that cells in which
spindle assembly is normally driven by centrosomes nonetheless have the ability to form anastral spindles. Similar
findings have been obtained with crane fly spermatocytes
(Dietz, 1966; Steffen et al., 1986
), but not with grasshopper
spermatocytes where both the chromosomes and the centrosomes are essential for spindle formation (Zhang and
Nicklas, 1995
). In addition, a series of studies has clearly
shown that spindle assembly during mitotic division of a
variety of vertebrate cell types invariably requires the
presence of functional centrosomes (reviewed in Rieder
et al., 1993
). Together, these findings raise the question of
why the ability to form anastral spindles in cells that normally contain centrosomes is restricted to a few meiotic
systems. It is possible that this property reflects different
types of interaction between chromosomes and microtubules. In vertebrate mitotic cells and in grasshopper spermatocytes, the chromosomes can only capture and stabilize the microtubules nucleated by the centrosomes, and do not appear to have the ability to stimulate microtubule
growth (Rieder et al., 1993
; Zhang and Nicklas, 1995
). In
contrast, in Drosophila male meiosis and most likely also
in Pales meiosis, the chromosomes act as microtubule-
organizing centers, even in the absence of centrosomes
(Fig. 3 B; see also Church et al., 1986
). Thus, we suggest
that anastral spindles are assembled only in those centrosome-containing systems where the chromosomes can induce formation of a sufficient number of microtubules.
In systems where the chromosomes are unable to promote
substantial microtubule growth, there would not be
enough microtubules to form a bipolar spindle.
asl Mutants Form a Normal Central Spindle that is Fully Able to Induce Cytokinesis
One of the most remarkable features of asl male meiosis is
the formation of a morphologically normal central spindle
in most ana-telophases. This finding challenges the classical view of central spindle assembly through interaction of
antiparallel polar microtubules. Our results argue for a
self-organization of the central spindle using either preexisting or newly formed microtubules (Masuda and Cande,
1987). Most likely, central spindle formation during male
meiosis is mediated by microtubule cross-linking, plus-end-directed kinesin-like motors (reviewed in Sawin and
Endow, 1993
; Ault and Rieder, 1994
; Hoyt, 1994
). This hypothesis is supported by the finding that mutations in
KLP3A, a Drosophila gene encoding a kinesin-like protein that concentrates in the central spindle midzone during male meiosis, disrupts central spindle formation and
cytokinesis (Williams et al., 1995
; Giansanti et al., 1998
).
An open question about cell cleavage in animal systems
is the source of signals that stimulates contractile ring formation and cytokinesis (reviewed by Fishkind and Wang,
1995; Glotzer, 1997
; Goldberg et al., 1997). It has been
suggested that these signals may be provided either by the
metaphase chromosomes (Earnshaw et al., 1991
) or the asters (Rappaport, 1961
; Hiramoto, 1971
; Rappaport, 1986
)
or the central spindle (Rappaport and Rappaport, 1974
;
Cao and Wang, 1996
; Fishkind et al., 1996
). Our results clearly show that the asters are not needed for the cytokinetic signal. Moreover, the fact that asl chromosomes
are scattered within the cell and never congress into a
metaphase plate strongly suggests that chromosomes cannot dictate the positioning of the cleavage furrow. This
conclusion agrees very well with the results of recent micromanipulation experiments showing that cytokinesis can
occur in the absence of chromosomes in grasshopper spermatocytes (Zhang and Nicklas, 1996
). Thus, of the three
components of the anaphase spindle
the asters, the chromosomes, and the central spindle
only the latter appears
to be required for signaling cytokinesis. In this respect, we
would like to point out that our findings rule out the possibility of the central spindle merely accumulating cytokinetic signals originating from the asters.
We have recently shown that during Drosophila male
meiosis, there is a cooperative interaction between the
central spindle and the contractile ring; when one of these
structures is disrupted the other one is also affected (Giansanti et al., 1998). Thus, the central spindle appears to play
an essential role during cytokinesis. The asters, however,
may be important for symmetrical positioning of the central spindle between the two daughter cells.
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Footnotes |
---|
Received for publication 10 March 1998 and in revised form 15 June 1998.
Address all correspondence to Silvia Bonaccorsi, Dipartimento di Genetica e Biologia Molecolare, Universita' di Roma La Sapienza, P.le Aldo Moro 5, 00185 Rome, Italy. Tel.: 39-6-49912593; Fax: 39-6-4456866; E-mail: bonaccorsi{at}axcasp.caspur.itWe thank B. Wakimoto for EMS-induced male sterile mutants; C. Field,
W.G. Whitfield, T.C. Kaufman, and B.C. Williams for anti-anillin, anti-
tubulin, anti-centrosomin, and anti-KLP3A antibodies, respectively; C. Goday, L. Lascari, and F. Pasquetti for advice and help with EM; and
M.L. Goldberg for comments on the manuscript.
This work was supported in part by a grant from Progetto Strategico del CNR Cell Cycle and Apoptosis.
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