1 Cancer Research UK, Cell Cycle Genetics Research Group, Department of
Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EN, UK
2 Drosophila Genetic Resource Center, Kyoto Institute of Technology,
Sagaippongi-cho, Ukyou-ku, Kyoto, 616-8354, Japan
* Author for correspondence (e-mail: dmg25{at}mole.bio.cam.ac.uk)
Accepted 29 November 2002
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SUMMARY |
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Key words: Orbit, Mast, CLIP-190, Anillin, Pavarotti-KLP, Fusome, Asymmetric division, CLASP, Cytokinesis, Oogenesis, Drosophila
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INTRODUCTION |
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Oogenesis in Drosophila is an attractive developmental process for
studying microtubule-based polarisation events, as it involves asymmetric cell
divisions that are important in establishing cell fate, and also polarised
inter- and intracellular transport phenomena required for the oocyte to
differentiate. A specialised organelle, the fusome (reviewed by Telfner, 1975;
Büning, 1994;
de Cuevas et al., 1997
;
McKearin, 1997
) plays a
determinative role in imposing asymmetry upon dividing stem cells and cysts,
as it associates with only one pole of the mitotic spindle at every mitosis
(Storto and King, 1989
;
Lin and Spradling, 1995
;
McGrail and Hays, 1997
) and
later is partitioned unequally between daughter cells (de Cuevas and Sradling,
1998). The fusome comprises membrane skeletal proteins such as the
-
and ß-spectrins, ankyrin, the adducin-like HtsF (the fusome specific
product of the hu-li tai shao gene)
(Yue and Spradling, 1992
), Bam
(bag-of-marbles) (McKearin and Ohlstein,
1995
), TER94 (León and
McKearin, 1999
), and motor molecules such as cytoplasmic dynein
encoded by the Dhc64C gene
(McGrail and Hays, 1997
).
Mutations in the genes hts (Yue
and Spradling, 1992
),
-spectrin
(de Cuevas et al., 1996
),
ovarian tumour (King et al.,
1978
) and Dhc64C
(McGrail and Hays, 1997
)
disrupt the fusome, leading to formation of a cyst with an abnormal number of
germ cells and the failure of any cystocyte to acquire oocyte identity. Thus,
the fusome is required for the formation of a polarised 16-cell cyst and for
oocyte specification, and fulfils this function by its regular and polarised
growth throughout the stem cell and cyst cell cycles
(Lin et al., 1994
;
Knowles and Cooley, 1994
;
Deng and Lin, 1997
;
de Cuevas and Spradling, 1998
;
Grieder et al., 2000
). During
each growth cycle, fusome spindle interactions specify the cleavage
plane. Cleavage is incomplete, with the furrow arresting upon contact with the
central spindle (spindle remnant). Concomitantly, fusome material is recruited
to lie between the arrested cleavage furrow (nascent ring canal) and spindle
remnant, and will transform into a fusome plug. The fusome plug, together with
its ring canal, moves centripetally, most likely facilitated by microtubules,
and gradually fuses with the pre-existing fusome, so changing the geometry of
the cyst. As a consequence, the fusome remains asymmetrically distributed
within the cyst: the older cells retain bigger, while the younger cells retain
smaller parts of fusome at the end of each cycle of cyst division. Upon
completion of cyst divisions, the fusome continues to play a crucial role in
polarising the 16-cell cyst by interacting with interphase-specific
microtubules that span across the ring canals, and as a result a selective,
microtubule-dependent transport is initiated (reviewed by Mahajan-Miklos and
Cooley, 1994; Navarro et al.,
2001
; Riechmann and Ephrussi,
2001
). Several mRNAs and proteins are transported along
microtubules, whose polarity is determined by the microtubule-organising
centre (MTOC) of the cyst situated in the oocyte. During mid-oogenesis, this
MTOC is disassembled, a process requiring the par-1 gene. This is a
precondition of the oocyte-specific anteroposterior and dorsoventral
polarisation events (Tomancak et al.,
2000
; Shulman et al.,
2000
). Thus, a plethora of cytoskeletal polarisation events
contribute to the differentiation of the oocyte.
As CLASPs and CLIP-170 had been shown to mediate interactions between
microtubules and the cortical cytoskeleton in cultured mammalian cells, we
wondered whether their Drosophila counterparts Orbit/Mast and
CLIP-190 (Lantz and Miller,
1998) could be involved in cytoskeletal polarisation events during
oogenesis. To this end, we have characterised the phenotype of newly isolated
orbit mutants (orbit5 and
orbit6) in oogenesis. Our findings point to the importance
of Orbit/Mast for many aspects of stem cell, cytoblast and cystocyte
divisions, and in the generation of polarised arrays of microtubules in the
16-cell cyst. We show that the Orbit/Mast and CLIP-190 proteins follow
specific localisation patterns in dividing germline cysts. Orbit/Mast is
initially found on the mitotic spindle, being concentrated at the poles, while
CLIP-190 accumulates on the spindle and at higher levels on fusome. Orbit/Mast
progresses onto the spindle remnant, from where it moves to the arrested
cleavage furrow and fusome. Concomitantly, CLIP-190 will show a uniform
distribution across the cyst as mitosis is completed. A role for the
Orbit/Mast protein in facilitating interactions between the organelles of the
cystocytes is indicated by the failure of the fusome to contact the mitotic
spindles, severe ring canal differentiation defects and disrupted fusome
organisation in newly isolated orbit mutant alleles. Moreover, in
mid-oogenesis, the polarised microtubule network that interconnects the oocyte
with the remaining nurse cells of the 16-cell fails to develop in the
orbit mutants.
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MATERIALS AND METHODS |
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Remobilisation of the P-lacW element
In order to revert the orbit5 mutation, the P-lacW
element was remobilised under dysgenic conditions. About 250 jump starter
males of genotype w1118/Y;
orbit5/TM3,Sb,ry,[2-3, ry+] were crossed
individually to w1118/w1118; TM3,Sb,Ser/TM6b,Tb
virgins. From their progeny the orbit5/TM3 and
orbit5/TM6b flies were scored for w-
or modified w+ expression when compared with original
w+ expression level seen in the eyes of
orbit5 flies. For each jump starter male, only one fly was
selected, showing the w- or the modified
w+ phenotype and for these revertants strains were
established over TM6b,Tb. A complementation test was performed with
orbit1, orbit5 and their revertant alleles.
Mutant phenotype analysis
The phenotypes of homozygous, hemizygous and transheterozygous
orbit5 and orbit6 mutants were
determined on a w1118;TM6b,Tb or y,w;TM6c,Tb,Sb
background (Table 1).
Individual sterility tests were performed for homozygous, hemizygous and
transheterozygous orbit5 and orbit6
females by crossing them to wild-type Canton-S males. The
[orbit+] transgene
(Inoue et al., 2000) was used
to rescue the orbit5- and
orbit6-associated homozygous mutant phenotypes.
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Germline autonomy of the orbit6 phenotype
To decide whether the orbit6 mutation affects the
female germline or soma, two types of germline chimeras were constructed
(Tables 2,
3). In the first experiment,
orbit6/orbit6 pole cells had been transplanted
into Fs(1)K1237/+ host females. The Fs(1)K1237 mutation
blocks germline function without affecting the soma
(Komitopoulou et al., 1983).
In the reciprocal experiment, wild-type pole cells, marked with y, v, f,
mal mutations, were transplanted into
orbit6/orbit6 host females, then mated with
y, v, f, mal males and the progeny scored for these marker mutations.
In both experiments, the orbit6/TM3, orbit6/TM6b and
TM3/TM6b sibling chimeras served as internal controls.
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Western blot analysis of ovarian extracts
Total ovarian protein extracts were prepared from 15 wild-type,
orbit1/orbit1,
orbit5/orbit5 or
orbit6/orbit6 females in standard 2x
Sigma protein loading buffer. Equal volumes of ovarian protein samples were
loaded onto 7.5 or 10% polyacrylamide gels and after electrophoresis
electroblotted onto Hybond ECL (Amersham-Pharmacia) nitrocellulose membranes.
Tubulin was used as a loading standard and detected using mouse monoclonal
-tubulin antibody (diluted 1:10; Amersham-Pharmacia). The Orbit protein
was detected with the affinity purified rabbit antibody described by Inoue et
al. (Inoue et al., 2000
),
diluted 10-3. Horseradish peroxidase-labelled anti-rabbit and
anti-mouse secondary antibodies were purchased Jackson Immunoresearch
Laboratories. The blots were developed using ECL or ECL plus kits
(Amersham-Pharmacia).
Immunoprecipitation
Ovarian lysates were prepared by dissecting and grinding 50 pairs of
ovaries in 150 ml of lysis buffer (0.3M sorbitol; 10 mM HEPES, pH 7.5; 10 mM
sodium azide; 1 mM PMSF; 0.5 mg/ml leupeptin; 0.7 mg/ml pepstatin). Two
volumes of IP dilution-1 buffer (125 mM Tris-HCl, pH 6.8; 20% glycerol; 20 mM
DTT; 0.02 Bromophenol Blue) were added. After 1 hour of incubation at room
temperature, four volumes of IP dilution-2 buffer (1.25% Triton X-100; 190 mM
NaCl; 6 mM EDTA; 60 mM Tris-HCl, pH 7.5) were added and the lysate spun for 5
minutes in a microcentrifuge at maximum speed. The supernatant was incubated
with either anti-Orbit or anti-CLIP-190 antibodies at 4°C overnight, after
which 100 ml of Dynabeads M-280 sheep anti-rabbit IgG (Dynal Biotech) was
added and the incubation continued for 3 more hours at room temperature. The
samples were washed three times in IP buffer (1% Triton X-100; 0.2% SDS; 150
mM NaCl; 5 mM EDTA; 50 mM Tris-HCl, pH 7.5) before using the magnetic particle
concentrator. After the last wash, the Dynabead particles were resuspended in
two volumes of protein gel loading buffer (4% SDS; 125 mM Tris-HCl, pH 6.8;
20% glycerol; 100 mM DTT; 0.02% Bromophenol Blue) and incubated for 1 hour at
room temperature. The cleared lysate was removed, boiled and loaded onto 7.5%
SDS-PAGE gels. Blotting and detection of proteins was carried out as described
above.
Immunocytochemistry and confocal microscopy
To study the germarial divisions in orbit mutants, ovaries of
newly eclosed, 1-2 and 3-day-old females were dissected in EBR buffer and
fixed as described by McGrail et al.
(McGrail et al., 1995). For
the phenotypic analysis of the orbit egg chambers, the ovaries were
dissected out from 2- and 3-day-old females that were mated to wild-type males
and fed with yeast paste, while the fixation was carried out as described by
Minestrini et al. (Minestrini et al.,
2002
). For rhodamine-phalloidin staining, the ovaries were
dissected out from females in EBR buffer and fixed according to the protocol
of McGrail et al. (McGrail et al.,
1995
), but the fixative and washing solutions (that were used
prior antibody incubation) were supplemented with 5 units/ml
rhodamine-phalloidin (Molecular Probes). All the samples were incubated
overnight with the primary antibodies at 4°C, while the incubations with
the secondary antibodies were for 4 hours at room temperature. Orbit/Mast
protein was detected with the affinity purified rabbit antibody described by
Inoue et al. (Inoue et al.,
2000
), and was diluted 1:200. Tubulins were detected with YL1/2
rat monoclonal anti-
-tubulin antibody (1:50; Sera Lab) or the
Bx69 mouse monoclonal anti-ß-tubulin antibody (1:2) and mouse
monoclonal anti-
-tubulin (1:50; Sigma GTU88). The T47 mouse monoclonal
anti-lamin antibody was used to visualise the nuclear lamina (1:10)
(Paddy et al., 1990
). The
fusome was stained using rabbit affinity-purified anti-
-spectrin
(1:200, Sigma), mouse monoclonal anti-HtsF antibody (1B1; 1:10; DSHB of Iowa
University). Ring canals had been stained with mouse monoclonal anti-HtsRC,
mouse anti-Kelch (1B, 1:1) (Xue and
Cooley, 1993
), rat anti-Filamin (1:200)
(Sokol and Cooley, 1999
), PY20
anti-phosphotyrosine (1:1000; ICN), rabbit affinity-purified
anti-Pavarotti-KLP antibody (1:200) (Adams
et al., 1998
) and the rabbit affinity-purified anti-Anillin
(1:2000) (Field and Alberts,
1995
). The rabbit anti-Inscuteable (1:100)
(Huynh and St Johnston, 2000
)
was used to visualise the synaptonemal complexes and the affinity purified
rabbit anti-CLIP-190 (1:1000) antibody was provided by K. Miller. The Alexa
488, 594 and 624-conjugated anti-rat, anti-mouse and anti-rabbit secondary
antibodies were obtained from Molecular Probes. DNA was visualised through
TOTO3 staining purchased from Molecular Probes. Digital images of serial
optical sections were collected with a BioRad 1024 confocal microscope,
merged, and then processed using Adobe Photoshop 5.5 (Adobe Systems).
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RESULTS |
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An orbit allele that disrupts oogenesis
In order to determine whether the distribution of Orbit/Mast protein on the
spindle, fusome and ring canal reflected a role in organising these
structures, we sought mutant alleles that might specifically affect these
structures. The original P-element insertion line orbit1
is hypomorphic and leads to maternal effect lethality as a result of the
abnormal mitotic spindles that form in syncytial embryos
(Inoue et al., 2000). Other
amorphic orbit alleles show late larval lethality with hyperploid
mitotic cells. We have identified a new P-lacW insertion line from the
collection of Deák et al.
(Deák et al., 1997
) that
had a P-element inserted in the opposite direction to that in the original
allele and 690 bp upstream from the first ATG of the orbit
open-reading-frame. As the associated mutant phenotype was completely rescued
by an [orbit+] transgene
(Table 1) we have named this
new allele orbit5. Through the remobilisation of the
P-element from the orbit5 line, we isolated a new allele,
orbit6; the mutant phenotype of
orbit6/orbit5 was again completely rescued by
the [orbit+] transgene
(Table 1). In this allele the
P-element was inserted into the identical site but with the opposite
orientation to the P-element in orbit5 and 60 bp of
downstream sequence were deleted. Most homozygous orbit5
individuals died as pharate adults, but 1-5% reached adulthood with females
and males being sterile. Homozygous orbit5 adults died
shortly after eclosion and females did not lay eggs. Hemizygous
orbit5 individuals showed third larval instar/pupal
lethality indicating the hypomophic nature of the mutation and suggesting
multiple somatic in addition to germline roles for the orbit/mast
gene. The orbit6 individuals were viable when homozygous,
hemizygous or transherozygous to orbit5 and both females
and males were sterile, suggesting that the mutation affects the function of
this gene in the germline. To confirm the female germline dependency of the
orbit6 mutation, we constructed two types of germline
chimeras. First, orbit6/orbit6 germline cells
were transplanted into K1237/+ hosts that have wild-type soma and
non-functional germline (Table
2). Eight such chimeras were identified that each manifested the
same mutant phenotype as orbit6/orbit6 females.
In the second experiment, wild-type (y,v,f,mal) germline cells were
transplanted into orbit6/orbit6 females, and
seven chimeras were recovered that produced wild-type (y,v,f,mal)
offspring (Table 3). Thus,
classical germline clonal analysis suggests that the
orbit6 mutation appears to affect only the female
germline.
To characterise the ovarian expression levels of the Orbit/Mast protein in
the new mutant alleles, we performed Western blot analysis on total protein
extracts from wild-type, orbit1/orbit1,
orbit5/orbit5 and
orbit6/orbit6 ovaries
(Fig. 2A). Although in
orbit1/orbit1 ovaries the level of Orbit/Mast
protein was slightly reduced when compared with the wild-type, in
orbit5/orbit5 ovaries it was reduced to 30%,
while in orbit6/orbit6 ovaries only traces of
protein were detectable. In all three mutants, the residual Orbit/Mast protein
had identical mobility to wild type. Disruption of oogenesis in
orbit5/orbit5 and
orbit6/orbit6 ovaries was further suggested by
the absence of Hts (Hu-li tai shao) isoform usually expressed during mid to
late oogenesis (Zaccai and Lipshitz,
1996) (Fig. 2B). In
agreement with this observation, we found that oogenesis never proceeds beyond
mid-oogenesis in the mutant orbit5/orbit5,
orbit6/orbit6, orbit6/orbit5
and orbit6/Df(3L)PC-MK ovaries. Moreover, in such
orbit mutant ovaries the levels of Orbit/Mast appeared to decrease as
the females aged. It was the highest at the time females emerged from pupae,
while from the third day onwards hardly any protein was found
(Fig. 2C). The ovaries of
orbit5/orbit5, orbit6/orbit6,
orbit6/orbit5 and
orbit6/Df(3L)PC-MK females were small and only a few egg
chambers were found in about one fifth of the mutant ovaries, suggesting the
potential involvement of a diminished production rate of 16-cell cysts.
Oocytes did not differentiate in these mutant orbit egg chambers
(about 700 were examined), and all the nuclei resembled those of nurse cells
but with poor nuclear lamina organisation
(Fig. 2D), a condition
potentially caused by abnormal germline divisions and/or the failure to
establish the correct architecture of the cyst required to support normal
oocyte differentiation. In the following, we describe the cytological
phenotype of orbit6 homozygotes, although we have observed
similar phenotypes in the above genotypic combinations with
orbit6.
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Stem cell maintenance and fusome growth are compromised in
orbit6 germaria
The finding of Orbit/Mast in the fusome in wild-type ovaries led us to
examine whether fusome integrity might be affected in orbit mutants.
The fusome is first evident in the ovariolar niche of wild-type germaria in
the asymmetric divisions of the stem cells. The proliferative activity and
identity of ovarian stem cells is regulated in this microenvironment, situated
at the tip of the germarium, where two stem cells interact with the terminal
filament and cap cells, both of somatic origin. We found defects in the
structure of ovariole niches in orbit6 females from their
emergence through three consecutive days of development
(Fig. 3) that increased in
severity as the amount of Orbit/Mast protein decreased
(Fig. 2C). In the wild-type
ovariolar niche, the position of two stem cells could be inferred from the
-spectrin positive spherical fusome situated close to the terminal
filament and cap cell (Fig. 3A,
arrowhead). More distally, the fusome branched, reflecting its growth in
successive germline division cycles (Fig.
3A, arrow). In about 50% of germaria of newly emerged and
1-day-old orbit6 females, fusome-containing stem cells
could be recognised that were associated with terminal filament and cap cells
in the ovariolar niche (Fig.
3B, arrowhead). Some cysts at later stages had developed branched
fusomes of normal appearance (Fig.
3B, arrow). In many other cysts, the fusome appeared as though it
had failed to branch suggesting its growth was compromised
(Fig. 3B, star), although this
structure could be a spherical fusome often seen after ring canals have
coalesced at the four-cell stage. The remaining 50% of germaria of 0- to
1-day-old females, showed no fusome material in the ovariolar niche and thus
would appear not to contain any stem cells, despite the presence of both
terminal filament and cap cells (Fig.
3C). Such ovaries would have appeared at one time to have had stem
cells since they frequently contained either aggregates or fragments of fusome
material often contacting irregular microtubule bundles located in more distal
cysts (Fig. 3C, star). Such
abnormal germaria predominated in 2- to 3-day-old orbit6
females (Fig. 3D). Thus,
neither the fusome organisation or cycling ability of stem cells appeared to
be maintained in the ovariolar niche of orbit6
germaria.
|
We further assessed the morphology and growth of the fusome in
orbit6 germaria by focusing on its interactions with ring
canals. In the wild-type ovary, the fusome plug first forms in each newly
assembled ring canal following mitosis of stem cells, cystoblasts and
cystocytes. These plugs then migrate and fuse with the pre-existing fusome.
The development of the spherical fusome of the stem cell and cystoblast into a
large branched structure that extends through the ring canals into every cell
of the cyst was revealed by HtsF or -spectrin staining
(Fig. 4;
Fig. 3A). We monitored the
progressive growth of the fusome in wild-type and orbit6
mutant females revealed by immunostaining HtsF with respect to Anillin and
Pav-KLP, two components of the ring canals that form from the cytokinetic
cleavage ring that in the germline does not undergo complete closure
(Fig. 4). Anillin accumulates
at the contractile ring and binds to actin filaments during cytokinesis in
many cell types including stem cells, cytoblasts and cystocytes
(de Cuevas and Spradling,
1998
). The protein was recruited to the contractile ring in
cystoblasts and cystocytes before fusome plug formation
(Fig. 4A, arrow), and was
present in the constricting and migrating ring canals
(Fig. 4B). Moreover, it
persisted in the ring canals that had reached the fusome until after the cyst
had left the germarium (Fig.
4C). In contrast to wild type, we found no Anillin stained ring
canals in orbit6 cysts although there were HtsF containing
fusome-like bodies (Fig. 4D,E).
Similar findings were obtained by monitoring Pav-KLP
(Fig. 4F-H). Pav-KLP acts in
the early stages of cytokinesis to organise the central spindle and persists
in the ring canals of germline cysts in both oogenesis and spermatogenesis
(Adams et al., 1998
;
Carmena et al., 1999
;
Minestrini et al., 2002
). In
wild-type germaria, we found higher levels of Pav-KLP in the nuclei of stem
cells and cystoblasts than in cystocytes. In mitotically dividing stem cells,
cystoblasts and cystocytes, Pav-KLP was present in the midzone of the central
spindle (data not shown). It was then to be incorporated into the cleavage
furrows and so the ring canals through which the fusome was seen to pass
(Fig. 4F,G). In
orbit6 cysts we could not assess the recruitment of
Pav-KLP to the central spindle as we were unable to find a single anaphase or
telophase spindle. However, in some mutant cysts, we were able to see
occasional ring-like, Pav-KLP-containing structures associated with
fusome-like bodies, while the other fusome-like pieces could be scattered
within a cyst and showed no Pav-KLP staining
(Fig. 4H). These multiple
fusome pieces appeared to have failed to fuse and form a properly branched
fusome. Thus, we conclude that not only is there a reduction in the formation
of fusomes, but those that do form fail to grow in part due to defects in ring
canal formation and migration.
|
orbit6 mutants disrupts the asymmetric
orientation of mitotic spindles
As aspects of the orbit phenotype described above suggested
defects in cell division of the germarium stem cells or cystocytes, we
searched for cells in the process of division in wild-type and
orbit6 ovaries, staining them to reveal DNA,
-tubulin and Orbit/Mast protein. In wild-type stem cells at prophase,
-tubulin was recruited to the centrosomes and the Orbit/Mast protein
accumulated on both centrosomes and the centrosome nucleated microtubules, its
staining increasing in intensity at metaphase
(Fig. 5A). Although the levels
of Orbit/Mast protein were dramatically reduced in total ovaries from 1- to
3-day-old orbit6 females, we were still able to detect
faint staining of spindle MTs by the anti-Orbit antibody within a small number
of cystoblasts and cytocytes of newly eclosed females. In such germaria the
structure of the cystoblast and/or cytocyte spindles was severely affected
(Fig. 5B,C). Spindles were much
shorter (average length 3 µm; arrowheads in
Fig. 5B,C) than the metaphase
spindles (average length 7 µm) observed in wild-type two- or four-cell
cysts, and could even be monopolar (Fig.
5B, arrow). This suggests that bipolar spindles might first form
and then collapse to monopolar structures as recently described in
orbit/mast-derived embryos and following orbit/mast RNAi in
cultured cells (Maiato et al.,
2002
). We analysed over 2000 wild-type germaria and identified 48
that contained mitotic stem cells, and 54 that had cystoblasts or cystocytes
at various stages of mitosis. The stem cell specific mitotic index (MI) of 2.4
was slightly higher, but still consistent with the observations of Deng and
Lin (Deng and Lin, 1997
). In
about 2000 germaria of orbit6/orbit6 females,
we found no stem cells in mitosis and only seven cystoblast and cytocyte
divisions. The mitotic index of orbit6
cystoblasts-cystocytes was thus dramatically reduced compared with wild type
(0.35 versus 2.7). Thus, unlike larval neuroblasts, cystocytes appear not to
arrest in mitosis, suggesting that if they have a mitotic checkpoint it is
over-ridden by the developmental process of oogenesis. However, reduced
proliferation ability of stem cells or their failure to be maintained in
orbit6 germaria as noted above would further account for
the reduction in mitotic index.
It is a characteristic of stem cell, cystoblast and cystocyte divisions that one of the poles of the mitotic spindles associates with the fusome leading to the asymmetry of division proposed to play a determinative role in generating and maintaining cyst asymmetry and to be a precondition for oocyte formation. In wild-type germaria we found the pole of each spindle most proximal to the terminal filament cells was attached to a spherical fusome (Fig. 5D). In mitotic cystoblasts, the fusome kept its spherical shape and was often located in the posterior half of the cell with the posterior spindle pole anchored to it (data not shown). In two-cell, four-cell and eight-cell mitotic cysts, we found spindles arranged in compact clusters with one pole of each spindle in close contact with a single branch of the fusome (Fig. 5E). To find examples of spindles in the orbit6 mutant, we examined about 2000 mutant germaria. In the most affected germaria, Orbit/Mast staining was very weak, and no spindles were visible. Whenever there was sufficient residual Orbit/Mast protein to allow some spindles to form, such spindles never made contact with the fusome. This can be seen in the cyst shown in Fig. 5F, where two bipolar and one monopolar (arrow) spindles lie in the vicinity of a fusome but show no association with it. In general, fusomes looked fragmented in most cysts (Fig. 5G, arrowhead). Taken together, our observations suggest that the orbit6 mutation affects the bipolar organisation of the spindles and also prevents the asymmetric interaction between the mitotic spindle and fusome.
orbit6 egg chambers have ring canals of abnormal
structure and number
The 16 cystocytes of each germline cyst are connected by 15 ring canals
representing the arrested cleavage furrows of the preceding four rounds of
germline cyst divisions. Once these divisions are completed and concomitant to
formation of the polarised microtubule network, the ring canals acquire outer
and inner rims by recruiting different actin binding proteins. Filamin is
recruited to both inner and outer rims of the ring canals
(Li et al., 1999;
Sokol and Cooley, 1999
),
whereas HtsRC (the ring canal specific product of the hts gene)
(Yue and Spradling, 1992
),
further F-actin (Theurkauf et al.,
1993
) and Kelch (Xue and
Cooley, 1993
) accumulate in the inner rims. The Orbit/Mast protein
did not associate extensively with the ring canals of wild-type egg chambers,
although some punctate accumulation may be inferred based on
coimmunolocalisation studies with other ring canal proteins
(Fig. 6E).
|
In orbit6 egg chambers, we observed a gradient of ring canals defects, the severity of which varied as a function of the age of the female (Fig. 6). Egg chambers from younger females (Fig. 6B,C,F) showed milder defects than older ones (Fig. 6D,G). Whereas F-actin and Filamin formed the expected overlapping rings in wild-type ring canals, in the egg chambers of younger orbit6 females, these proteins appeared disorganised, extending into and obstructing partially if not totally the lumen of the canals (Fig. 6B,C). The inner rim proteins Filamin and HtsRC extended to the very centre of the canal (insets to Fig. 6C,F). We also noted these orbit6 egg chambers showed varying loss of actin at the nurse cell boundaries indicating a disruption to the cortical cytoskeleton within these cells (Fig. 6B-D). In older orbit6 females, the egg chambers often had no ring canals, although ring canal specific proteins such as Pav-KLP (not shown), phosphotyrosine protein(s) (data not shown), actin, Filamin (Fig. 6D) and HtsRC (Fig. 6G) were detected in the form of aggregates. Surprisingly, we were unable to detect any Kelch protein in the ring canals of either younger or older orbit6 females (data not shown). Together this suggests defects in the differentiation of ring canals from the cleavage furrows of the cystocytes.
In wild-type cysts, the number of ring canals is one fewer than the cell number providing a record of each incomplete cytokinesis involved in the formation of the 16-cell cyst. The egg chambers of younger orbit6 females frequently had seven ring canals and eight nurse cell nuclei, suggesting that they might have had developed from eight-cell germline cysts that fail to execute the fourth round of mitotic division (Fig. 6B). However, in other egg chambers the number of ring canals and nurse cell nuclei did not seem to correlate and it was often impossible to determine their exact number, suggesting a more extensive failure of cell division. Many egg chambers contained between one and six nurse nuclei and no ring canals, which suggested that germline cyst mitoses had occurred but that cytokinesis had not been completed correctly. Thus, defects in the structural integrity of eggs chambers in the absence of Orbit/Mast protein appeared to be due in part to cytokinesis defects. Moreover, the defects seen in ring canal differentiation in orbit6 egg chambers further suggest that during cytokinesis ring canal fusome interactions were affected.
The posterior MTOC fails to develop correctly in
orbit6 egg chambers and the microtubule network is
perturbed
In wild-type ovaries, egg chambers form and mature along the length of the
ovariole as the germline cysts complete their divisions and are encapsulated
by follicle cells of somatic origin. One of the 16 cystocytes will acquire
oocyte identity as the `stage 1' egg chamber buds off the germarium and enters
the vitellarium. Here, the oocyte differentiates at the expense of nurse cells
and surrounding follicle cells. After completion of cyst divisions, the
Orbit/Mast protein showed punctate localisation pattern within the 16-cell
cyst, and co-localised with the regressing fusome and microtubules but
disappeared from the differentiating ring canals (data not shown). As
oogenesis progressed, the Orbit/Mast protein became concentrated in the oocyte
cytoplasm of stage 1 egg chambers and persisted until stage 6-7 of oogenesis,
when it translocated into the oocyte nucleus
(Fig. 7A,D). The localisation
of the Orbit/Mast protein in the oocyte cytoplasm during stages 1 to 7 of
oogenesis is largely coincident with the single MTOC located behind the oocyte
nucleus. Microtubule bundles nucleated at this centre extend through the ring
canals to form a network that interconnects the oocyte-nurse cell complex
within each egg chamber. The punctate accumulations of Orbit/Mast appeared
only in part connected to microtubule bundles with a substantial amount of
protein remaining apparently free in the cytoplasm. To demonstrate that
localisation of Orbit/Mast protein in egg chambers was dependent upon
microtubules, we used colchicine treatment to disassemble microtubules after
the completion of cyst divisions, as described by Theurkauf and colleagues
(Theurkauf et al., 1993). When
wild-type flies were fed with 20-50 µg/ml colchicine for 16-48 hours, the
newly produced egg chambers had 16 nurse cell nuclei indicating that oocyte
fate was not properly established and/or maintained. Moreover, in such egg
chambers the Orbit/Mast protein failed to accumulate at any site equivalent to
a MTOC and the punctate nature of its localisation was diminished
(Fig. 7C). Together, these
observations indicate that the microtubule network is essential for
recruitment of Orbit/Mast protein to the posterior of the stage 1-6 egg
chamber and for maintaining the identity of the oocyte.
|
In contrast to wild-type egg chambers, the microtubule network of orbit6 mutants was severely disrupted or completely abolished (Fig. 7B). The severity of the observed defects reflected the age of the female and paralleled the levels of Orbit/Mast protein in ovaries. The egg chambers of younger females had reduced numbers of microtubules in irregular bundles that were associated with some residual Orbit/Mast protein. Weak Orbit staining of a body apparently equivalent to the MTOC could be seen in such egg chambers of younger females, even though they had reduced numbers of nurse cells and had not specified an oocyte nucleus. In such cases, the MTOC varied in its position. In older females, Orbit/Mast protein, microtubule bundles and MTOC were not detected. Thus, the Orbit/Mast protein appears to play a role in maintaining microtubules in the post-mitotic egg chamber and in establishing the MTOC that is usually formed adjacent to the oocyte nucleus.
CLIP-190 interacts with Orbit/Mast during oogenesis
In mammalian cells, CLASP, the counterpart of Orbit/Mast, has been shown to
interact with CLIP-170, a protein implicated in the interphase functions of
microtubules (Akhmanova et al.,
2001). We therefore wished to know whether Orbit/Mast protein
interacted with CLIP-190 (the fly orthologue of CLIP-170) (Lanz and Miller,
1998) and whether the localisation of CLIP-190 in ovaries was in any way
dependent upon Orbit/Mast. To this end, we carried out immunoprecipitation
experiments on total protein extracts made from wild-type ovaries with
antibodies to the Orbit/Mast protein and found that CLIP-190 coprecipitated.
Similarly, immunoprecipitations with anti-CLIP-190 antibodies also
demonstrated an interaction with Orbit/Mast
(Fig. 8G). Moreover, in
wild-type germaria, CLIP-190 followed a similar localisation pattern to
Orbit/Mast protein: it associated with spindle MTs and fusomes during stem
cell and cystoblast-cystocyte mitosis (Fig.
8A): it was present in punctate masses contacting the microtubule
network of newly formed egg chambers (Fig.
8B); and in egg chambers that had left the germaria, it followed a
punctate distribution along the cytoplasm of the nurse cells and oocyte that
seemed to associate with the interconnecting microtubule bundles
(Fig. 8C and inset). In
contrast to Orbit/Mast, CLIP-190 also accumulated in the apical region of the
follicle cells and did not show any localisation to the oocyte-associated
MTOC. In orbit6 germaria, CLIP-190 was not observed on the
spindles, although occasionally in younger females local accumulations of it
were seen in close contact with some spindle poles
(Fig. 8D, arrowhead). CLIP-190
was also absent from the irregular microtubule bundles surrounding nurse cells
of younger orbit6 females
(Fig. 8E). However, the
follicle cells of such egg chambers contained normal levels of CLIP-190,
indicating that its localisation in these cells was independent of Orbit/Mast.
In older orbit6 females, few germline cysts had completed
their divisions and these rarely showed microtubule bundles. In such cysts
there was very weak accumulation of CLIP-190 in the cytoplasm apparently not
associated with microtubules (Fig.
8F). Thus, CLIP-190 and Orbit/Mast show considerable overlap in
their subcellular distribution. Moreover, the dependence of CLIP-190
localisation on Orbit/Mast function suggests that colocalisation seen in these
regions is likely to be mediated through the interaction between the two
proteins. The localisation of the two proteins to both the fusome and the
polarised MT network, structures that are aberrant in orbit mutants,
suggests a role for the complex in maintaining these structures.
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DISCUSSION |
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As orbit6 females age they ultimately show a complete
failure of egg chamber production, reflecting an eventual loss of stem cells
from the ovariolar niche. The wild-type ovariolar niche consists of two
germline stem cells and the somatic terminal filament and cap cells thought to
cooperate in establishing and maintaining stem cell identity (reviewed by
Spradling et al., 2001). This
process requires that stem cells divide asymmetrically along the
anteroposterior axis of the germarium such that only their anterior daughter
cells inherit cap cell contact and a larger piece of fusome. The posteriorly
situated daughter cells that inherit a smaller piece of fusome loose contact
with cap cells and differentiate into cytoblasts. The
orbit6 phenotype indicates that the gene product is
required for stem cell maintenance. One possible explanation may be offered
for this that the absence of Orbit/Mast protein leads to stem cell division
defects that cause stem cells to lose contact with the cap cells and the
terminal filament cells.
The ovaries of younger females do have stem cells that are able to produce
egg chambers, although these have reduced numbers of nurse cells. This
reflects directly the requirement of Orbit/Mast protein for cell division
and/or fusome growth. During mitosis of the germline cells, we found that the
Orbit/Mast protein associated with the spindle microtubules and spindle poles,
and that towards the completion of cell division it became concentrated in the
spindle remnants, as previously reported in other cell types
(Inoue et al., 2000;
Lemos et al., 2000
). Indeed,
mitotically dividing orbit6 cystoblasts showed a variety
of abnormalities consistent with defects seen in dividing somatic cells of
other orbit alleles (Inoue et
al., 2000
; Lemos et al.,
2000
). These included short bipolar and monopolar spindles,
supporting recent observations that the mitotic requirement for Orbit/Mast is
to maintain spindle bipolarity (Maiato et
al., 2002
). Orbit/Mast protein is also found in the fusome that,
in wild-type cells, is always associated with one pole of each mitotic spindle
in the cyst of dividing cells. By contrast, the orbit6
spindles never made contact with fusome fragments. Loss of connections between
spindles and fusome have been previously described in mutants for the heavy
chain of the minus end directed motor dynein (Dhc64C)
(McGrail and Hays, 1997
) and
in germline clones of null alleles of Lis1, which encodes the
Drosophila homologue of the lissencephaly disease gene, the
product of which interacts with dynein
(Liu et al., 1999
;
Swan et al., 1999
). The
phenotypes in each case have some similarities to orbit6:
the spindles often fail to attach to fusomes, cysts are produced with fewer
than 16 cells, and the oocyte is not specified. It has been suggested that
these defects were due to interactions between fusome and spindles or
interphase microtubules. Although the defects seen in
orbit6 could arise in this way, the mitotic defects seen
in the mutant give greater emphasis to the necessity to maintain correct
interactions between the metaphase spindle and the fusome and suggest that
Orbit/Mast protein may have a direct role in ensuring such connections.
The fusome provides a physical basis to ensure asymmetry of the developing
cyst that is a precondition for cyst polarisation and oocyte specification
(Lin et al., 1994;
Theurkauf, 1994
;
deCuevas and Spradling, 1998
;
Grieder et al., 2000
). Fusome
material does develop in the cysts of younger orbit6
females, but tends to remain as separate fragments. The failure of correct
fusome development is most likely to be the underlying cause behind the
failure to specify the oocyte in orbit6 mutants. Our data
suggest that not only is Orbit/Mast required for interactions of the fusome
with spindles, but it is also needed for its subsequent interactions with
spindle remnants and the ring canals. We confirm that fusome material
accumulates in the vicinity of the spindle remnants in wild-type cysts. These
spindle remnants have been suggested to help restrict constriction of the
cleavage furrows (Mahajan-Miklos and Cooley, 1994) and so guide fusome growth
by supporting the migration of fusome plugs and their ring canals towards the
pre-existing fusome (Storto and King,
1989
). The absence of spindle remnants in the mutant
orbit6 cysts may therefore also contribute to the lack of
fusome growth. One possibility is that the transfer of Orbit/Mast protein from
the degenerating spindle remnants to the fusome plugs inside the ring canals
themselves may facilitate migration of fusome plugs along interphase
microtubule bundles to fuse with pre-existing fusome. This would ensure the
regular and polarised growth of the fusome.
The defects we observe in ring canal formation in
orbit6 mutants are almost certainly secondary to the
failure of the fusome to develop correctly. The central spindle (spindle
remnant) late in mitosis is important to arrest closure of the cleavage
furrows so they may be transformed into ring canals. However, the ring canals
continue to differentiate even after completion of cystocyte divisions
(Robinson et al., 1994).
Anillin (de Cuevas and Spradling,
1998
), glycoprotein D-mucin
(Sokol and Cooley, 1999
),
Orbit/Mast and Pav-KLP (this study) are the earliest known proteins to
associate with the ring canals during germline cell divisions. Upon completion
of these divisions, Anillin and Orbit/Mast disappear from the ring canals,
while glycoprotein D-mucin and Pav-KLP will be joined by Filamin, HtsRC and
Kelch. The ring canals that did form in younger orbit6
females were occluded by Filamin, F-actin and HtsRC. A failure to recruit
Kelch may be a secondary consequence of the formation of such aberrant
structures. Ring canal occlusion could be the result of the irregular
structure of the spindle remnant in dividing orbit6
germline cysts and the failure to plug the newly formed ring canal with fusome
components. Mutant egg chambers from older females showed stronger defects;
often they contained irregular structures that contained HtsRC, Filamin and
F-actin but did not resemble ring canals. This gradient of phenotype along the
ovariole may reflect once again the exhaustion of a diminished pool of
Orbit/Mast protein as development proceeds.
Molecules found in the fusome are normally found in the cortical
cytoskeleton of most other cell types. Thus, the involvement of Orbit/Mast in
the interactions of microtubules with the fusome may be akin to the interphase
functions ascribed to CLASP, its counterpart in mammalian cells
(Akhmanova et al., 2001).
Interactions between the CLASP, CLIP-170, EB1 and APC proteins have been
proposed to mediate the crosstalk between cellular structural elements,
particularly actin filaments, microtubules and the plasma membrane
(Sisson et al., 2000
;
Akhmanova et al., 2001
;
Rosin-Arbesfeld et al., 2001
;
Schuyler and Pellman, 2001
).
The notion of Orbit/Mast having an equivalent role in the Drosophila
egg chamber is supported by our finding that the Orbit/Mast protein exists in
a complex with CLIP-190, the counterpart of CLIP-170. The two proteins can be
co-immunoprecipitated and overlap considerably in their pattern of subcellular
localisation throughout oogenesis.
A further interphase role for the Orbit/Mast protein in oogenesis would
seem to be in facilitating the organisation of the polarised network of
microtubules essential for transport of mRNAs and proteins from the nurse
cells to the growing oocyte at stages 1 to 7 (for a review, see
Johnstone and Lasko, 2001).
The minus ends of microtubules in this network are nucleated by a unique MTOC
situated at the posterior of the oocyte. The Orbit/Mast protein is present in
punctate bodies along these microtubules and accumulates at this MTOC. Its
accumulation at the MTOC requires functional microtubules, as it fails to
accumulate in females treated with a microtubule depolymerising drug. This is
one aspect of its localisation in which it forsakes its partner CLIP-190,
which (although present in punctate bodies associated with the microtubule
network) is absent from the MTOC. The mechanisms that regulate the association
of these two proteins with respect to the polarity of microtubules are likely
to be significant in the full understanding of their biological roles. The
polarised microtubule network is disrupted and the MTOC is reduced or absent
from egg chambers of orbit6 females. Once again this
phenotype is age dependent: younger mutant females feature only irregular MT
bundles and diminutive irregularly placed MTOCs whereas the bundles and MTOC
are totally absent in egg chambers of older females. The localisation of
CLIP-190 to the fusome and to the polarised microtubule array is also
prevented in the orbit6 mutant, suggesting that it
requires complexing to the Orbit/Mast protein to achieve this
distribution.
Taken together, our observations strongly argue that the Orbit/Mast protein is required for major polarisation events in wild-type egg chambers: the establishment of the polarised fusome that ultimately enables a single cell within the 16-cell cyst to be specified as the oocyte and in the formation of the polarised MT network in mid-oogenesis. Further studies are needed to determine the precise mechanism whereby the Orbit/Mast protein participates in the organisation of the mitotic spindle, fusome and ring canals, on the one hand, and in the organisation of polarised arrays of interphase microtubules, on the other; and to determine whether the other Drosophila orthologues that are members of the CLASP complex are also involved in these processes.
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ACKNOWLEDGMENTS |
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