RanGTP is required for meiotic spindle organizationand the initiation of embryonic developmentin Drosophila

J. Cesario and K. S. McKim*Waksman Institute and Department of Genetics, Rutgers, the State University of New Jersey, 190 Frelinghuysen RD, Piscataway NJ 08854-8020,USA

*Author for correspondence (mckim@rci.rutgers.edu)

Accepted 4 July 2011Journal of Cell Science 124, 3797–3810� 2011. Published by The Company of Biologists Ltddoi: 10.1242/jcs.084855

SummaryRanGTP is important for chromosome-dependent spindle assembly in Xenopus extracts. Here we report on experiments to determine therole of the Ran pathway on microtubule dynamics in Drosophila oocytes and embryos. Females expressing a dominant-negative form of

Ran have fertility defects, suggesting that RanGTP is required for normal fertility. This is not, however, because of a defect inacentrosomal meiotic spindle assembly. Therefore, RanGTP does not appear to be essential or sufficient for the formation of theacentrosomal spindle. Instead, the most important function of the Ran pathway in spindle assembly appears to be in the tapering of

microtubules at the spindle poles, which might be through regulation of proteins such as TACC and the HURP homolog, Mars. Oneconsequence of this spindle organization defect is an increase in the nondisjunction of achiasmate chromosomes. However, the meioticdefects are not severe enough to cause the decreased fertility. Reductions in fertility occur because RanGTP has a role in microtubule

assembly that is not directly nucleated by the chromosomes. This includes microtubules nucleated from the sperm aster, which arerequired for pronuclear fusion. We propose that following nuclear envelope breakdown, RanGTP is released from the nucleus andcreates a cytoplasm that is activated for assembling microtubules, which is important for processes such as pronuclear fusion. Aroundthe chromosomes, however, RanGTP might be redundant with other factors such as the chromosome passenger complex.

Key words: Meiosis, Mitosis, Microtubule, Drosophila melanogaster, Acentrosomal spindle, Chromosome segregation

IntroductionRan is a member of the Ras family of small GTP-bindingproteins. It was originally discovered for its role in shuttlingproteins with nuclear localization sequences (NLS) into the

nucleus (Moore and Blobel, 1993). Ran cycles from an activestate, RanGTP, to an inactive state, RanGDP. The conversion ofRanGDP to RanGTP is stimulated by the chromatin bound

guanine nucleotide exchange factor RCC1 (Bischoff andPonstingl, 1991). Conversely, the conversion of RanGTP toRanGDP is facilitated by the cytoplasmic GTPase-activating

protein RanGAP (Bischoff et al., 1994). During G2, the nuclearenvelope creates a barrier where active RanGTP can only befound within the nucleus, because RCC1 is chromatin bound. Theimportin complex, which consists of importin a and importin b,

is capable of binding proteins with NLS and transporting theminto the nucleus. Once inside the nucleus, RanGTP bindsimportin b, releasing importin a and NLS containing proteins

(Clarke and Zhang, 2008).

Ran also has a role in spindle assembly by releasing spindleassembly factors from the repressive importin complex (Kalab

and Heald, 2008). The production of RanGTP near chromatin andconversion to RanGDP in the cytoplasm can lead to the formationof a gradient of active Ran that is capable of triggering

chromosome-mediated spindle assembly (Caudron et al., 2005).The role of RanGTP in chromosome-mediated spindle assemblyhas been most clearly shown by its activity in Xenopus laevis egg

extracts that lack centrosomes. Chromatin-mediated microtubuleassembly depends on the presence of RanGTP in Xenopus

extracts (Carazo-Salas et al., 1999). Similarly, depletion of RCC1results in a failure to form microtubule asters. Addition of

RanGTP to these RCC1-depleted eggs is sufficient to induce self-

organization of microtubule asters (Ohba et al., 1999). Disruptionof RanGTP levels also affects mitotic spindle assembly in

mammalian (Clarke and Zhang, 2008; Kalab et al., 2006),Drosophila melanogaster (Silverman-Gavrila and Wilde, 2006)

and Caenorhabditis elegans (Askjaer et al., 2002; Bamba et al.,2002) cells. These results suggest that RanGTP is a major

contributor to spindle assembly.

We have undertaken an analysis of Ran function in the

Drosophila oocyte because several aspects of oogenesis and

embryogenesis depend on microtubule dynamics (Dix and Raff,2007; Roth and Lynch, 2009). In Drosophila oocytes, as in many

oocytes, meiosis is acentrosomal. Spindle assembly occurswithout the guidance of the microtubule organizing centers at

the poles. In this situation, the chromosomes play an important

role in spindle assembly. Nuclear envelope breakdown (NEB) isfollowed by the accumulation of microtubules around the

chromosomes (Matthies et al., 1996; Theurkauf and Hawley,1992). The subsequent bundling and tapering of these

microtubules by motor proteins results in a bipolar spindle.

Thus, Drosophila oocyte chromosomes carry a signal thatpromotes spindle assembly when released into the cytoplasm

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upon NEB. However, it is unclear, what are the components of

this signal.

Meiosis in Drosophila arrests at the first division (Theurkauf

et al., 1993). When the oocyte then moves down the oviduct, it

becomes activated and the two meiotic divisions are completed.

Independently, fertilization occurs and the sperm centriole

recruits microtubules that are required to bring together the

male and female pronuclei. Finally, the nuclear membranes of the

two nuclei fuse prior to the first mitotic division. All these events

depend on maternally contributed proteins and are thus a function

of the oocyte. We have examined the role of the Ran pathway in

these early developmental processes. We found that RanGTP has

a role in pronuclear fusion in the embryo, is active in promoting

microtubule assembly in the oocyte cytoplasm, but it might not

be required for their recruitment of microtubules by the meiotic

chromosomes.

ResultsGeneration of dominant ran mutants

Ran is required for mitosis (Silverman-Gavrila and Wilde, 2006)

and a mutation in the ran gene (G0075) causes lethality in

Drosophila (Peter et al., 2002). Furthermore, mutations in the

Drosophila RCC1 homolog, Bj1, cause lethality, and germline

clones do not make oocytes (Shi and Skeath, 2004) (K.S.M.,

unpublished results). Because these properties make it impossible

to study ran mutations in oocytes, we generated mutations

predicted to be dominant alleles of ran. Previous studies in a

variety of systems have characterized mutations that lock Ran in

either the GDP (inactive) or GTP (active) states (Kahana and

Cleveland, 1999; Trieselmann and Wilde, 2002). Because Ran is

highly conserved, these same changes can be made in

Drosophila. Transgenes were made by fusing the coding region

of the wild-type ran or mutant variants to three copies of the HA

epitope tag at the N-terminus. They were also put under the

control of the UASP promoter, which allows for inducible

germline expression regulated by a second transgene expressing

GAL4 (Rorth, 1998). To generate a GDP-locked mutant, the

P{w+; UASP:ranT24N} transgene was constructed with an amino

acid substitution of threonine to asparagine at position 24,

hereafter referred to as ranT24N. To generate a GTP-locked

mutant, the P{w+; UASP:ranQ69L} transgene was constructed

with an amino acid substitution of glutamine to leucine at

position 69, hereafter referred to as ranQ69L. For each allele, at

least three transgenic lines were examined for expression levels

and phenotypes. For each experiment, flies with these mutations

were compared with flies expressing a wild-type transgene

(P{w+; UASP:ran+}), hereafter referred to as ran+.

Expression of ranT24N has a dominant-negative effect

To determine whether expression of the mutant forms of ran

would cause lethality similar to the loss-of-function mutant, weexpressed the transgenes using P{tubP-GAL4}, which inducesubiquitous expression of UAS transgenes (Lee and Luo, 1999).

Expression of ran+ had no effect on viability and was able torescue the lethality of a ran mutation (Table 1). Furthermore,

dividing neuroblasts from third instar larvae exhibited properlyassembled spindles with no detectable abnormalities during

metaphase and anaphase (supplementary material Fig. S1). Thelocation of wild-type Ran was examined using antibodies to theHA epitope tag that was fused at the N-terminus of the ran

transgenes. Ran was nuclear during interphase and thenoverlapped with the spindle during metaphase and anaphase

(supplementary material Fig. S1). Similar to the localizationpattern in embryos (Trieselmann and Wilde, 2002), Ran was notdetected on the chromosomes. Unlike ran+, ubiquitous

expression of ranT24N or ranQ69L resulted in lethality of theembryos or at an early stage of larval development (Table 1).

These results suggest that expression of ranT24N or ranQ69L has adominant effect and disrupts the normal functioning of the Ranpathway.

Maternal expression of ranT24N and ranQ69L causes sterility

To examine the function of Ran in oogenesis and embryogenesis,

the ran transgenes were expressed using the P{GAL4:VP16-

nos.UTR}MVD1 driver, and then genetic assays were performed

to measure fertility and the frequency of X-chromosomenondisjunction. This driver typically overexpresses UASPtransgenes in oocytes (Jang et al., 2007; Van Doren et al.,

1998). Differences in expression levels between differentinsertion lines were assayed by western blotting and found to

be minimal (data not shown). When the wild-type ran transgenewas expressed, levels of fertility were normal (Table 2).

Therefore, expressing wild-type Ran does not have deleteriouseffects on embryonic development.

Expression of ranT24N in the female germline caused a drastic

reduction in fertility, with an average of only 6.8 progeny perfemale parent compared with 62.1 progeny per female expressing

wild-type Ran (Table 2). P{GAL4:VP16-nos.UTR}MVD1 alsodrives expression in the male germline. Males carrying this driverand ranT24N were sterile, suggesting that RanGTP has an essential

role in male meiosis. Expression of ranQ69L in the oocyte resultedin complete sterility (Table 2). These effects of ranT24N and

ranQ69L on fertility indicate that there is an important role forRanGTP in either meiosis, fertilization or the embryonicdivisions. Therefore, we examined oocytes and embryos in

more detail in order to determine why the ran mutants are sterile.

Table 1. Effect of ran mutants on viability

Transgene Progeny expressing Ran Progeny not expressing Ran

ran+ a 755 326ranT24N a 0 464ranQ69L a 0 1405ranG0075/Y; ran+ b 66 307

aEach transgene was expressed by crossing to P{tubP-GAL4}/TM6, Tb. Progeny expressing a ran transgene were Tb+. For each transgene at least twoindependent insertions were scored, both of which gave similar results. Stocks containing the ran mutant transgenes are viable and fertile, indicating that thelethality is specific and depends on the presence of the Gal4 driver.

branG0075/FM7 females were crossed to P{w+; UASP:ran+}/+; P{tubP-GAL4}/+ males. 25% of the progeny were expected to inherit both the driver andtransgene.

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Two components of the Ran pathway, RCC1 and RanGAP,are present in the Drosophila oocyte

The Drosophila oocyte develops in a cyst along with 15 nurse

cells. Over the course of 3–5 days, the oocyte grows in size whilein a diplotene–diakinesis-like state, eventually receiving most ofthe nurse cell components by the time they reach stages 13 to 14.

Following NEB in the oocyte, microtubules accumulate aroundthe chromosomes, which are bundled together into a karyosome

(Matthies et al., 1996; Theurkauf and Hawley, 1992). This isfollowed by the extension of poles and lengthening of the spindle.

Our previous work has shown that the central spindle is importantfor organizing bipolarity (Jang et al., 2005), which can be

detected by staining for Subito, a kinesin 6 that localizes to theantiparallel microtubules of the central spindle. Meiosis arrests atmetaphase I until the oocyte passes down the oviduct and

becomes activated, at which point the two meiotic divisionsoccur.

A gradient of RanGTP with the concentration highest near thechromosomes can be established if RCC1 is enriched on the

chromatin and RanGAP is in the cytoplasm. We stained matureoocytes with an antibody raised against RCC1 (Frasch, 1991) andfound that it localized around the outside of the karyosome

(Fig. 1A). By contrast, RanGAP (Kusano et al., 2001) waslocalized to globular structures throughout the oocyte cytoplasm

(Fig. 1B). Although the oocyte contains many vesicles, thelocalization pattern of RanGAP did not correspond to structures

detected by a Lamin antibody (supplementary material Fig. S2).These results show that RCC1 and RanGAP are located indiscrete locations within the oocyte during assembly of the

meiotic acentrosomal spindle. However, because the oocyte islarge relative to the meiotic spindle and RanGAP appears in

globular structures, a gradient of RanGTP, with the highestconcentration around the chromosomes, might not exist.

Expression of RanT24N affects spindle pole organization inDrosophila oocytes

To examine the effect of Ran on meiotic spindle assembly, wild-type and mutant UASP:ran transgenes were expressed usingthe P{GAL4:VP16-nos.UTR}MVD1 driver. Immunofluorescence

assays of mature oocytes showed that wild-type Ran surroundsthe metaphase I spindle (Fig. 2A). Interestingly, this localizationpattern showed almost no overlap with the spindle microtubules,unlike the pattern observed in mitotically dividing neuroblasts

and embryos. Additional accumulations of Ran were foundadjacent to the clusters of RanGAP that form throughout thecytoplasm, but there was generally no overlap (Fig. 3A). In these

oocytes expressing wild-type Ran, spindle and karyosomemorphology were normal (Fig. 2A; Table 3). A low frequencyof abnormal-looking spindles is expected because some of the

oocytes are in early prometaphase when the spindle is firstassembling. In addition, Subito localized normally to the centralspindle (supplementary material Fig. S3). Therefore, expression

of the HA-tagged wild-type Ran does not grossly affect spindlebipolarity or morphology.

Ran that was locked in the inactive GDP state had a different

localization pattern to wild-type Ran in mature oocytes. RanT24N

accumulated closely around the chromosomes (Fig. 2B), ratherthan around the outside of the spindle as does wild-type Ran. Thislocalization is similar to the localization of RCC1 (Fig. 3B),

consistent with the RanT24N protein binding to RCC1 but notbeing converted into the GTP form. There was some variation inthe RanT24N staining pattern; although it was always tight around

Table 2. Fertility and nondisjunction phenotypes by ran transgenes

Transgenea Regular progenyX-chromosome

nondisjunction progeny Progeny/female parent (n) Nondisjunction (%)

ran+ 2481 1 62.1 (40) 0.1ran+ (no driver) 1301 0 54.2 (24) 0.0ranT24N 498 2 6.8 (74) 0.8ranT24N (no driver) 1542 0 51.4 (30) 0.0y w; ranT24N 619 7 14.6 (43) 4.1Bwinscy/w 1596 0 61.4 (26) 0.0Bwinscy/w; ranT24N 408 61 3.6 (56) 35.6ranQ69L 0 0 0.0 (80) –ranQ69L (no driver) 2163 0 80.1 (27) 0.0subDNT 0 0 0.0 (20) –subDNT; ran+ 0 0 0.0 (20) –subDNT; ranT24N 338 3 34.1 (10) 1.7

aEach transgene was expressed by crossing to the P{GAL4::VP16-nos.UTR}MVD1 driver and then crossed as described in the Materials and Methods.For each transgene at least two independent insertions were scored.

Table 3. Characterization of meiotic figures in wild-type and mutant ran oocytes

Transgenea Oocytes Abnormal spindle Abnormal karyosome z-value

ran+ 39 4 10% 0 0%ranT24N 16 8 50% 8 50% 2.9b

ranQ69L 18 11 61% 4 22% 3.7b

mars1 11 9 82% 0 0 4.9b

aEach transgene was expressed by crossing to the P{GAL4::VP16-nos.UTR}MVD1 driver.For each transgene at least two independent insertions were scored, both of which gave similar results.bP#0.01.

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the karyosome (Fig. 3B), in some images there was staining away

from DNA as well (Fig. 2B). Oocytes expressing ranT24N did not

appear to have a problem initiating the assembly of microtubules

around the chromosomes or building a bipolar spindle. However,

the ranT24N-expressing oocytes had an increased frequency of

abnormal spindle and karyosome organization (Table 3). The

microtubules were often not tapered at the spindle poles

(Fig. 2B). Furthermore, the chromosomes were frequently

disorganized and failed to condense into a single round or oval

karyosome (supplementary material Fig. S4). By contrast, Subito

localized correctly (supplementary material Fig. S3), suggesting

the central spindle was able to form in these mutants. These

results suggest that the Ran pathway has a role in organizing the

meiosis I spindle poles, but it might not be essential for initiating

chromosome-based microtubule assembly or for regulating

central spindle proteins like Subito.

Because RanGTP promotes spindle assembly through the

release of spindle assembly factors, we tested whether ranT24N

mutant oocytes showed evidence of downregulating proteins

known to be regulated by the Ran pathway. Mars is the

Drosophila homolog of HURP, a spindle assembly factor

regulated by RanGTP (Wilde, 2006) and has been shown to

have a role in the attachment of the centrosome to the mitotic

spindle during Drosophila embryogenesis (Tan et al., 2008; Yang

and Fan, 2008; Zhang et al., 2009). To study the role of Mars in

meiotic spindle assembly, we examined mar1, which is a null

allele that deletes part of the coding region (Tan et al., 2008).

Like ranT24N mutants, mature oocytes homozygous for the mars1

mutation were able to assemble a bipolar spindle but failed to

properly taper the microtubules at the poles (Fig. 2E). To test

whether these similarities could be the result of ranT24N mutant

oocytes failing to activate Mars, we stained ranT24N mutant

oocytes with an antibody against Mars (Tan et al., 2008). In wild-

type or ran+-expressing oocytes, Mars colocalized with tubulin,

except at the spindle poles and the central spindle (Fig. 4A). By

contrast, approximately 50% of oocytes expressing ranT24N failed

to localize Mars to the meiotic spindle (Fig. 4B–D), which was

significantly different from ran+ oocytes (z53.6, P#0.01).

Therefore, a Mars localization defect might contribute to the

spindle-tapering defect observed in ranT24N mutant oocytes.

These results are consistent with the conclusion that the RanGTP

pathway is not essential for the initiation of acentrosomal spindle

assembly in Drosophila oocytes, but might have a role in tapering

the poles.

Another spindle assembly factor regulated by the RanGTP

pathway is the microtubule-associated factor Transforming acidic

coiled-coil, or TACC (Kalab and Heald, 2008) (see Discussion).

TACC localizes to the poles of the meiotic (Cullen and Ohkura,

2001) and mitotic spindle (Giet et al., 2002) where it contributes

to the localization of Msps (Minispindles). To examine the

localization of TACC during female meiosis, we expressed a

GFP fusion gene under the control of a ubiquitin promoter

(Gergely et al., 2000). As expected, in wild-type oocytes TACC

localized to the poles of most metaphase I spindles (Fig. 4E,F).

There was some variation in this pattern, with TACC tending to

be less focused at the poles of shorter spindles. By contrast, in all

Fig. 1. Localization of RCC1 and RanGAP in mature (stage 14) oocytes. DNA is in blue and tubulin is in green. (A) Wild-type oocyte stained with RCC1

antibody (red). (B) Wild-type oocyte stained with RanGAP antibody (red). (C) A low magnification view of the same oocyte as in B showing the clusters of

RanGAP located throughout the ooplasm. The arrow points to the karyosome. Scale bars: 10 mm.

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ranT24N oocytes TACC failed to show enrichment towards the

poles and was present next to the DNA (Fig. 4G,H). The failure

to properly localize both Mars and TACC in ranT24N mutant

oocytes is consistent with loss of RanGTP activity, but the

mislocalization of spindle assembly factors is not severe enough

to prevent spindle assembly.

Achiasmate chromosome segregation is abnormal in

ranT24N mutants

To determine whether the spindle organization defects in the ran

mutant females were associated with errors in chromosome

segregation, two genetic crosses were performed to measure the

frequency of X-chromosome nondisjunction. These experiments

measured the frequency of chiasmate and achiasmate chromosome

nondisjunction. Segregation of chiasmate chromosomes was

measured in females homozygous for normal sequence

X-chromosomes that were expected to have a crossover in

greater than 95% of meioses (Baker and Hall, 1976). In these

experiments, we found a low frequency of nondisjunction among

the few progeny from ranT24N-expressing mothers, showing that

X-chromosome segregation was not substantially affected by

loss of RanGTP (Table 2). These results are consistent with

fluorescence in situ hybridization (FISH) experiments, which

showed that ranT24N and ranQ69L mutants were able to properly

orient their homologous chromosomes at metaphase I

(supplementary material Fig. S5). Thus, although the spindles

are not properly tapered and the karyosome is disorganized in ran

mutants, this does not affect biorientation or segregation of

chiasmate chromosomes.

The achiasmate system in Drosophila females efficiently

segregates homologous chromosomes lacking a crossover

(Hawley and Theurkauf, 1993). For example, in a female

Fig. 2. Effect of Ran on spindle

morphology in mature oocytes. The

transgenes in these and all subsequent

experiments were expressed using the

P{GAL4::VP-nos.UTR}MVD1 driver.

DNA is in blue, Ran proteins are in red

and tubulin is in green. Ran was

detected using an antibody to the HA

tag fused to either wild-type ran

(A), ranT24N (B) or ranQ69L (C,D). The

images in A–C are high magnification

images centered on the karyosome.

The image in D is of the same oocyte

as in C but at lower magnification to

show the localization of mutant

RanQ69L in the oocyte cytoplasm. The

arrow in D points to the karyosome.

(E) In mars1 mutant oocytes, the

microtubules often fail to be properly

tapered at the spindle poles. Scale bars:

10 mm.

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heterozygous for a balancer, crossing over is drastically reduced

between the homologs. The effect of ranT24N on the achiasmate

system was tested in heterozygotes for the X-chromosomes

balancer Bwinscy. In contrast to the ranT24N females with normal

sequence X-chromosomes, Bwinscy heterozygous females showed

a high frequency of X-chromosome nondisjunction (Table 2).

These results were confirmed with FISH experiments using

heterochromatic probes to detect centromere orientation. In the

presence of Bwinscy, expression of ranT24N caused orientation

defects of the X-chromosome but not an autosome (Fig. 5).

Spindle morphology was similar to that of the ranT24N oocytes

with normal X chromosomes. These results indicate that in

ranT24N mutant oocytes, the achiasmate system of chromosome

segregation is disrupted.

Ran locked in the GTP form does not promote

spindle assembly

High levels of RanGTP will induce chromatin-independent spindle

assembly in Xenopus oocytes (Carazo-Salas et al., 1999). By

contrast, the expression of ranQ69L did not result in the formation

of ectopic spindles in the oocyte, as would be expected if RanGTP

is sufficient to initiate spindle assembly (Fig. 2D). Instead,

expression of ranQ69L in oocytes caused abnormal spindle

assembly reminiscent of ranT24N oocytes; they failed to properly

taper microtubules at the poles (Fig. 2C) and the karyosome was

disorganized (Table 3). Despite the similar spindle phenotype,

RanQ69L protein had a localization pattern in oocytes that was

strikingly different from Ran+ or RanT24N. Whereas Ran and

RanGAP do not colocalize in wild-type oocytes, RanQ69L protein

and RanGAP colocalized in clusters throughout the ranQ69L oocyte

(Fig. 3C), suggesting that RanQ69L could be locked in an

interaction with RanGAP. Expression of ranQ69L also changed

the localization pattern of other proteins, such as nuclear Lamin

(supplementary material Fig. S2). These observations suggest that

the expression of RanQ69L in the oocyte affects how nuclear

envelope proteins interact with the Ran pathway.

Maternal expression of ranT24N blocks pronuclear fusion

and embryogenesis

The spindle organization and chromosome segregation defects

we observed in dominant ran mutants were unlikely to be the

cause of the sterility in ranT24N females. To determine whether

the low fertility of the ranT24N mutant females was due to

an embryonic defect, we examined embryos from mothers

expressing the mutant versions of ran. Expression of ran+

resulted in zygotes that underwent normal synchronous divisions,

with spindle assembly and chromosome organization

characteristic of wild-type embryonic divisions (Fig. 6A).

Furthermore, Ran+ localized to the mitotic spindle. This pattern

of localization is similar to that found in neuroblasts, and

Fig. 3. Colocalization of RanT24N with RCC1, and RanQ69L with RanGAP. Wild-type and mutant variants of Ran were detected using an antibody to the HA

tag. In all images, Ran, tagged with HA, is in red, RCC1 or RanGAP are in green and DNA is in blue. (A) In ran+ oocytes, Ran and RanGAP appear to be closely

associated, but examination of individual optical sections shows they do not colocalize. (B) In ranT24N oocytes, RanT24N and RCC1 colocalize around the

karyosome. (C) In ranQ69L oocytes, RanQ69L and RanGAP colocalize in many clusters throughout the cytoplasm. The arrow points to the karyosome. Scale bars:

10 mm.

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Fig. 4. Localization of Mars and TACC to the meiotic

spindle is defective in ranT24N mutant oocytes. Mars or

TACC-GFP staining (P{[w+]5Ubi-tacc.GFP}1) (Gergely

et al., 2000) is in red, DNA is in blue and tubulin is in green

except in G where HA is in green. (A) In ran+ oocytes

Mars localizes to most microtubules in the meiotic spindle,

with the possible exception of the poles and the central

spindle. (B–D) The localization of Mars in ranT24N mutant

oocytes falls into three categories: present (B), completely

absent (C) and reduced (D). (E,F) In wild-type, TACC

localizes at the poles and is more concentrated towards the

poles. The arrows point to the gap between the TACC

staining and the chromosomes. (G,H) In ranT24N oocytes,

TACC is not restricted to the poles. The arrows point to

where the TACC staining meets the chromosomes. Scale

bars: 10 mm.

Fig. 5. FISH on Bwinscy/+ oocytes.

(A–D) Oocytes in a wild-type

background (A); expressing wild-type

ran (B); and expressing ranT24N(C,D).

Hybridization was performed using

probes that bind to highly repeated

sequences in the centromeric

heterochromatin region of both the

X (red) and third (not shown)

chromosomes. Tubulin is in green and

DNA is in blue. Proper orientation of

the homologous chromosomes was

scored as the separation of two FISH

signals, one signal on each half of the

spindle, towards opposite poles. The

data are summarized in the Table

below. Scale bars: 10.

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previously by the injection of fluorescently labeled Ran protein

into embryos (Trieselmann and Wilde, 2002). Thus, expression

of ran+ produces no deleterious effects on the mitotic divisions of

the embryo and recapitulates the known localization pattern to

spindle microtubules.

The majority of zygotes expressing ranT24N arrested

development without any evidence of the embryonic mitotic

divisions. In wild-type zygotes, the two meiotic divisions are

completed without the formation of polar bodies (Demerec, 1950).

Therefore, prior to pronuclear fusion, the oocyte contains four

female meiotic products and the sperm nucleus. Three of the

female products fuse, while the fourth fuses with the sperm

nucleus. The ranT24N zygotes were of two types. Approximately

half of them contained the unfused female and male meiotic

products and were devoid of organized microtubules (Fig. 6B). In

these cases, RanT24N protein was closely associated with the

chromosomes, as would be expected if it was bound to RCC1. The

remaining half of the zygotes had no visible nuclei. These results

suggest that meiosis can be completed in the ranT24N zygotes but

the female and male pronuclei do not fuse and the three remaining

female meiotic products fail to aggregate. Consistent with this

conclusion, we have observed normal meiosis II spindles in

ranT24N embryos (data not shown). Expression of ranT24N in

embryos appears to disrupt the assembly of the microtubule

network nucleated by the sperm centrosome that brings together

the female and male pronuclei. The failure to observe any nuclei in

half the embryos could indicate a failure to reform the nuclear

envelope following completion of meiosis (Ciciarello et al., 2007)

or a failure of pronuclear fusion.

Similar to the effect of ranT24N, maternal expression of ranQ69L

led to a failure to initiate embryonic development. In most of the

mutant zygotes, a cluster of DNA and microtubules was observed

in the center of the cell and there was no evidence of any mitotic

divisions (Fig. 6C). This phenotype was different from the two

observed with ranT24N and might be due to a defect shortly after

pronuclear fusion (see Discussion).

ran is required for cytoplasmic microtubule assembly

The lack of pronuclear fusion in the ranT24N mutant suggests

that RanGTP is required for microtubule assembly that occurs in

the cytoplasm. To test this possibility, we determined whether

RanGTP has a role in another example of microtubule assembly

that does not involve direct interactions with the chromatin.

Such ‘cytoplasmic microtubule assembly’ occurs in Drosophila

oocytes expressing a mutation in subito (sub) that removes the

N-terminal domain of the protein. As observed previously,

expression of P{UASP:subDNT} resulted in the formation of

ectopic spindles in the oocyte (Table 4; Fig. 7) (Jang et al.,

2007). These ectopic spindles do not form until after NEB,

consistent with a diffusible nuclear factor being required for

Fig. 6. Ran is required to initiate embryonic development. DNA is in blue, Ran tagged with the HA epitope is in red, tubulin is in green, (A) Mitotic spindles

form normally when wild-type Ran is expressed. (B) Embryogenesis is blocked when RanT24N is expressed. The presence of a separate male pronucleus (arrow)

and no organized microtubules indicates pronuclear fusion has not occurred. The cluster of four DNA masses is the female pronuclei, three of which normally fuse

into a polar body. (C) Embryogenesis is blocked when RanQ69L is expressed. These embryos contain a disorganized mass of DNA and tubulin. All transgenes were

under the control of the UASP promoter and were expressed in zygotes using the P{GAL4::VP-nos.UTR}MVD1 driver. Scale bars: 10 mm.

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their formation. Ectopic spindles cluster and form in many

regions of the mutant oocytes without direct contact with

chromosomes.

We tested whether RanGTP in the cytoplasm stimulates

microtubule assembly by constructing a double mutant with the

N-terminal deletion mutation using subDNT and ranT24N.

Typically, two to four clusters of ectopic spindles could be

observed in subDNT mutant oocytes, such as at the posterior tip

and the region near the karyosome (Fig. 7A,B). Any oocyte

containing more than one cluster of spindle formation was

considered to have the ectopic spindle phenotype. The frequency

of subDNT oocytes expressing ran+ with ectopic spindles was

similar to that with subDNT alone (96.8% and 97.9%, respectively;

Table 4). Strikingly, the dominant-negative mutation ranT24N

completely suppressed the ectopic spindle phenotype (Fig. 7C,D;

Table 4). The only spindle that formed in ranT24N; subDNT

oocytes was around the karyosome. These results suggest that

RanGTP is required for the interaction between SubitoDNT and

microtubules that occurs in the absence of the chromosomes. In

other words, RanGTP could be required for microtubule

assembly that does not depend on direct contacts with the

chromosomes. Another surprising finding was that the

suppression was reciprocal. The ranT24N; subDNT double mutant

had increased fertility relative to the two single mutants (Table 2)

and this correlated with an increased frequency of embryos

undergoing mitosis (Fig. 7E).

DiscussionThe Ran pathway has a variety of targets, which leads to effects

on kinetochores, centrosomes and microtubule-associated

proteins (Kalab and Heald, 2008). RanGTP is potentially an

important molecule for spindle assembly in acentrosomal oocytes

because it has been identified as a key factor for chromatin-

induced spindle formation in Xenopus extracts (Carazo-Salas

et al., 1999; Kalab et al., 1999; Karsenti and Vernos, 2001; Ohba

et al., 1999). Surprisingly, our results suggest that RanGTP might

be more important for microtubule assembly in other

circumstances, such as when centrosomes are present or when

microtubules assemble without direct contact with the

chromosomes.

Regulators of RanGTP, RCC1 and RanGAP, during meiosis

in Drosophila females

Diffusion of RanGTP from its source, the chromatin, into the

cytoplasm, where it is converted into RanGDP, can create a

gradient that regulates microtubule organization (Caudron et al.,

2005; Kalab et al., 2006). Drosophila oocytes contain two key

regulators of the Ran pathway in distinct locations. RCC1, as

expected, is located tightly around the karyosome in mature

oocytes. RanGAP localization is more complex than expected

because it is present in many clusters, possibly vesicles, within

the oocyte, suggesting that conversion of RanGTP to RanGDP

might be regulated and only occur in certain locations. This could

mean that a gradient of RanGTP (Clarke and Zhang, 2008; Kalab

and Heald, 2008) is not established in the oocyte. A candidate

protein responsible for generating the concentrations of RanGAP

is Ran binding protein 2 (RanBP2; also known as Nup358). This

protein is found within the nuclear envelope and binds to

RanGAP (Hutten et al., 2008). Following NEB, RanGAP could

be anchored to RanBP2-containing cytoplasmic vesicles.

Ran has an unusual localization pattern in oocytes;

concentrating around the outside of the spindle. By contrast,

Ran overlaps with the spindle in Drosophila mitotic cells (this

work) (Silverman-Gavrila and Wilde, 2006; Trieselmann and

Wilde, 2002). We have not determined whether these

concentrations of Ran are in the GDP or GTP state. However,

we can speculate on the basis of the localization patterns of wild-

type and mutant proteins. From this type of evidence,

Trieselmann and Wilde suggested that the bulk of Ran on the

embryonic spindle is in the GTP state. (Trieselmann and Wilde,

2002) Similarly, the bulk of the Ran localized around the outside

of the meiotic spindle might be in the GTP form. The pattern of

mutant RanQ69L staining suggests it enters RanGAP-containing

vesicles but does not leave because it is not hydrolyzed. Thus, the

wild-type Ran that localizes adjacent to the clusters of RanGAP

could be the GDP form of the protein that has left RanGAP-

containing vesicles.

RanGTP has a role in organizing the acentrosomal

spindle poles

RCC1 and RanGTP were found to be required for chromatin-

induced spindle assembly in Xenopus extracts (Carazo-Salas

et al., 1999; Kalab et al., 1999). In such extracts, expression of

RanT24N blocks spindle assembly (Ohba et al., 1999) and high

concentrations of RCC1 or expression of a GTP-locked form of

Ran leads to spindle formation in the absence of chromosomes

and centrosomes (Carazo-Salas et al., 1999).

Our analysis of RanGTP function in Drosophila oocytes is

based on these and numerous other studies in which expression of

the ranT24N mutation effectively reduces the concentration of

RanGTP. We believe that the ranT24N mutant had the desired

effect of reducing RanGTP production, for four reasons. First,

expression of RanT24N in somatic cells caused embryonic or early

larval lethality. Second, RanT24N localized tightly to the meiotic

chromosomes, consistent with the expectation that this form of

Ran remains bound to RCC1 because it has a low rate of GTP

exchange. The high affinity of RanT24N for RCC1 causes a

reduction in the production of RanGTP (Dasso et al., 1994).

Third, the spindle organization defects observed in ranT24N

oocytes were similar to defects seen in mars1 mutant oocytes, a

Table 4. Characterization of ectopic spindle phenotype in subDNT double mutants

Genotype Total oocytes Oocytes containing ectopic spindles Ectopic spindle (%)a

subDNT 93 91 98subDNT; ran+ 63 61 97ranT24N 16 0 0subDNT; ranT24N 28 0 0

Each genotype was expressed by crossing to P{GAL4::VP16-nos.UTR}MVD1 driver.aEctopic spindle (%) is equal to the number of oocytes with ectopic spindles divided by the total number of oocytes.

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protein known to be regulated by the Ran pathway. Fourth,

ranT24N caused dramatic disruptions in chromosome-independent

microtubule assembly assays, such as pronuclear fusion (see

below). Because these chromosome-dependent (meiotic spindle)

and -independent functions occur in the same cytoplasm, we

conclude that the reduction in RanGTP levels sufficient to block

pronuclear fusion were not sufficient to block acentrosomal

spindle assembly.

Unlike the results in Xenopus extracts, expression of the

dominant-negative GDP-locked variant of Ran had relatively

Fig. 7. Ran is required for ectopic spindle formation caused by subDNT, a mutation that deletes the N-terminal non-motor domain of the kinesin 6, Subito.

In all the images, SubitoDNT is a GFP fusion protein and is shown in red, tubulin is in green and DNA is in blue. (A) Ectopic spindles form in subDNT oocytes

expressing Ran+. This is an example where several spindles have formed nears the chromosomes. (B) Low magnification image of ectopic spindles in subDNT

oocytes expressing Ran+. There are two clusters of ectopic spindles in the oocyte. (C) Expression of RanT24N in oocytes suppresses the ectopic spindle phenotype

of subDNT. There are several masses of DNA in the subDNT single mutant but only a single karyosome and spindle in the double mutant. (D) A low magnification

image of the same oocyte shown in B. The arrow in D points to the chromosomes and spindle. (E) subDNT suppresses the arrest in embryonic development caused

by ranT24N. Scale bars: 10 mm.

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mild effects on Drosophila oocyte spindle assembly andkaryosome organization. Meiosis I spindles were bipolar in

ranT24N oocytes. Indeed, reducing the RanGTP concentration inthe oocyte was not sufficient to severely affect either meioticdivision, because meiosis II spindles (data not shown) and femalemeiotic products could be seen in the embryos. The most

important defect was that the meiosis I spindle often had non-tapered poles, and was associated with the abnormal localizationof proteins necessary for pole formation such as Mars or TACC

(Cullen and Ohkura, 2001). These results are consistent withexperiments in embryos that found depletion of RanGTP causesdefects in spindle pole organization and chromosome

organization and congression (Silverman-Gavrila and Wilde,2006). Abnormal spindle morphology in oocytes could be thereason for the disorganized karyosome phenotype andnondisjunction of achiasmate chromosomes. Loss of RanGTP

could result in a failure to activate Aurora A, whichphosphorylates TACC (Barros et al., 2005; Kalab and Heald,2008). In embryos, TACC localization to the centrosomes

depends on phosphorylation by Aurora A (Barros et al., 2005).TACC initially binds all microtubules, but as the spindle matures,TACC is phosphorylated and localizes to the poles. Expression of

ranT24N in oocytes might cause a reduction in Aurora A activity,resulting in a failure to phosphorylate TACC and localize it to thepoles. Further studies are needed, however, because the role of

Aurora A in Drosophila female meiosis is not known. In addition,Mars might have a role in promoting the dephosphorylation ofTACC (Tan et al., 2008). Overall, RanGTP might have a specificrole in organizing spindle poles but might not be required for

chromosome-promoted spindle assembly in oocytes.

Expression of ranT24N did not block spindle assembly.Conversely, expression of the GTP-locked mutant, ranQ69L, did

not induce an uncoupling between spindle assembly and thechromosomes, as it does in Xenopus oocytes (Carazo-Salas et al.,1999). Thus, RanGTP might not be sufficient to initiate spindle

assembly in Drosophila oocytes. Surprisingly, ranQ69L oocytesshowed loss-of-function spindle phenotypes similar to ranT24N

mutant oocytes. Interestingly, manipulation of RanGTP levelswith T24N or Q69L mutations in mammals has similar

phenotypes. For example, the expression of either form of Ranin mouse oocytes resulted in similar meiosis II spindlephenotypes (Dumont et al., 2007). These results suggest that

the effects of manipulating RanGTP levels in an intact oocyte arenot easily predicted by experiments in Xenopus extracts. Otherfactors such as protein localization might play important roles in

regulating the Ran pathway. We also cannot rule out thepossibility that the Ran pathway functions differently in oocytemeiosis, such as if active Ran is not GTP dependent.

We suggest there could be two reasons for the similarity of the

ranQ69L and ranT24N phenotypes. First, expressing the GTP-locked ranQ69L mutation can inhibit the binding of RCC1 to thechromatin (Zhang et al., 2002), causing a reduction in RanGTP

near the chromatin. Alternatively, the phenotypes of the ranQ69L

mutant oocyte might be associated with defects in theorganization of membranes or vesicles. For example,

expression of the ranQ69L mutation caused Lamin, RanGAP andRanQ69L to colocalize in globular structures throughout theoocyte. Kramer and Hawley (Kramer and Hawley, 2003) have

proposed that the transmembrane protein Axs is a component of amembranous structure surrounding the meiotic spindle. With thecaveat that the link between membranous structures and spindle

assembly is not known, the ranQ69L mutation might cause defects inmembranous structures that have a role in spindle organization. Wehave found that Ran and Axs are closely associated, although at the

light microscope level it is difficult to determine if Ran is inside oroutside the Axs staining (J.C. and K.S.M., unpublished results).

Similar to the oocytes, the phenotype of the ranQ69L mutantzygotes might be associated with defects in membrane structure.

In ranQ69L mutants, a single cluster of DNA and microtubulescould be observed in the center of the zygote. A strikingly similarphenotype has been observed in dominant-negative Ketel

mutants; Ketel is the Drosophila homolog of importin-b(Timinszky et al., 2002; Tirian et al., 2000). In the Ketel

dominant mutants, meiosis I and II occur and the female andmale pronuclei come together, but they interact abnormally

because of defects in the nuclear envelopes. Subsequently, thefirst mitotic division fails and the chromosomes disintegratewithin a large aggregate of microtubules. Similar to the Ketel

mutant, ranQ69L could cause abnormal interactions amongnuclear envelope proteins in the zygote, causing a failure in thefirst mitotic division.

RanGTP is required for achiasmatechromosome segregation

There are two chromosome segregation mechanisms inDrosophila females. The first is the segregation of bivalentsconnected by chiasmata (Hawley, 1988), which is how most

chromosomes segregate. The second is of the chromosomes thatlack chiasmata. This includes the small fourth chromosome,which always lacks crossovers, and larger chromosomes, which

lack a crossover in approximately 5% of meioses. Homologouspairs can be forced into the achiasmate system with balancers thatsuppress crossing over. In all these cases, homologous

chromosomes segregate correctly even though they are notconnected by chiasmata. Expression of ranT24N had only mildeffects on chiasmate segregation, but had a severe effect on thesegregation of achiasmate X-chromosomes. These results suggest

that the spindle pole organization defects caused by low RanGTPlevels affect chromosome segregation.

RanGTP is required for chromosome independentmicrotubule assembly

Unlike assembly of the meiosis I spindle, expression of ranT24N

blocked two other types of microtubule assembly. First, ranT24N

mutants had a defect in the fusion of the female and male

pronuclei. This was the most probable cause of the fertilitydefect in ranT24N mutants. Several genes with roles inmicrotubule assembly are also required for pronuclear fusion,including subito (Giunta et al., 2002). This process depends on

the assembly of a microtubule array that is nucleated by thecentrosome donated by the sperm, and acts to draw the femalepronucleus towards the male pronucleus. Second, ranT24N

suppressed the formation of the ectopic spindles that form in aneomorphic subito mutant (subDNT) (Jang et al., 2007). Theformation of these spindles occurs after NEB, consistent with a

dependence on release of RanGTP from the nucleus. Both ofthese examples involve assembly of microtubules without directinteraction with the chromosomes and suggest that the assembly

and bundling of microtubules in the oocyte cytoplasm depend onRanGTP.

One characteristic of the ectopic spindles in subDNT mutants isthat they form in discrete clusters within the oocyte. Because

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RanGAP appears in clusters, it is possible that RanGTP is not in agradient or distributed evenly in the cytoplasm. Thus, aninteresting possibility is that the regions containing ectopicspindles are where the concentration of RanGTP-dependent

spindle assembly factors are at their highest.

These experiments revealed a surprising mutual suppression by

the subDNT and ranT24N mutations. Although both mutants havedecreased fertility, the double mutant is fertile. One interpretationis that RanGTP regulates Subito, and the subDNT mutation

bypasses the dependence on RanGTP. However, we found thatran mutations did not affect Subito localization or the formationof the central spindle. A more probable explanation is that there

are two independent spindle assembly pathways in the oocyte andthe loss of spindle assembly factors in ranT24N zygotes isbalanced by the enhanced spindle assembly activity present in thesubDNT mutant. Expression of ranT24N might suppress the sterility

phenotype of subDNT by abolishing ectopic spindles, whereassubDNT might suppress the reduced fertility phenotype of ranT24N

by overcoming the defects in microtubule assembly needed for

processes such as pro-nuclear fusion.

ConclusionsUsing dominant-negative mutations, we have found that RanGTP is

required for the fertility of Drosophila females. We found no evidencethat RanGTP is required, or sufficient, for the initiation of acentrosomalspindle assembly in Drosophila oocytes. We did detect a role inorganizing the spindle poles that could be explained by RanGTP

regulation of proteins such as Mars/Hurp, TACC and Aurora A. Thesedefects, however, would not be expected to have a severe effect onfertility. A similar conclusion was drawn from expressing a dominant-

negative form of Ran in mouse oocytes or when RCC1 was depletedfrom Xenopus oocytes (Dumont et al., 2007). By analyzing mutantssimilar to the ones we used here, only mild defects in meiosis I spindle

assembly were found, such as a delay establishing bipolarity. Thefailure to observe evidence supporting a role for RanGTP inacentrosomal spindle assembly might be explained by a predominant

chromosome-dependent pathway in oocytes involving thechromosome passenger complex (CPC). The CPC is required forchromosome-dependent spindle assembly in Xenopus egg extracts(Maresca et al., 2009; Sampath et al., 2004) and Drosophila oocytes

(Colombie et al., 2008) (S. Radford and K.S.M., unpublished results).

Our results suggest that, compared with chromosome-mediated

spindle assembly, RanGTP has a greater role in microtubuleorganization when centrosomes are present or when at a distancefrom the chromosomes. We suggest that NEB preceding the

assembly of the meiosis I spindle releases RanGTP into thecytoplasm, which results in a cytoplasm enriched for activespindle assembly factors. The restriction of RanGAP to vesicle-like structures could leave a considerable amount of RanGTP in

the cytoplasm. This activity has only a minor role in meioticspindle assembly, but is crucial for the early events ofembryogenesis. The microtubule array facilitating pronuclear

fusion assembles while the nuclear envelope is intact. Therefore,the oocyte might accumulate and store RanGTP during themeiotic divisions when there is no nuclear envelope in order to

support pronuclear fusion, when the nuclear envelope is intact.

Materials and MethodsGeneration and analysis of transgenic linesFull-length and substitution derivatives of ran were amplified by PCR. The cloneswere verified by sequencing and then the fragments were cloned into the pENTR4vector (Gateway). The fragment was then recombined using Clonase (Invitrogen)

into the pPHW vector that encodes three copies of the HA epitope at theN-terminus of the coding region in a pUASP backbone (Rorth, 1998). Amino acidsubstitutions were made by modifying the wild-type ran clone in pENTR4 usingthe Change-IT mutagenesis kit (USB) and the appropriate primers. For the ranT24N

transgene, an asparagine was substituted for a threonine at amino acid 24. For theranQ69L transgene, a leucine was substituted for a glutamine at amino acid 69.

For ubiquitous expression in somatic tissues, males carrying a ran transgene,P{UASP::ran}, were crossed to females carrying a GAL4 transgene with atubulin promoter (P{tubP-GAL4}) (Lee and Luo, 1999). A cross with the driverheterozygous to a balancer that provides a Tubby phenotype visible in larvae,P{tubP-GAL4}/T(2;3)B3, CyO: TM6B, Tb, results in two genotypes:P{UASP::ran}/P{tubP-GAL4} and P{UASP::ran}/T(2;3)B3, CyO: TM6B, Tb.The percentage survival was calculated as (Tb+ flies)/(total flies). For expressionin the germline and early embryo, males carrying a ran transgene were crossed tofemales carrying a GAL4 transgene with a nanos promoter, P{GAL4::VP16-nos.UTR}MVD1 (Van Doren et al., 1998). To measure fertility and chromosomesegregation during meiosis, females carrying a transgene and the nanos driverwere crossed to either y w/BSY or C(1;Y)v f B, c(4) ci eyR males. Thenondisjunction frequency was calculated as 2(BS R+B+ =)/[B+ R+BS =+2(BS

R+B+ =)].

Antibodies and immunofluorescence microscopy

Mature (stage 14) oocytes were collected from 50–200 yeast-fed females that wereaged 3–4 days by physical disruption in a common household blender (McKimet al., 2009; Theurkauf and Hawley, 1992). The oocytes were fixed in modifiedRobb’s medium and cacodylate–formaldehyde fixative for 8 minutes and then theirouter membranes were removed by rolling the oocytes between the frosted part ofa slide and a coverslip.

Embryos were collected by placing females and males in cages with grape juiceplates for 2 hours to enrich for those undergoing the syncytial divisions. Embryoswere removed from the grape juice plates with water and placed in 50% bleach for90 seconds to remove the chorion. They were then thoroughly washed with waterto remove all traces of bleach. The embryos were fixed using heptane andmethanol (Rothwell and Sullivan, 2000).

For squashed neuroblast preparations, the third instar larval brains weredissected in saline and the brains were fixed in 3.7% formaldehyde in 16 PBS for30 minutes. The brains were then placed in 45% acetic acid for 3 minutes beforetransferring to ,8 ml of 60% acetic acid on a siliconized coverslip where theywere firmly squashed between the coverslip and slide. The slides were brieflyfrozen in liquid nitrogen and the coverslips were flicked off with a razor blade. Theslides were placed in ethanol at 220 C̊ (chilled on dry ice) for 10 minutes, thentransferred to a slide chamber containing 0.1% Triton X-100 in PBS for 10minutes. Rubber cement was used to form wells on the slides and the preparationswere washed twice for 5 minutes each with PBS. The tissue was blocked with 1%BSA in PBS for 45 minutes.

Oocytes, embryos and neuroblasts were stained for DNA with Hoechst 33342 ata 1:1000 dilution (10 mg/ml solution) and for microtubules with mouse anti-a-tubulin monoclonal antibody DM1A (1:50), directly conjugated to FITC (Sigma)or rat anti-a-tubulin monoclonal antibody (1:75; Millipore). The primaryantibodies were rat anti-SUB antibody (used at 1:75) (Jang et al., 2005), ratanti-HA (Roche, clone 3F10: 1:25), rat anti-INCENP (1:500) (Wu et al., 2008),mouse anti-RCC1 (1:20) (Frasch, 1991), rabbit anti-RanGAP (1:800) (Kusanoet al., 2001), rabbit anti-Mars (Tan et al., 2008) and mouse anti-Lamin Dm0

(1:800) (Klapper et al., 1997). These primary antibodies were detected with eithera Cy3 or Cy5 secondary antibody preabsorbed against a range of mammalianserum proteins (Jackson Labs) and Drosophila embryos. TACC was detected usinga GFP fusion protein (Gergely et al., 2000). Images were collected on a Leica TCSSP2 confocal microscope with a 636, 1.3 NA lens. Images are maximumprojections of image stacks with the individual channels merged and then croppedin Adobe Photoshop.

Fluorescent in situ hybridization

Stage 14 oocytes were collected as described above and then processed for bothimmunofluorescence and fluorescent in situ hybridization (FISH) as describedpreviously (Dernburg, 2000; McKim et al., 2009). Oligonucleotidescorresponding to the satellite sequence AACAC for the second chromosomecentric heterochromatin or CCCGTACTCGGT (Dodeca) for the thirdchromosome centric heterochromatin were end-labeled with Cc3-dCTP or Cc5-dCTP (GE Healthcare) by Terminal Deoxynucleotidyl Transferase (Invitrogen).A probe to the 359 bp repeat on the X-chromosome was amplified by PCR andend labeled as described previously (Dernburg, 2000). Oocytes weresubsequently stained for microtubules and DNA as described above.

AcknowledgementsWe are grateful to Li Nguyen for technical assistance, DaimarkBennett for providing Mars antibodies and the mars1 mutant andJanet Jang for Fig. 2A. Some stocks used in this study were obtained

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from the Bloomington Stock Center and some antibodies were fromDevelopmental Studies Hybridoma Bank at the University of Iowa,developed under the auspices of the National Institute of ChildHealth and Human Development.

FundingThis work was supported by a fellowship from the Busch foundation(to J.C.); and a grant from the National Institutes of Health [grantnumber GM 067142 to K.S.M.]. Deposited in PMC for release after12 months.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.084855/-/DC1

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