Separase is required for normal chromosome segregation during Drosophila male meiosis

Drosophila Separase (SSE) does not have a large N-terminal regulatory region as typically observed in other species [42]. However, it forms a complex with the product of the three rows (thr) gene which appears to have resulted from a separase gene split during Drosophila evolution [43, 44]. Moreover, the product of the pimples (pim) gene also needs to accumulate during interphase and join the SSE-THR complex for eventual separase function during mitosis [42, 45]. Therefore, embryos lacking pim function zygotically also display a separase loss-of-function phenotype. This mutant phenotype does not reveal that PIM actually also has an additional, separase-inhibitory role. However, PIM is known to be the Drosophila securin homolog that prevents premature separase activity until late metaphase [46, 47]. While PIM is required initially for separase complex formation, it has to be degraded again eventually during late metaphase via activation of the ubiquitin ligase APC/C. The meiotic function of Sse, thr and pim cannot be studied in zygotic null mutants. They do not develop to the developmental stages where progression through meiosis starts because of sister chromatid separation failure during the earlier mitotic divisions [42, 45, 48]. To investigate meiotic functions, we applied transgenic RNAi expressed specifically in spermatocytes using the bam-GAL4-VP16 (bG) driver. Knock down of thr was found to be most effective, causing male sterility (S1 Table).

To characterize the effects of THR depletion at a cellular level, we analyzed testis squash preparations after staining with a DNA stain and anti-tubulin. Anti-tubulin labeling facilitates identification of meiotic cells and discrimination of meiosis I and II also when chromosome segregation is abnormal. Cells during the meiotic divisions have spindles that change in a characteristic manner during progression through these divisions. During late anaphase and telophase for example, formation of a prominent central spindle occurs. Anti-tubulin labeling also revealed the overall cell size which is halved by cytokinesis first during meiosis I and once again during meiosis II. Conversely, cell number per cyst increases and also provided information whether cells are in meiosis I or II. Preparations with testes from bG males with and without UASt-thr^RNAi transgene displayed normal prometaphase I figures (Fig 1A).

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Fig 1. Depletion of separase complex subunits results in chromosome bridges during telophase I and II.

(A) Testes were isolated from males without (+) or with spermatocyte-specific THR depletion by transgenic RNAi (thr-RNAi). Squash preparations were labeled with anti-tubulin (tubulin) and a DNA stain (DNA). Single spermatocytes at the indicated meiotic stages are displayed. Scale bar = 5 μm. (B) Bars represent the percentage of spermatocytes in telophase I that displayed a chromosome bridge. Testes analyzed were from males without (+) or with spermatocyte-specific THR (thr) or PIM (pim) depletion by RNAi, as well from males without (+) or with spermatocyte-specific SSE depletion by deGradFP (Sse). In case of THR depletion, we also analyzed testes, in which transgenic RNAi was accompanied by expression of an RNAi-resistant thr transgene (thr + resc). (C) Bars represent the percentage of cysts with spermatocytes that displayed chromosome bridges during telophase II. The same genotypes as in (B) were analyzed. Precise genotype descriptions and the number of samples analyzed for the bar diagrams (B, at least 145 telophase I figures per genotype; C, at least 10 telophase II cysts) are given in S1 Table.

https://doi.org/10.1371/journal.pgen.1005996.g001

Up to four DNA masses could be resolved corresponding to the bivalents with the sex chromosomes (XY) and the three autosomes (2^nd, 3^rd, and small 4^th chromosome). In the control preparations, telophase I figures and the figures from prometaphase II and telophase II were normal as expected (Fig 1A). Telophase cells contained two round daughter nuclei of comparable size close to the two spindle poles. In contrast, after THR depletion 86% of the telophase I figures were clearly abnormal (Fig 1A and 1B). Bi-lobed DNA staining with a connecting chromosome bridge was observed (67%), as well as cases with a single DNA mass that was no longer bi-lobed (19%). Subsequent meiotic stages were also abnormal after THR depletion (Fig 1A). Prometaphase II cells contained highly variable amounts of chromatin. Moreover, chromosome separation failure was again apparent during telophase II in 100% of the cysts (Fig 1C).

Time-lapse analysis of progression through the meiotic divisions without and with THR depletion (S1 Movie and S2 Movie) fully confirmed the findings from squash preparations. THR depletion did not affect meiosis I up to anaphase onset. However, chromosome separation during anaphase I did not succeed. Subsequent cytokinesis was irregular as well, producing a pair with a nucleate and an anucleate cell in some cases, or cutting through the undivided mass of chromatin in other cases. As a result, meiosis II spindles were often abnormal. But even in cases with normal meiosis II spindles, chromosome separation during anaphase II never occurred normally. Time-lapse imaging also revealed that THR depletion did not severely affect the dynamics of progression through meiosis I. However, the number of our movies that start before nuclear envelope breakdown in meiosis I is low (only two cysts from independent preparations) because time-lapse imaging was usually started only after finding a cyst within the testis preparation which already had initiated meiosis I. Starting time-lapse imaging earlier at a stage where cysts are still in the long premeiotic G2 phase allows analysis of progression through meiosis only in very rare fortuitous cases because cyst viability deteriorates in most of the cases before entry into meiosis. In the two completely tracked THR depleted meiotic cysts, the duration of prometaphase I, metaphase I, and anaphase I was 12/18, 12/16, and 11/12 minutes, respectively. The average duration of these meiosis I phases in controls was 12.3 ± 1.8, 13.3 ± 3.7, and 10.7 ± 1.2 minutes respectively (± s.d.; n = 6 cysts from independent preparations). Additional movies allowing determination of the duration of metaphase I (n = 2) and anaphase I (n = 4) after THR depletion provided further support for our conclusion that the temporal dynamics of congression of bivalents and onset of anaphase during meiosis I were not severely altered by THR depletion, although subtle effects cannot be excluded. As a result of the chromosome separation defects caused by THR depletion during meiosis I, meiosis II was severely affected in various variable ways and therefore, analysis of temporal dynamics during interkinesis and meiosis II after THR depletion was not attempted.

As bG-directed expression starts already during the final mitotic division cycles that generate the cysts of 16 interconnected spermatocytes, the meiotic defects described above might arise in principle as secondary consequences from earlier division abnormalities. However, several observations indicated that the cysts entering into meiosis after bG-directed THR depletion were normal. Cysts still comprised 16 cells as expected. Moreover, the number of centromeres detected during meiosis indicated that all spermatocytes were euploid. FISH with X and Y probes, as well as MNM/SNM immunolocalization also confirmed euploidy (see below). However, testes of bG males with UASt-thr^RNAi were smaller than those of control males. Therefore, some germline stem cell or gonial cyst depletion might occur as a result of leaky expression during earlier stages.

To rule out off-target effects, we introduced into the males with bG and UASt-thr^RNAi also an UASt-thr^Rr transgene predicted to be RNAi-resistant as a result of silent mutations. While the RNAi-resistant transgene was unable to restore a completely normal meiosis, it reduced the frequency of telophase I abnormalities fourfold (Fig 1B). Moreover, the residual chromosome bridges during telophase I were less massive and less stable when the RNAi-resistant transgene was present (S1 Fig).

For further confirmation that the abnormalities resulting after THR depletion reflect a loss of separase function, we depleted other separase complex subunits. Analogous bG directed expression of UASt-Sse^RNAi and UASt-pim^RNAi resulted in no or milder abnormalities, respectively. Abnormalities after PIM depletion were also first observed during telophase I, as in case of THR depletion. Chromosome bridges were apparent in 56% of the telophase I figures (Fig 1B) and all cysts during telophase II also displayed chromosome bridges. To achieve SSE depletion, we applied an alternative method, deGradFP which allows regulated proteolytic degradation of GFP fusion proteins [49]. Therefore, we first generated a line in which the lethality associated with hemizygosity for an Sse null mutation (Sse^13m/Df(3L)SseA) was prevented by a transgene driving expression of EGFP-SSE under control of the Sse cis-regulatory region (gEGFP-Sse). The resulting flies were also fertile (S1 Table). However, introduction of an additional transgene directing NSlmb-vhh-GFP4 expression by the bam regulatory region led to complete sterility (S1 Table). NSlmb-vhh-GFP4 has been shown to result in polyubiquitination and proteasomal degradation of GFP fusion proteins [49]. Analyses of testis squash preparations revealed that SSE depletion by deGradFP resulted in abnormalities that were indistinguishable from those caused by thr-RNAi (Figs 1B, 1C and S2).

In summary, spermatocyte-specific depletion of the three separase complex subunits, THR, PIM or SSE, with different strategies (transgenic RNAi and deGradFP) was found to cause the same specific defect during meiosis. Importantly, defects start already during anaphase I. Although the degree of interference was lower in case of PIM, presumably as a result of incomplete depletion, chromosome segregation during anaphase I was affected in each case. We conclude that separase function is required for chromosome segregation during both meiotic divisions in Drosophila males.

Separase is required for the release of alternative homolog conjunction proteins during meiosis I

To determine whether separase is required for the removal of alternative homolog conjunction proteins before chromosome segregation during meiosis I in Drosophila males, we analyzed the effects of THR depletion on the subcellular localization of MNM and SNM with the help of a fully functional mnm-EGFP transgene [35] and anti-SNM antibodies [35]. As previously described [35], co-localized MNM-EGFP and anti-SNM signals were detected primarily in a highly prominent spot on the XY bivalent during prometaphase and metaphase of meiosis I (Fig 2A).

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Fig 2. THR depletion causes failure of chromosomal MNM/SNM release and homolog separation during meiosis I.

(A) Testes were isolated from mnm-EGFP males without (+) or with spermatocyte-specific THR depletion by transgenic RNAi (thr-RNAi). Squash preparations were labeled with anti-SNM (SNM) and a DNA stain (DNA). Single spermatocytes at the indicated meiotic stages are displayed. Scale bar = 5 μm. (B) Still frames after time-lapse imaging of progression through meiosis I with testes expressing mnm-EGFP (MNM) and His2Av-mRFP (His2Av). Moreover, testes were from males without (+) or with spermatocyte-specific THR depletion by transgenic RNAi (thr-RNAi). Time in minutes is indicated in the lower right corner of the merged images with t = 0 representing the time when chromatin decondensation had progressed to the point where chromosome arms could no longer be resolved individually. In the control (+), the last metaphase I frame was at -9 minutes and the MNM-EGFP dot disappeared within the first 3 minutes of anaphase I. After thr-RNAi, the MNM-EGFP dot persisted throughout exit from meiosis I. Scale bar = 5 μm. (C) Squash preparations were labeled with anti-tubulin (tubulin) and a DNA stain (DNA). The images on the left side are from mnm mutant males without (mnm) or with spermatocyte-specific THR depletion by transgenic RNAi (thr-RNAi mnm). The images on the right side are from tef mutant males without (tef) or with spermatocyte-specific THR depletion by transgenic RNAi (thr-RNAi tef). Single spermatocytes at the indicated meiotic stages are displayed. Scale bar = 5 μm. (D) Squash preparations of testis from tef mutant males with thr-RNAi were labeled with anti-tubulin (tubulin) and a DNA stain (DNA). In addition, FISH with a red fluorescent X chromosome probe (X) and a green fluorescent Y chromosome probe (Y) was performed in case of the images on the left side, and anti-SNM (SNM) labeling in case of the images on the right side. Scale bar = 5 μm.

https://doi.org/10.1371/journal.pgen.1005996.g002

In contrast, no MNM-EGFP/anti-SNM spot was detected during telophase I (n = 47) (Fig 2A) and later meiotic stages in control testis where meiosis I is normal [35]. However, after THR depletion, MNM-EGFP/anti-SNM spots were not only present early during meiosis I, but also during telophase I (n = 209 spermatocytes) (Fig 2A) and all subsequent stages up to the postmeiotic stages of sperm tail elongation (S3 Fig). In telophase I cells with chromosome bridges, the MNM-EGFP/anti-SNM spots were usually associated with the bridge (94%). Telophase I cells without obvious chromosome bridges (6%) also had always an MNM-EGFP/anti-SNM spot in one of the two daughter nuclei. For confirmation, we also performed time-lapse imaging with testis expressing MNM-EGFP and a red fluorescent histone (Fig 2B, S3 Movie and S4 Movie). In control spermatocytes, the MNM-EGFP spot was observed to disappear during early anaphase I within 2–3 minutes (n = 18 spermatocytes). In contrast, after THR depletion chromosome separation failure was accompanied by persistence of the MNM-EGFP spot during exit from meiosis I (n = 76 spermatocytes).

By anti-SNM staining of testis squash preparations, we evaluated whether PIM depletion by RNAi or SSE depletion by deGradFP also resulted in SNM perdurance beyond anaphase I (S4 Fig). These stainings clearly revealed anti-SNM signals during telophase I and meiosis II in spermatocytes depleted for these other separase complex subunits. We conclude that separase function is required for the release of alternative homolog conjunction proteins from chromosomes during progression through meiosis I.

The observed failure of MNM and SNM release from chromosomes at anaphase I onset after THR depletion is likely responsible for the associated failure of homolog separation during meiosis I. Accordingly, when alternative homolog conjunction fails to be established, THR depletion is no longer expected to cause chromosome bridging during telophase I. To evaluate this prediction, we depleted THR in mnm and snm mutants. However, we first confirmed that mutations in mnm and snm do not cause chromosome bridges during telophase I independent of THR depletion. Consistent with earlier reports [35], instead of four bivalents as in wild type, we observed up to eight chromatin masses during prometaphase I in mnm and snm mutants (Figs 2C and S5), indicating the known defect in homolog conjunction. In addition, daughter nuclei were often unequal in size and DNA content (Figs 2C and S5), reflecting the known random distribution of chromosomes during meiosis I [35]. Chromosome bridges were very rare in telophase I (Fig 3B).

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Fig 3. THR and SSE are required for sister chromatid separation during meiosis II.

(A) Testis squash preparations were labeled with anti-tubulin (tubulin) and a DNA stain (DNA). Single spermatocytes at the indicated stages during meiosis II are displayed. Testes were isolated from mnm mutant males with spermatocyte-specific THR depletion (top row, thr-RNAi mnm), from solo mutant males (middle row, solo), and from mnm solo double mutant males with spermatocyte-specific THR depletion (bottom row, thr-RNAi mnm solo). Mutations in solo but not mnm suppress chromosome bridges induced by THR depletion during meiosis II, indicating that these bridges represent stretched univalents. Scale bar = 5 μm. (B) Bars represent the percentage of spermatocytes in telophase I that displayed a chromosome bridge. Testes analyzed were from males with the genotypes indicated below the bars. thr indicates spermatocyte-specific THR depletion by RNAi, and Sse spermatocyte-specific depletion of SSE by deGradFP. mnm, snm, tef, and solo indicate loss-of-function mutants. (C) Bars represent the percentage of cysts with spermatocytes that displayed chromosome bridges during telophase II. The same genotypes as in (B) were analyzed. Precise genotype descriptions and the number of samples analyzed for the bar diagrams (B, at least 82 telophase I figures per genotype; C, at least 5 telophase II cysts) are given in S1 Table.

https://doi.org/10.1371/journal.pgen.1005996.g003

The few bridge-like structures in telophase I that were observed in mnm and snm mutants presumably represent univalents lagging in the division plane rather than stretched bivalents [35]. The low frequency of chromosome bridges during meiosis I in mnm and snm mutants also confirms that co-orientation of sister kinetochores during meiosis I does not depend on homolog conjunction during male meiosis [35]. THR depletion in mnm mutants did not induce any phenotypic change during meiosis I (Figs 2C and S5). Importantly, it did not cause chromosome bridges during telophase I (Figs 2C and 3B), in striking contrast to the effect of THR depletion in spermatocytes with functional alternative homolog conjunction (Fig 1). Moreover, THR depletion did also not result in chromosome bridges during telophase I in snm mutants (Figs 3B and S5). In addition, SSE depletion by deGradFP did also no longer cause chromosome bridges during telophase I when performed in mnm or snm mutants (Figs 3B and S5). In summary, the fact that mnm and snm mutations, which abolish the alternative homolog conjunction in male meiosis, suppress chromosome bridge formation during telophase I in spermatocytes progressing through meiosis in the absence of separase function indicates that separase is required for the resolution of homolog conjunction during meiosis I.

Our conclusion that absence of separase function during Drosophila male meiosis I results in a specific failure of homolog separation is further supported by analyses with an additional mutant. tef mutants were chosen because alternative homolog conjunction is also defective in these mutants but not completely. While autosomes fail to pair into bivalents, formation of the X-Y bivalent is not affected [38]. THR depletion in tef mutants, therefore, is predicted to result in a chromosome bridge during telophase I that always represents a stretched XY bivalent, if resolution of homolog conjunction during male meiosis I requires separase function. As in case of nmn and snm mutants, chromosome bridges during telophase I in tef mutants with undisturbed separase function (Figs 2C and 3B) were infrequent and presumably represent occasional lagging autosomes. THR depletion in tef mutants resulted in an increased frequency of chromosome bridges during telophase I (Figs 2C and 3B). However, these bridges were fewer and less massive compared to those induced by THR depletion in spermatocytes with entirely normal homolog conjunction (Figs 1, 2A and 2B), consistent with the notion that only the XY bivalent forms bridges in tef mutants, while all bivalents form bridges in case of normal homolog conjunction. To demonstrate directly that the bridges resulting from THR depletion in tef mutants represent stretched XY bivalents, we performed fluorescence in situ hybridization (FISH) with a red fluorescent probe for the X and a green fluorescent probe for the Y chromosome (Fig 2D). After THR depletion in tef mutants, the red and green signals were invariably observed on the bridge (Fig 2D). Moreover, anti-SNM staining also resulted in a dot on the non-segregating DNA mass resulting after THR depletion in tef mutants (Fig 2D).

FISH was also applied to analyze the segregation of the X and Y chromosome after THR and SSE depletion in spermatocytes with normal homolog conjunction, and also in mnm and snm mutants after SSE depletion (S6 Fig). These analyses confirmed that disjoining of X and Y was inhibited in the absence of separase function and that the suppression of chromosome bridges resulting from a lack of separase function by inactivation of the alternative homolog conjunction system was paralleled by random segregation of X and Y during meiosis I. Finally, live imaging of progression through meiosis in spermatocytes with green fluorescent centromeres and red fluorescent chromosomes indicated that THR depletion causes chromosome separation failure directly and not indirectly via impairment of spindle or kinetochore function or premature exit from meiosis I (S7 Fig and S5 and S6 Movies).

Separase is required for sister chromatid separation in meiosis II

While mutations inactivating the alternative homolog conjunction system during Drosophila male meiosis very effectively suppressed chromosome bridges during telophase I after THR depletion (Figs 2C and 3B), they completely failed to do so during telophase II (Fig 3A and 3C). These telophase chromosome bridges presumably reflect a failure to separate sister centromeres during meiosis II in the absence of separase function. To confirm this notion, we performed experiments with mutations in sisters on the loose (solo). SOLO has no sequence homology to known proteins [50, 51]. However, analysis of the solo mutant phenotype revealed that it provides a function analogous to that of Rec8 in other eukaryotes. Rec8 is a meiosis-specific α-kleisin. Pericentric cohesin with Rec8 instead of the non-meiosis-specific Rad21 α-kleisin is protected from separase-dependent cleavage during meiosis I but no longer during meiosis II [6]. The Drosophila genome does not contain an obvious Rec8 homolog. But SOLO expression is also meiosis-specific [50, 51]. It interacts physically and functionally with the SMC1 core cohesin subunit and it is present at meiotic centromeres until anaphase II. Mutations in solo result in premature separation of sister chromatids. The chromosome bridges observed during telophase II after THR depletion in mnm mutants are therefore predicted to be abolished when the spermatocytes also lack solo function, if the bridges reflect stretched sister chromatids. Indeed, chromosome bridges during telophase II were not only absent in solo single mutants but also after THR depletion in solo mnm double mutants (Fig 3A and 3C). Chromosome bridges during telophase II were also missing after THR depletion in solo snm double mutants (Figs 3C and S8). These results demonstrate that Separase is required for resolution of sister chromatids in meiosis II.

We point out that mutations in solo, in contrast to mutations in mnm and snm, did not abolish the THR depletion-induced chromosome bridges during meiosis I (Figs 3B and S9), indicating that the alternative homolog conjunction system functions independent of sister chromatid cohesion. Consistent with this conclusion, earlier data reported as unpublished [50] indicated that MNM and SNM are present on bivalents in solo mutants where the large majority of bivalents also remain intact through metaphase I. Anti-SNM staining revealed that the prominent SNM dot on the XY bivalent cannot be detected any longer in telophase I and subsequent stages in solo mutants (S10 Fig), indicating that the inactivation of the alternative homolog conjunction during meiosis I occurs normally. However, THR depletion in solo mutants effectively prevented the disappearance of the SNM dot during exit from meiosis I (S10 Fig). Intense SNM dots were still present during meiosis II and during the early postmeiotic stages (S10 Fig). This perdurance of the alternative homolog conjunction presumably also explains the high frequency of chromosome bridges that was observed after THR depletion in solo mutants during meiosis II (Fig 3C).

Biorientation of sister kinetochores during meiosis II does not depend on normal separase function

The fact that separase is no longer required for chromosome separation during meiosis I in mutants with a non-functional alternative homolog conjunction system facilitates the analysis of a putative separase requirement for sister centromere individualization during progression through meiosis I. Since centromeric cohesin is thought to unite sister centromeres into a functional unit before meiosis I [12], destruction of centromeric cohesin by separase during meiosis I might enable sister centromere individualization and biorientation during meiosis II. After THR depletion in mnm mutants, univalents are randomly distributed into the two daughter cells. If separase function during meiosis I is required for sister centromere individualization, biorientation of these univalents in the meiosis II spindle is expected to fail and co-orientation of sister kinetochores as in meiosis I will also occur during meiosis II. To monitor sister centromere behavior we performed live imaging with spermatocytes expressing green fluorescent CID/Cenp-A as well as red fluorescent histone His2Av. Splitting of sister centromeres as well as sister kinetochore biorientation in meiosis II was clearly observed not only in control spermatocytes but also after THR depletion in mnm and snm mutants (Fig 4A and S7 and S9 Movies).

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Fig 4. Sister kinetochore biorientation during meiosis II is not affected by THR depletion.

(A) Still frames after time-lapse imaging of progression through meiosis II with testes expressing cid-EGFP (CID) and His2Av-mRFP (His2Av). Testes were from males without (+) or with spermatocyte-specific THR depletion by transgenic RNAi (thr-RNAi) as well as from mnm mutant males with identical spermatocyte-specific THR depletion (thr-RNAi mnm). During interkinesis before the onset of meiosis II, sister centromeres are not resolved and appear as a single Cid-EGFP dot. After sister kinetochore biorientation in the metaphase II spindle, sister centromeres are stretched apart along the spindle axis (double arrow). In control (+, top panel), sister centromeres are segregated far apart to opposite poles during exit from meiosis II; only one of the two postmeiotic nuclei with four centromeres is shown. However, after THR depletion (middle and bottom panel), sister centromere segregation to opposite poles during exit from meiosis II fails and each pair coalesces again into a single dot on the bridged (middle panel) or totally unpartitioned (bottom panel) chromatin mass present after meiosis II. Note that in the middle panel, a poorly visible fine chromatin bridge between two homologs is retracted at onset of the second meiotic division, and therefore one Cid-EGFP dot (arrowheads) joins the other dots in the upper cell (broken circle). Scale bar = 5 μm (B) Bars representing the percentage of single Cid-EGFP dots (n ≥ 43 Cid-EGFP dots) present before meiosis II that were separated along the spindle axis into two dots during metaphase II. The analyzed genotypes are indicated below the bars. thr-RNAi indicates spermatocyte-specific THR depletion. mnm and snm indicate loss-of-function mutations. +/+ indicates the presence of two functional thr gene copies, while P/+ and Df/+ indicate heterozygosity for either a P-element disrupted thr gene or a thr deficiency, respectively. Precise genotype descriptions are given in S1 Table. (C) The distance between pairs of Cid-EGFP dots representing sister centromeres was measured during metaphase II. Bars represent average separation (n ≥ 34 pairs of Cid-EGFP dots), whiskers indicate s.d. The same genotypes and movies as in (B) were analyzed. (D) Bar diagram indicating average duration (± s.d.) of the indicated phases during meiosis II as revealed by time-lapse imaging of secondary spermatocyte cysts. The same genotypes and movies as in (B) were analyzed. The numbers of analyzed secondary spermatocytes were 5 (control), 8 (thr-RNAi in mnm mutants with only one functional thr⁺ gene copy), and 8 (thr-RNAi in smn mutants with only one functional thr⁺ gene copy).

https://doi.org/10.1371/journal.pgen.1005996.g004

In fact, sister centromere splitting and biorientation could also be clearly detected after THR depletion in spermatocytes with functional homolog conjunction although meiosis II was often highly irregular as a result of the meiosis I chromosome separation failure (Fig 4A and S8 Movie). Importantly, sister centromere individualization and biorientation during meiosis II after THR depletion was detectable only transiently during metaphase II. During progression into metaphase II, the single centromere dots present in a secondary spermatocyte were split into a pair of dots along the spindle axis. However, the resolved sister kinetochores did not move apart towards opposite poles during anaphase II in the absence of separase function as a result of the failure in releasing sister chromatid cohesion.

While the sister chromatid separation failure indicated effective THR depletion, the presence of residual separase function still sufficient for sister centromere individualization during meiosis I cannot be ruled out definitively. To address this possibility, we carefully compared the effects of THR depletion in spermatocytes with either two or only one functional thr gene copy quantitatively. A reduction of the thr gene copy number is predicted to increase the efficiency of THR depletion and hence sister centromere splitting and kinetochore biorientation might be more compromised, if separase function during meiosis I is crucial for these processes. Therefore, we also performed THR depletion in spermatocytes heterozygous for either a P-element insertion within thr (thr^k07805b) or a deficiency that deletes thr (Df(2R)BSC338). These THR depletion experiments were again performed in a background with mutations in mnm or snm, where meiosis II is not accompanied by spindle irregularities resulting from chromosome separation failure during meiosis I. We quantified the fraction of secondary spermatocytes that displayed centromere splitting during metaphase II (Fig 4B). Moreover, we also determined the inter-sister kinetochore distance during metaphase II (Fig 4C) because partial centromere individualization during meiosis I might result in a reduced inter-sister kinetochore distance during metaphase II. These measurements did not reveal a difference between metaphase II spermatocytes in controls and after THR depletion in spermatocytes with only one functional thr gene copy. Finally, by estimating the duration of the different phases (prometa-, meta-, ana-, telophase) in the time-lapse movies, we assessed whether the temporal dynamics of progression through meiosis II was affected by THR depletion in either mnm or snm mutants that had only one functional thr⁺ gene copy (Fig 4D). Compared to controls, the average time interval between nuclear envelope breakdown and anaphase onset was found to be slightly extended in the two THR depletion cases (28.4 ± 6.2 and 26. 1 ± 4.2 versus 22.5 ± 2.5 min; n = 8, 7 and 5, respectively) but not in a statistically significant manner. Similarly, prometaphase appeared to be extended in the two THR depletion cases (Fig 4D) although variablity in these two cases was considerable. Therefore, it is conceivable that separase makes a contribution to chromosome biorientation during meiosis II but it does not appear to be essential.

Drosophila lines

The following lines with previously characterized mutations or transgenes were used: Sse^13m and Df(3L)SseA [42], thr^k07805b [65], Df(2R)BSC338 [66], P{ry+, hsp70-mnm-EGFP}, mnm^Z3-3298, mnm^Z3-5578, snm^Z3-0317, and snm^Z3-2138 [35], solo^Z2-0198 and solo^Z2-0338 (Yan et al., 2010), tef^Z2-4169 and tef^Z2-3455 [67], P{w⁺, bamP-GAL4-VP16}III [68], P{w⁺, His2Av-mRFP}II.2 and P{w⁺, gcid-EGFP-cid}II.1 [69], P{w⁺, pUbi-EGFP-alphaTub84B}II [70], and P{w⁺, pUbi-EYFP-asl} [71].

Lines for transgenic RNA interference, UAS-V20thr^shmiR9, UAS-V20thr^shmiR10 and UAS-W20thr^shmiR45, were generated by integrating the pVALIUM20 and pWALIUM20 constructs (see below) into the attP2 landing site. For production of P{w⁺, UASt-thr^Rr}attP40, allowing expression of a thr cDNA with silent mutations in the regions targeted by thr^shmiR9, thr^shmiR10 and thr^shmiR45, a pUASt-attB construct (see below) was integrated into the attP40 landing site. For pim-RNAi, we used y w¹¹¹⁸; P{w⁺, KK106514}VIE-260B (v100534). PBac{3xP3-ECFP, gEGFP-Sse}III.1 was generated by germline transformation with a PiggyBac construct. P{w⁺, bamP-NSlmb-vhh-GFP4}II.1 was isolated after P-element-mediated germline transformation with a pCaSpeR4 construct.

For all experiments, flies were cultured at 25°C. Detailed genotypes of the flies analyzed are provided in the supplemental material (S1 Table).

Plasmids

For the production of transgenic lines allowing GAL4-dependent expression of short hairpin microRNAs (shmiRs), we generated constructs using the vectors pVALIUM20 and pWALIUM20 [72]. Inserts were generated by annealing the following oligonucleotides: 5'-ctagcagt-CCCTTGGAAGCTACAAGTCAA-tagttatattcaagcata-TTGACTTGTAGCTTCCAAGGG-gcg-3' and 5'-aattcgc-CCCTTGGAAGCTACAAGTCAA-tatgcttgaatataacta-TTGACTTGTAGCTTCCAAGGG-actg-3' for thr^shmir9, 5'-ctagcagt-AACGCTTCTAGTTCAACTAAA-tagttatattcaagcata-TTTAGTTGAACTAGAAGCGTT-gcg-3' and 5'-aattcgc-AACGCTTCTAGTTCAACTAAA-tatgcttgaatataacta-TTTAGTTGAACTAGAAGCGTT-actg-3') for thr^shmir10, 5’-ctagcagt-AAGAAGTAGATCATTCTTCAA-tagttatattcaagcata-TTGAAGAATGATCTACTTCTT-gcg-3’ and 5’-aattcgc-AAGAAGTAGATCATTCTTCAA-tatgcttgaatataacta-TTGAAGAATGATCTACTTCTT-actg-3’ for thr^shmir45. Capital letters indicate the regions corresponding to sequences within the thr coding sequence.

For the production of a transgenic line allowing expression of EGFP-Sse under control of the Sse cis-regulatory region, we generated pBac{3xP3-ECFP-gEGFP-Sse} using a modified version of the previously described gSse transgene construct [42]. During construction, an Sse cDNA fragment replacing the Sse genomic region containing the first two introns was introduced as well as the EGFP coding sequence fused at the N-terminus.

For the construction of pCaSpeR4-bamP-NSlmb-vhh-GFP4, the fragment coding for NSlmb-vhh-GFP4 was amplified from pUASt-NSlmb-vhh-GFP4 [49] using primers SCH1 (5’-GACTACCGGTATGATGAAAATGGAGACTGAC-3’) and SCH2 (5’-GACTGCGGCCGCTTAGCTGGAGACGGTGAC-3’). After digestion with AgeI and NotI, the insert was used to replace the GAL4-VP16 coding region released by the same restriction enzymes in pCaSpeR4-bamP-GAL4-VP16 [68].

A modified thr cDNA was inserted into pUASt-attB [73] for the production of pUASt-attB-thr^Rr. The thr cDNA [48] was modified by replacing the region containing shmiR target sequences with a synthetic variant (GenScript, Piscataway, NJ 08854, USA).

Fertility tests

The fertility of 5–10 single males per genotype was assayed in parallel. Each single male was allowed to mate with three w virgin females for two days. Flies were transferred into a fresh vial and discarded after two more days. Flies eclosing from this vial were counted.

Testis preparations

Testis squash preparations were done as described [74]. For immunolabeling, mouse monoclonal anti-α-tubulin DM1A (Sigma) was used at 1:10’000 and affinity-purified rabbit polyclonal anti-SNM [35] at 1:250. Secondary antibodies were Alexa488- or Alexa568-conjugated goat antibodies against mouse or rabbit IgG diluted 1:1000.

Fluorescence in situ hybridization (FISH)

Dissection of testis, fixation with 4% PFA, permeabilization with PBST-DOC and anti- α-tubulin staining were done as described (protocol 3.2.2, steps 1–14) [74]. Cy5-conjugated goat anti-mouse IgG diluted 1:1000 was used as secondary antibody. Ethanol incubations and dehydration with a formamide series were also done as described (immuno-FISH protocol 3.2, steps 10–26) [75]. An oligonucleotide (5'-TTTTCCAAATTTCGGTCATCAAATAATCAT-3') with Atto-565 on 5’ and 3’ end (Integrated DNA Technologies, B-3001 Leuven Belgium) was used for detection of the X-specific 359 bp repeats at a concentration of ≈ 1 ng/μl in hybridization Buffer. An oligonucleotide (5'-AATACAATACAATACAATACAATACAATAC-3') with Alexa-488 fluorophore at the 3’ end (Sigma-Aldrich, 8107 Buchs, Switzerland) was used for detection of the Y-specific AATAC repeats at a concentration of ≈ 2 ng/μl in hybridization buffer. The denaturation step was performed at 98°C for 6 min, and hybridization over night at 16°C. Slides were washed twice in 50% formamide, 2x SSCT at 16°C for 1 hour each. Thereafter, additional washes were performed at room temperature in 25% formamide, 2x SSCT for 10 min and three times in 2x SSCT for 10 min each. DNA was stained with Hoechst 33258 (1 μg/ml) for 10 min and slides were washed twice in PBS for 5 min. Slides were mounted in 70% Glycerol, 50 mM Tris-HCl pH 8.5, 10 mg/ml propyl gallate, 0.5 mg/ml phenylendiamine.

Microscopy and image analysis

Image stacks with 250 nm spacing between focal planes were acquired with a 63×/1.4 oil immersion objective on a Zeiss Cell Observer HS microscope. If not stated differently, the images displayed in the figures represent maximum intensity projections. The data used for statistical analyses of a particular genotype was obtained from multiple slides and each slide was prepared with about 14 dissected testes.

Time-lapse imaging

Testes from pupal or adult males were dissected in Schneider’s Drosophila Medium (Invitrogen, #21720), 10% fetal bovine serum (Invitrogen), 1% penicillin/streptomycin (Invitrogen, #15140). The dissected testes were transferred into 40 μl of medium in a 35 mm glass bottom dish (MatTek Corporation, #P35G-1.5-14-C), and opened with fine tungsten needles to release the cysts. To reduce movements within the sample, methylcellulose (Sigma) was added. A wet filter paper was placed inside along the dish wall before sealing the lid with parafilm. Time-lapse imaging was performed with a spinning disc confocal microscope (Visitron) with a 60×/1.4 oil immersion objective. Image stacks with 24–45 focal planes spaced by 0.5–1 μm were acquired with a time interval of 30–60 sec. Precise numbers are specified in the legends for each supplementary movie. The distance between sister centromeres during metaphase II was measured in 3D using Imaris software (Bitplane). To exclude that the measurements actually represent early anaphase time points, the last three time points before anaphase onset were excluded from consideration.