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Research Article
Different roles for Aurora B in condensin targeting during mitosis and meiosis
Karishma S. Collette, Emily L. Petty, Netta Golenberg, Joshua N. Bembenek, Gyorgyi Csankovszki
Journal of Cell Science 2011 124: 3684-3694; doi: 10.1242/jcs.088336

Summary

Condensin complexes are essential for mitotic and meiotic chromosome segregation. Caenorhabditis elegans, like other metazoans, has two distinct mitotic and meiotic condensin complexes (I and II), which occupy distinct chromosomal domains and perform non-redundant functions. Despite the differences in mitotic and meiotic chromosome behavior, we uncovered several conserved aspects of condensin targeting during these processes. During both mitosis and meiosis, condensin II loads onto chromosomes in early prophase, and condensin I loads at entry into prometaphase. During both mitosis and meiosis, the localization of condensin I, but not condensin II, closely parallels the localization of the chromosomal passenger kinase Aurora B (AIR-2 in C. elegans). Interestingly, condensin I and AIR-2 also colocalize on the spindle midzone during anaphase of mitosis, and between separating chromosomes during anaphase of meiosis. Consistently, AIR-2 affects the targeting of condensin I but not condensin II. However, the role AIR-2 plays in condensin I targeting during these processes is different. In mitosis, AIR-2 activity is required for chromosomal association of condensin I. By contrast, during meiosis, AIR-2 is not required for condensin I chromosomal association, but it provides cues for correct spatial targeting of the complex.

Introduction

Condensin is a five-subunit complex that functions in the formation, compaction and segregation of mitotic and meiotic chromosomes (Hirano, 2005; Hudson et al., 2009). Condensin has been isolated in eukaryotic organisms ranging from yeast to humans. Two structural maintenance of chromosome (SMC) subunits of the SMC2 and SMC4 classes form the enzymatic core of the complex. In addition, condensin contains three regulatory CAP (for chromosome-associated polypeptide) proteins. Whereas yeast has a single condensin complex, higher eukaryotes possess two, condensins I and II, with condensin I being more similar to the single yeast condensin (Hirota et al., 2004; Ono et al., 2004; Ono et al., 2003). Condensins I and II have identical SMC proteins and distinct, yet similar, CAP components: CAP-G, -D2 and -H in condensin I, and CAP-G2, -D3 and -H2 in condensin II. In mammalian cells, condensins I and II associate with chromosomes at different times in the cell cycle: condensin II early in prophase and condensin I after nuclear envelope breakdown (NEBD). The two complexes also occupy distinct domains on mitotic chromosomes and perform non-redundant functions (Hirota et al., 2004; Ono et al., 2004; Ono et al., 2003).

How the two condensin complexes are targeted to distinct chromosomal domains in mitosis is not known. Studies in yeast identified cis-acting sites that play a role in targeting the single yeast condensin to chromosomes (D'Ambrosio et al., 2008). If cis-acting elements also play a role in condensin targeting in metazoans, the elements must be different for the two condensin complexes. Alternatively, differential targeting might be achieved by trans-acting factors differentially regulating the two complexes. One candidate for such a regulator is the mitotic kinase and chromosomal passenger complex (CPC) member Aurora B. In some experimental systems, Aurora B depletion has no effect on the chromosomal targeting of condensin(s) (Losada et al., 2002; MacCallum et al., 2002; Ono et al., 2004). However, in other studies, depletion of Aurora B leads to defects in loading of the single yeast condensin in fission yeast (Petersen and Hagan, 2003) and defects of condensin I loading in Drosophila (Giet and Glover, 2001), Xenopus (Takemoto et al., 2007) and HeLa cells (Lipp et al., 2007). Interestingly, condensin II in HeLa cells was unaffected (Lipp et al., 2007), indicating that Aurora B might preferentially affect condensin I targeting in mitosis.

We are using Caenorhabditis elegans as a model to study the function and differential regulation of condensin complexes. C. elegans chromosomes are holocentric, and the kinetochores assemble along the entire length of chromosomes, rather than being localized to a single site as on monocentric chromosomes. Despite this difference, chromosomal proteins and their functions are conserved between worms and other eukaryotes (Maddox et al., 2004). C. elegans also has two mitotic and meiotic condensin complexes, and an additional third condensin, condensin IDC, which functions in the hermaphrodite and X-chromosome-specific process of dosage compensation (Csankovszki et al., 2009; Mets and Meyer, 2009). The two mitotic complexes have identical SMC subunits, SMC-4 and MIX-1, and distinct sets of CAP proteins. Condensin I and condensin IDC only differ in their SMC4 subunits (Fig. 1A). Whereas condensins I and II associate with all chromosomes, condensin IDC only binds to the X chromosomes in hermaphrodites to halve gene expression, equalizing X-linked product in XX hermaphrodites and XO males (Csankovszki et al., 2009).

Fig. 1.
Fig. 1.

Condensin I and II during mitosis. (A) Subunit composition of human (in some cases designated with an h prefix) and C. elegans condensin complexes. (B) In early prophase, condensin I (CAPG-1, green) is not detected on chromosomes, whereas condensin II (HCP-6, green) localizes to the centromeres. From prometaphase onwards, condensin I discontinuously coats mitotic chromosomes, and condensin II maintains its centromere-enriched localization. Nuclear pore complex staining is shown in red, DAPI in blue. All images are from 2-cell to 8-cell stage embryos. (C) Longer exposure of an anaphase figure from an 8-cell embryo reveals condensin I (CAPG-1, green) staining on spindle midzone microtubules (red). (D) Live imaging of CAPG-1::GFP in an 8-cell embryo reveals similar patterns of chromosomal association at metaphase and spindle localization during anaphase (arrowhead). Scale bars: 5 μm.

Some aspects of condensin loading onto mitotic chromosomes are conserved between monocentric mammalian chromosomes and holocentric worm chromosomes. In both systems, condensin II is enriched at the centromeres (Hagstrom et al., 2002; Ono et al., 2004; Stear and Roth, 2002). C. elegans AIR-2 (the Aurora B homolog) has also been reported to affect chromosomal association of MIX-1 and SMC-4, components of both condensins I and II (Hagstrom et al., 2002; Kaitna et al., 2002). However, in a different study, recruitment of SMC-4 and the condensin II subunit CAPG-2 appeared to be unaffected by depletion of AIR-2 (Maddox et al., 2006). These C. elegans studies were conducted before the identification of two distinct mitotic complexes. Given that SMC proteins are common to condensins I and II, it remains to be determined whether AIR-2/Aurora B is needed for recruitment of one or both condensins.

Compared with mitosis, relatively little is known about condensin distribution and regulation in meiosis. In C. elegans meiosis, the two condensin complexes associate with chromosomal domains that are different from those they occupy in mitosis. During meiosis, condensin II localizes to an interior domain within sister chromatids, whereas condensin I is found between homologs in meiosis I and between sister chromatids in meiosis II (Csankovszki et al., 2009). The differences between mitotic and meiotic localization patterns probably reflect differences in chromosome behavior during these processes. These differences also raise the question of whether recruitment mechanisms are comparable between mitosis and meiosis.

The differences between mitotic and meiotic chromosome behavior arise from the unique events during meiosis I, when homologs are separated while sister chromatids stay together. In monocentric organisms, the centromere plays a central role in the coordination of these meiotic activities (reviewed by Sakuno and Watanabe, 2009). In meiosis I, cohesion between sister centromeres is preserved, whereas cohesion along chromosome arms is released to allow separation of homologs. In addition, cohesion at the centromeres ensures that microtubules attached to sister kinetochores connect to the same pole, whereas microtubules attached to kinetochores of homologs are attached to opposite poles to establish tension (Sakuno et al., 2009).

On the holocentric chromosomes of worms, the lack of a localized centromere necessitates coordination of meiotic events in a different manner (reviewed by Schvarzstein et al., 2010). During worm meiosis, the site of the crossover, and not a localized region of centromeric CENP-A-containing chromatin, ultimately determines the plane of chromosome orientation and the site of cohesion release (Monen et al., 2005; Nabeshima et al., 2005). Worm chromosomes typically have a single site of crossover, located in an off-center position. During meiosis I prophase, paired homologs (bivalents) are restructured into cross-shaped figures, in which the short arm corresponds to the region between the crossover and the closer chromosome end, and the long arm corresponds to the region between the crossover and the more distant chromosome end (Chan et al., 2004; Nabeshima et al., 2005). During metaphase, the short arms of bivalents are lined up along the metaphase plate and the long arms point towards opposite poles. Cohesin along the short arm will be released during meiosis I to separate homologs, and the remaining cohesin will be released in meiosis II to separate sisters (see Fig. 3). Because the crossover can happen at either end of the chromosome, the identity of short and long arms is different for the same chromosome in different meioses.

During worm meiosis, condensin I is restricted to the short arm of bivalents (Csankovszki et al., 2009). Because the short arm can correspond to either end of the chromosome, condensin I is targeted to different DNA sequences in different meioses. This observation makes it unlikely that cis-acting DNA elements provide the primary targeting cue. A more probable targeting signal originates from other chromosomal proteins localizing to the same region. Interestingly, AIR-2/Aurora B, the protein implicated in condensin I targeting during mitosis in various systems, also localizes to the short arm of bivalents (Kaitna et al., 2002; Rogers et al., 2002). How AIR-2/Aurora B activity influences condensin loading in meiosis has not been addressed.

In this study we investigated the timing and regulation of condensin recruitment in mitosis and meiosis in C. elegans with particular attention to the role of AIR-2/Aurora B. We found that the need for AIR-2 for correct condensin I targeting is conserved between mitosis and meiosis, but the exact role AIR-2 plays differs between the two processes.

Fig. 2.
Fig. 2.

Condensin I, but not II, depends on AIR-2 for mitotic recruitment. (A) Condensin I (CAPG-1, green) and AIR-2 (red) colocalize on mitotic chromosomes at metaphase. At anaphase, AIR-2 dissociates from DNA and localizes to the spindle midzone. Condensin I remains on DNA but also colocalizes at the midzone with AIR-2. (B) Chromosomal association of condensin I (CAPG-1, green) depends on the activity of AIR-2. In air-2 mutant embryos, the H3S10-P (H3S10Ph) mark (red) is undetectable, condensin I is not recruited to metaphase chromosomes, but condensin II (HCP-6, green) recruitment is less affected. All examples are from the first, second or third mitotic division. Scale bars: 2 μm.

Results

Condensin complexes in mitosis

Because condensin I and II are loaded onto chromosomes at different times in mammalian cells (Hirota et al., 2004; Ono et al., 2004; Ono et al., 2003), we set out to determine the timing of condensin loading during C. elegans mitosis using immunofluorescence microscopy. In mammalian cells, NEBD marks the entry into prometaphase. By contrast, in C. elegans, nuclear pore complexes (NPCs) break down in prometaphase, but the nuclear envelope does not fully disassemble until anaphase (Lee et al., 2000). We monitored the breakdown of NPCs using an antibody (mAb414) that recognizes a subset of nucleoporins (Davis and Blobel, 1986). Using our fixation conditions, the NPC signal greatly diminishes by prometaphase in embryos of all stages, as judged by chromosome morphology and microtubule staining (Fig. 1 and data not shown). To investigate the timing of condensin loading onto chromosomes, we used antibodies against CAPG-1, DPY-26, or DPY-28 to mark condensin I, and antibodies against HCP-6 or KLE-2 to mark condensin II (Fig. 1A). CAPG-1, DPY-26 and DPY-28 are components of both condensins I and IDC. Given that condensin IDC is absent from mitotic chromosomes in early embryos before the onset of dosage compensation (Csankovszki et al., 2009), we performed all our analysis in one-cell to eight-cell embryos to focus on the chromosomal targeting of condensin I.

In early prophase, only condensin II associated with chromosomes. Condensin I loaded onto chromosomes after NPC disassembly in prometaphase (Fig. 1B). These data indicate that the timing of condensin loading onto chromosomes is conserved between worm and mammalian mitotic cells. From prometaphase to anaphase, the spatial patterns of condensins I and II were different, with condensin II in a centromere-like pattern (Hagstrom et al., 2002; Stear and Roth, 2002) and condensin I diffusely coating all chromosomes (Fig. 1B). During anaphase, condensin I was also seen colocalizing with microtubules at the spindle midzone, using both immunofluorescence microscopy and live imaging of GFP-tagged CAPG-1 (Fig. 1C,D; supplementary material Movie 1), in addition to the chromosomal signal. The other condensin I CAP subunits, DPY-26 and DPY-28, associated with chromosomes at the same time and with similar patterns to that of CAPG-1 (supplementary material Fig. S1A). Furthermore, the chromosomal association of CAPG-1 was dependent on the presence of DPY-26 and DPY-28 (supplementary material Fig. S1B), indicating that the condensin I CAP subunits associate with mitotic chromosomes as a complex.

AIR-2 is needed for mitotic recruitment of condensin I but not condensin II

The pattern of condensin I, but not condensin II, on mitotic chromosomes resembles the distribution of AIR-2, the Aurora B homolog in C. elegans (Schumacher et al., 1998). Indeed, during metaphase CAPG-1 colocalized with AIR-2 on chromosomes, and during anaphase the spindle localization of condensin I was coincident with AIR-2 (Fig. 2A). These observations prompted us to examine whether AIR-2 is needed for condensin recruitment in C. elegans mitosis.

To inactivate AIR-2 in mitotic cells, worms homozygous for the temperature-sensitive loss-of-function allele air-2(or207) (Severson et al., 2000) were shifted to the restrictive temperature. Control wild-type worms were subjected to the same temperature shift. We monitored levels of histone H3 phosphorylated on S10 (H3S10-P), a mark deposited by AIR-2, to assess the efficiency of AIR-2 inactivation, and used anti-tubulin antibody as a staining control and to mark mitotic cells. We restricted our analysis to metaphase, at which point both condensin complexes are normally associated with chromosomes. A total of 29 out of 29 control wild-type metaphases had condensin I staining, and 20 out of 20 had condensin II staining (Fig. 2B). In 14 out of 18 metaphases from air-2(or207) embryos with no H3S10-P staining, condensin I was lost or greatly reduced. We attribute the weak condensin I staining on some metaphase figures to residual AIR-2 activity, which probably remains even after the temperature shift. By contrast, on most (25 out of 37) mitotic figures with no detectable H3S10-P, condensin II levels were comparable to those in wild-type controls (Fig. 2B), whereas on the remaining figures condensin II levels were reduced, but not absent. We conclude that, similar to what has been observed in mammalian cells (Lipp et al., 2007), Aurora B activity preferentially affects the mitotic recruitment of condensin I to chromosomes. Note that the or207 allele at the restrictive temperature produces a catalytically inactive AIR-2 protein that does still associate with chromosomes (Severson et al., 2000). Our results indicate that the mutant protein is not sufficient for condensin I recruitment.

Fig. 3.
Fig. 3.

Overview of C. elegans meiosis. (A) A diagram of the adult hermaphrodite gonad. Nuclei enter meiosis in the transition zone (TZ, leptotene and zygotene), and proceed through pachytene, diplotene and diakinesis before fertilization. Sperm are stored in the spermatheca (sp). The most proximal oocyte is designated −1. Oocytes move through the spermatheca, are fertilized and then complete meiotic divisions (MI and MII). A set of homologs is extruded as the first polar body (PB1) during MI, and a set of sister chromatids is extruded as the second polar body (PB2) during MII. O, Oocyte pronucleus; S, sperm-derived pronucleus. (B) Meiotic chromosomes are extensively restructured between pachytene and diakinesis. During pachytene, replicated chromosomes are held together by the SC (i). A single off-center crossover divides the paired homologs into two domains. At pachytene exit, HTP-1 and LAB-1 are retained between the crossover and the more distant chromosome end, and SYP-1 is retained between the crossover and the closer chromosome end. AIR-2 is recruited to the domain where SYP-1 is retained (ii). The AIR-2-bound domain becomes the short arm, and the HTP-1- and LAB-1-bound domain becomes the long arm of the bivalent (iii). Bivalents undergo extensive condensation (iv and v). At metaphase of meiosis I, bioriented bivalents are aligned with their long arms parallel to spindle microtubules and the AIR-2-occupied domain at the metaphase plate (the spindle pole axis is indicated by arrows). In meiosis I, AIR-2 promotes cohesion loss at the short arm and homologs move away from each other (v). In meiosis II, the AIR-2-occupied domain at the sister chromatid interface is aligned at the metaphase plate and sister chromatids become bioriented and eventually separated (vi).

Condensin I and II during oocyte meiosis

We next investigated chromosomal targeting of condensin complexes during meiosis in worms. In the C. elegans germline, syncitial nuclei are organized in a temporal-spatial array of meiotic stages (Schedl, 1997) (Fig. 3A). Mitotic nuclei in the distal germline are followed by a meiotic transition zone (leptotene and zygotene) where homologs begin pairing and alignment. In pachytene, homologs are synapsed through the synaptonemal complex. During late pachytene and diplotene, the synaptonemal complex disassembles, and chromosome pairs are condensed and restructured into compact bivalents (Fig. 3B) (Chan et al., 2004; de Carvalho et al., 2008; Martinez-Perez et al., 2008; Nabeshima et al., 2005). In hermaphrodites, oocytes in the proximal germline arrest at diakinesis with homolog pairs organized into six bivalents. The most proximal oocyte, referred to as −1, undergoes maturation followed by fertilization (McCarter et al., 1999). After fertilization, the oocyte-derived nucleus completes meiosis giving rise to two polar bodies and the haploid maternal pronucleus.

To characterize the timing of condensin loading onto chromosomes during oocyte meiosis, we compared CAPG-1 (condensin I) and HCP-6 (condensin II) patterns in the hermaphrodite germline and fertilized embryos. Whereas HCP-6 staining was apparent by early diplotene, as reported previously (Chan et al., 2004), chromosomal association of CAPG-1 was not seen until late diakinesis. During meiosis, NEBD occurs in the −1 oocyte at the time of maturation, immediately preceding fertilization (McCarter et al., 1999). We observed strong CAPG-1 staining only after, but not before, NEBD in the −1 oocytes, whereas HCP-6 staining was apparent both before and after NEBD (Fig. 4A). These results indicate that the timing of condensin loading is conserved between mitosis and meiosis in C. elegans. After NEBD, the two condensin complexes occupied distinct domains. Condensin I was found at the interface between homologs marked by reduced DAPI staining (‘DAPI-free zone’), whereas condensin II localizes to sister chromatids throughout meiosis (Fig. 4A). During anaphase, condensin I localized on the acentrosomal meiotic spindle between separating chromosomes (Fig. 4A–C; supplementary material Movie 2). As in mitosis, condensin I colocalized with AIR-2 on chromosomes during prometaphase and metaphase of meiosis, and on the spindle during anaphase (Fig. 4D).

Condensin I localizes to the short arms of bivalents, where cohesion between the exchanged parts of sister chromatids holds homologs together. Viewed from the side, it appeared as a straight line intersecting the bivalent along its shorter axis. Viewed from the end, CAPG-1 appeared as a ring around the center of the bivalent (Fig. 4E), a pattern that has also been seen for AIR-2 and the chromokinesin KLP-19 (Dumont et al., 2010; Wignall and Villeneuve, 2009). This ring-like appearance was observed both in mature oocytes and in fertilized embryos in metaphase of meiosis I, as well as at the sister chromatid interface in meiosis II (Fig. 4E). This ring-shaped midbivalent domain precedes the formation of anaphase linker structures between chromosomes that might function to drive chromosome separation (Dumont et al., 2010; Wignall and Villeneuve, 2009). The localization of condensin I on the meiotic anaphase spindle might be related to these previously observed central spindle structures (Fig. 4C).

Fig. 4.
Fig. 4.

Condensin I and II in oocyte meiosis. (A) Condensin I (CAPG-1, green) associates with the short arm of bivalents only after NEBD in the −1 oocyte. During metaphase I, condensin I localizes between homologous chromosomes, at anaphase I it localizes between separating homologs, and at metaphase II it localizes between sister chromatids and on the polar body (PB). Condensin II (HCP-6, green) associates with chromosomes before NEBD and remains at the core of sister chromatids throughout meiosis. The nuclear pore complex is shown in red, DAPI in blue. (B) Live imaging of CAPG-1::GFP showing condensin I on chromosomes and between separating chromosomes in a fertilized oocyte undergoing meiosis I. (C) Condensin I (CAPG-1, green) and tubulin (red) localization patterns. During acentrosomal meiosis, microtubules on the side towards the pole disassemble after metaphase and are seen primarily between separating chromosomes during anaphase. Condensin I colocalizes with microtubules during anaphase I and anaphase II. (D) Condensin I also colocalizes with AIR-2 at the metaphase plate and on the anaphase I spindle. (E) Enlarged images of a diakinesis bivalent (meiosis I) and pair of sister chromatids during meiosis 2. Viewed from the side, condensin I (CAPG-1, green) and AIR-2 (red) appear as a line between chromosomes. Viewed from the end, they appear as a ring encircling the chromosomes. Scale bars: 2 μm (A–D); 1 μm (E).

Before NEBD in oocytes, CAPG-1 was present in the nucleus, but it did not associate with chromosomes. The intensity of nucleoplasmic staining diminished after NEBD, representing diffusion of the protein into the much larger volume of the oocyte cytoplasm (Fig. 4A). The same pattern is also seen for the unique condensin IDC subunit DPY-27 and therefore represents the loading of condensin IDC into oocytes in preparation for dosage compensation in fertilized embryos (Chuang et al., 1994; Csankovszki et al., 2009). We used two methods to ensure that the diffuse nuclear staining was not obscuring chromosomal CAPG-1 association. First, we used detergent extraction to reduce nucleoplasmic CAPG-1 staining. In extracted oocytes, chromosomal association of CAPG-1 was still not observed before NEBD, even though chromosomal staining after NEBD remained comparable to that in unextracted nuclei (supplementary material Fig. S2A). Second, to reduce condensin IDC levels in oocytes, we examined worms carrying the partial loss-of-function mutation dpy-27(y57). In these worms, nucleoplasmic staining of CAPG-1 in oocytes was greatly reduced, corresponding to a reduction in condensin IDC, but chromosomal association was still only detected after NEBD (supplementary material Fig. S2B). On the basis of these results, we conclude that maximal enrichment of CAPG-1 on chromosomes occurs after NEBD in the −1 oocyte.

To investigate whether all condensin I CAP subunits associate with chromosomes in a similar pattern, we performed immunofluorescence microscopy using anti-DPY-26 and anti-DPY-28 antibodies. We observed DPY-26 and DPY-28 at the DAPI-free zone at the short arms of diakinesis bivalents and also at the chromosome interface in fertilized embryos (supplementary material Fig. S3A,B). CAPG-1 localization depended on the presence of DPY-26 and DPY-28 because the short-arm staining of CAPG-1 was undetectable in oocytes homozygous for the strong loss-of-function alleles dpy-26(n199) and in dpy-28(s939) (supplementary material Fig. S3C,D), or upon RNA interference (RNAi) depletion of DPY-26 or DPY-28 (data not shown). Therefore, the condensin I CAP subunits appear to localize to the short arm as a complex.

Condensin localization in the male germline followed the same general pattern (supplementary material Fig. S4A). Both condensins I and II began to accumulate in nuclei by late pachytene. Condensin II associated with chromosomes before NEBD, by the karyosome stage, a stage unique to the male germline in worms (Shakes et al., 2009), but condensin I chromosomal enrichment was not seen until after NEBD in late diakinesis (supplementary material Fig. S4A,B). Following NEBD, condensin I was between aligned homologs (meiosis I) and between sisters (meiosis II), whereas condensin II remained associated with sister chromatids throughout. Interestingly, condensin I behavior in sperm and oocytes differed at anaphase. In oocytes, condensin I colocalized with microtubules and AIR-2 between separating chromosomes during anaphase (Fig. 4C,D). In sperm, AIR-2 and microtubules were not prominent between separating chromosomes, and condensin I was absent from this region. Instead, condensin I and AIR-2 colocalized on the inner edges of separating chromosomes (supplementary material Fig. S4C).

AIR-2 restricts condensin I to the short arm of the bivalent

The chromosomal association patterns of condensin complexes were dissimilar in mitosis and meiosis. However, in both cases, condensin I localization closely paralleled that of AIR-2, whereas condensin II occupies a distinct domain (Figs 1,4). This observation, coupled with the finding that condensin I requires AIR-2 for mitotic chromosomal association, prompted us to investigate the potential role for AIR-2 in condensin I recruitment in meiosis.

To deplete AIR-2 levels, we used RNAi in the air-2 temperature-sensitive mutants shifted to the restrictive temperature. We needed to use this combination because the mutation alone or RNAi alone did not completely eliminate the H3S10-P signal in meiotic tissues. We limited our analysis to oocytes in which H3S10-P levels were reduced to below the level of detection by immunofluorescence microscopy. As a control, wild-type worms fed empty vector RNAi were shifted to the same temperature. In control oocytes after NEBD, we observed condensin I at the short arm of the bivalent, whereas condensin II associated with the four sister chromatids (Fig. 5A). In AIR-2-depleted oocytes, condensin II staining on the four sister chromatids was discernable, despite the somewhat disorganized structure of the bivalent. Similar results were obtained with the condensin II subunits HCP-6 (Fig. 5A) and KLE-2 (data not shown). By contrast, condensin I appeared mislocalized. In AIR-2-depleted oocytes, CAPG-1 occupied a cross shape, as though localizing to both arms of the bivalents (Fig. 5A). Co-staining with CAPG-1- and HCP-6-specific antibodies demonstrated that, in AIR-2-depleted oocytes, the organization of the sister chromatids into four quadrants remained intact, as evidenced by four distinct HCP-6 signals. CAPG-1 occupied the domains between these chromatids in a cross shape (Fig. 5B). Taken together, similar to mitosis, AIR-2 influences the chromosomal localization of condensin I, but not condensin II, during meiosis. However, unlike in mitosis, AIR-2 activity is not needed to load condensin I onto chromosomes in meiosis, indicating that AIR-2, or H3S10-P, is unlikely to serve as a direct recruiter of condensin I.

Fig. 5.
Fig. 5.

AIR-2 activity is needed for correct targeting of condensin I, but not condensin II, in meiosis. Enlarged bivalents from oocytes after NEBD. On control bivalents, condensin I (CAPG-1, green) is restricted to the short arm, and H3S10-P (H3S10Ph, red) is seen on both sides of the condensin I domain. In AIR-2-depleted oocytes, the H3S10-P signal is absent, and condensin I mislocalizes to both arms of the bivalents and appears in a cross shape. By contrast, the chromosomal association of condensin II (HCP-6, green) appears similar on control and AIR-2-depleted bivalents. DAPI is shown in blue and gray. Scale bars: 1 μm.

AIR-2 provides spatial cues for condensin I targeting in meiosis

In worms, crossovers divide bivalents into highly asymmetric structures, with AIR-2 targeted to the short arms. The AIR-2-occupied domain dictates not only the plane of cohesin release (Kaitna et al., 2002; Rogers et al., 2002), but also the plane of chromosome orientation. At metaphase I, the short arms are lined up at the metaphase plate, whereas the long arms are parallel to spindle microtubules (Albertson and Thomson, 1993; Wignall and Villeneuve, 2009). To investigate further how condensin I is targeted to the short arm, we analyzed mutant backgrounds in which the activity of AIR-2 is not restricted to this specialized domain or in which this domain does not exist due to lack of chiasma formation.

HTP-1, a HORMA-domain-containing protein, and LAB-1, a worm-specific chromosomal protein containing a PP1 phosphatase interaction domain, assume a reciprocal localization pattern with respect to AIR-2 and are restricted to the long arm of diakinesis bivalents (de Carvalho et al., 2008; Martinez-Perez et al., 2008) (Fig. 3B). Complete depletion of these proteins led to a defect in pairing and chiasma formation, and oocytes contained 12 univalents rather than six bivalents. However, in some htp-1(gk174) oocytes (homozygous for a deletion of the gene), the two X chromosomes form a bivalent. These rare bivalents lose their asymmetric features, many appear less elongated than in wild type and AIR-2 localizes in a cross shape on both bivalent arms (Martinez-Perez et al., 2008). Similarly, when LAB-1 is partially depleted using RNAi, such that most oocytes still contain six bivalents, AIR-2 localizes to both arms in a cross shape (de Carvalho et al., 2008). We observed AIR-2 spreading on some, but not all htp-1(gk174) and lab-1(RNAi) bivalents. On bivalents with a cross-shaped AIR-2 domain, CAPG-1 also spread onto both arms of the bivalent, whereas bivalents that do not show spreading of AIR-2 also did not show spreading of CAPG-1 (Fig. 6A). Interestingly, the H3S10-P domain spread out all over the bivalent, over a much larger domain than that occupied by AIR-2 on all bivalents, perhaps reflecting transient AIR-2 association at these regions (Fig. 6B). However, condensin I spreading was only observed in the more restricted domain occupied by AIR-2. These data indicate that stable AIR-2 association with chromosomes can be sufficient to guide condensin I localization, but H3S10-P is not.

We next analyzed mutant backgrounds in which homologs are not held together in meiosis I and instead of six bivalents they form twelve univalents. Therefore, there is no homolog interface to which both AIR-2 and condensin I would be normally targeted. In the spo-11(ok79) (deletion allele) background, chiasmata do not form owing to a defect in double-strand break formation (Dernburg et al., 1998). Because crossovers are required for the orderly asymmetric organization of bivalents, in spo-11 mutants, AIR-2 and HTP-1 are localized in a stochastic, rather than an orderly, manner. By diplotene or diakinesis they acquire mutually exclusive localization patterns, with some spo-11 univalents staining only with HTP-1, and others only with AIR-2 (Martinez-Perez et al., 2008; Nabeshima et al., 2005). We observed AIR-2 localization on approximately half of the spo-11(ok79) univalents, and CAPG-1 and AIR-2 always colocalized (Fig. 7A). These results indicate that crossover formation is not necessary for condensin I targeting to chromosomes and that AIR-2 is sufficient to dictate the spatial distribution of condensin I, even when AIR-2 localization is stochastic. Note that on some univalents, AIR-2 and condensin I colocalized along a faint DAPI-light zone intersecting the univalent. Most univalents had H3S10-P staining of various intensities, but condensin I only localized to those with most intense staining, presumably reflecting stable AIR-2 association (Fig. 7B).

Fig. 6.
Fig. 6.

Spreading of condensin I and AIR-2 on meiotic bivalents. Enlarged bivalents from oocytes after NEBD, stained with antibodies specific to condensin I (CAPG-1, green) and either AIR-2 (red) (A) or H3S10-P (H3S10Ph, red) (B). On wild-type bivalents, condensin I, AIR-2 and H3S10-P localize to the short arm. On some htp-1(gk174) and lab-1(RNAi) bivalents, H3S10-P spreads to cover the bivalent surface, but AIR-2 either remains at the short arm or spreads to both arms. When AIR-2 spreads away from the short arm, condensin I follows AIR-2 and extends onto the long arm. When AIR-2 remains restricted to the short arm, condensin I also remains restricted to this region. Spreading of condensin I and AIR-2 is most pronounced on more rounded and less asymmetric bivalents.

Fig. 7.
Fig. 7.

Condensin I localization on meiosis I univalents. Enlarged univalents from oocytes after NEBD, stained with antibodies specific to condensin I (CAPG-1, green) and either AIR-2 (red) (A) or H3S10-P (H3S10Ph, red) (B). On some spo-11 univalents, condensin I and AIR-2 colocalize to a DAPI-light line, whereas on others neither condensin I nor AIR-2 are detected. Most spo-11 univalents have H3S10Ph staining of varying intensity, and condensin I only localizes to univalents with brighter H3S10-P staining. On all rec-8 univalents, condensin I and AIR-2 colocalize at the interface between sister chromatids and H3S10-P forms a boundary on either side of condensin I. Scale bars: 1 μm.

In rec-8(ok978) (deletion) mutants, sister chromatids are held together by the REC-8 paralogs COH-3 and COH-4 until anaphase I, forming 12 univalents. rec-8 univalents biorient at metaphase I, and sisters will prematurely separate toward opposite spindle poles at anaphase I (Severson et al., 2009). Unlike on the cooriented spo-11 univalents, AIR-2 consistently localized to a prominent DAPI-free zone between sisters on all 12 rec-8 bioriented univalents. H3S10-P intensity was also uniform among univalents. In these oocytes, condensin I colocalized with AIR-2 between sister chromatids, similar to what we observe in wild-type meiosis II (Fig. 7A). Taken together, our data suggest that although AIR-2 is not required for recruitment of condensin I, it provides spatial cues that determine the localization of condensin I on meiotic chromosomes. AIR-2, an important determinant of bivalent asymmetry and chromosome orientation, is also responsible for guiding condensin I to the chromosomal domain that will be aligned at the metaphase plate, whether on wild-type meiosis I bivalents, wild-type meiosis II sister chromatids or rec-8 mutant meiosis I univalents.

Discussion

Timing of condensin loading to chromosomes in mitosis and meiosis

Meiosis includes a prolonged prophase I during which homologous chromosomes pair, synapse and exchange genetic material. By contrast, prophase in mitosis is relatively brief. Despite these differences, condensin complexes load at analogous time points: condensin II as chromosomes begin to condense in early prophase, and condensin I in prometaphase. This time point in worm oocytes coincides with maturation and fertilization. The timing of condensin I and II recruitment in mitosis is conserved between worms and mammals (Ono et al., 2004), raising the possibility that it is also conserved in meiosis in all metazoans. Consistent with that, condensin I loads onto chromosomes by prometaphase in mouse spermatocytes (Viera et al., 2007). However, condensin II or NEBD were not analyzed in that study.

What triggers condensin I loading at NEBD and prometaphase is unclear. All components of condensin I are present in the nucleoplasm before NEBD, yet they do not associate with chromosomes. This is clearly demonstrated in the male germline, where condensin CAP subunit staining cannot be attributed to the presence of condensin IDC. Aurora B activity and H3S10-P staining is also apparent on both mitotic and meiotic chromosomes before prometaphase, excluding the possibility that H3S10 phosphorylation triggers condensin I assembly. Future studies will be needed to determine how the timing of condensin I loading is coordinated with other cell cycle events.

Mitotic recruitment of condensin complexes

Similar to what has been observed in mammalian cells (Lipp et al., 2007), AIR-2 inactivation in worms disrupts the efficient recruitment of condensin I to mitotic chromosomes, but condensin II recruitment is unaffected. It remains unclear whether this reflects a direct recruitment by the kinase or its chromatin mark H3S10-P or, alternatively, whether it reflects a need for an AIR-2-mediated change in chromatin structure. Our results also resolve previous conflicting data in the field. Previous studies have concluded that AIR-2 is required for loading of the SMC proteins MIX-1 and SMC-4 (shared between condensins I and II) onto mitotic chromosomes (Hagstrom et al., 2002; Kaitna et al., 2002). However, a different study failed to detect a noticeable change in SMC-4 and CAPG-2 (the condensin II CAP subunit) recruitment (Maddox et al., 2006). We suggest that the observed reduced recruitment of SMC-4 and MIX-1 upon AIR-2 depletion reflects a loss of condensin I from chromosomes. Because condensin II recruitment is unaffected, its subunits remain on chromosomes. Our results also explain the findings that MIX-1 function before prometaphase is AIR-2 independent, yet its chromosomal association in metaphase is (at least partially) AIR-2 dependent (Kaitna et al., 2002). Before prometaphase, condensin II complexes containing MIX-1 associate with chromosomes in an AIR-2-independent manner and facilitate chromosome condensation. By contrast, after prometaphase, condensin I complexes also containing MIX-1 associate with chromosomes in an AIR-2-dependent manner.

Meiotic recruitment of condensin complexes

Despite the differences in mitotic and meiotic chromosome architecture, condensin I occupies the same domains as AIR-2 in both processes (this study), and condensin II colocalizes with centromeric protein CENP-A in both processes (Chan et al., 2004; Hagstrom et al., 2002; Stear and Roth, 2002). Consistently, AIR-2 is required for condensin I recruitment in mitosis and for correct condensin I localization in meiosis, but not for condensin II targeting in either process. By contrast, CENP-A is needed for recruitment of condensin II in mitosis but not during meiosis (Chan et al., 2004; Stear and Roth, 2002). The fact that condensin II can load onto meiotic chromosomes in the absence of CENP-A, is consistent with CENP-A function being dispensable during C. elegans meiosis (Monen et al., 2005).

Our data is consistent with a wild-type AIR-2 protein or its chromatin mark H3S10-P serving as a direct recruiter for condensin I in mitosis. However, an inactive kinase is not sufficient for recruitment (see Fig. 2). In meiosis, AIR-2 plays a different role. In meiosis, condensin I can associate with chromosomes in the absence of AIR-2, but without targeting cues from AIR-2, it localizes to both bivalent arms. Interestingly, we observed similar spreading of condensin I in oocytes in which the AIR-2 domain is expanded, indicating that, when present, AIR-2 is sufficient to dictate condensin I localization. Consistent with that, condensin I also colocalizes with AIR-2 on spo-11 univalents (where AIR-2 distribution is stochastic) and at the sister chromatid interface on bioriented rec-8 univalents.

The more limited localization of AIR-2 compared with the broader distribution of H3S10-P in the lab-1(RNAi) and the htp-1(gk174) backgrounds is reminiscent of what was observed for some histone modifying enzymes and their modification in the context of gene silencing (Kahn et al., 2006; Papp and Muller, 2006; Schwartz et al., 2006) or activation (Gelbart et al., 2009; Parker et al., 2008). It is unclear whether it represents a transient spreading of AIR-2 to phosphorylate H3S10 in a broader region or transient looping of other chromosomal territories into the AIR-2 occupied domain for modification. In any case, H3S10-P was not sufficient to mislocalize condensin I. Only where AIR-2 was detectable by immunofluorescence, could we see a spreading of the condensin-I-occupied domain.

Condensin I and chromosomal passengers

It is intriguing that condensin I colocalizes with AIR-2 not just on chromosomes but also on the anaphase spindle, both in mitosis and in meiosis. Localization on the anaphase spindle is most prominent during the acentrosomal oocyte meiotic anaphase (Fig. 4A–C), when spindle microtubules are found predominantly between chromosomes (Dumont et al., 2010; Wignall and Villeneuve, 2009) but can also be detected during mitosis (Fig. 1B–D). During centrosome-based sperm meiosis, midbody microtubules are not prominent and cytokinesis is sometimes incomplete until spermatids bud off from the residual body (Shakes et al., 2009). Under these circumstances, condensin I and AIR-2 levels are also low between separating chromosomes. The spindle localization of condensin I mirrors Aurora B kinase and other CPC components (Rogers et al., 2002), and it will be interesting to determine whether condensin I contributes to Aurora B function at this stage. Condensin localization to the midzone has also been seen in yeast mitosis (Nakazawa et al., 2011).

Is the role of Aurora B in condensin I targeting during meiosis conserved?

Aurora B regulates many events to coordinate cell division, including kinetochore microtubule attachments, chromosome orientation, cohesion release and cytokinesis. Most of these functions are conserved between monocentric and holocentric organisms, with some important differences in meiosis (Fig. 8). On monocentric chromosomes, Aurora B is needed for coorientation of sister kinetochores and biorientation of kinetochores of homologs by destabilizing improper kinetochore–microtubule attachments at the centromeres (Hauf et al., 2007; Monje-Casas et al., 2007). In holocentric organisms, such as C. elegans, localized centromeres are lacking, and instead the location of crossover determines which end of the chromosome will form the short arm of the bivalent (Nabeshima et al., 2005), which in turn determines the plane of chromosome orientation (Albertson and Thomson, 1993; Wignall and Villeneuve, 2009). In both monocentric and in holocentric organisms, Aurora B is located in an ideal position to monitor homolog biorientation and sister co-orientation: at the centromeres in monocentric organisms and at the bivalent short arm in holocentric organisms.

Fig. 8.
Fig. 8.

Model for AIR-2 activity on monocentric and holocentric chromosomes during meiosis I. On holocentric chromosomes, AIR-2 is in an ideal position (i.e. at the short arm of the bivalent) to promote both homolog biorientation and sister coorientation by ensuring that microtubules do not cross the AIR-2 zone, thereby keeping sisters together and homologs apart. The AIR-2 zone is also the region where sister chromatid cohesion must be released in meiosis I to allow homolog separation. At the short arm of bivalents, AIR-2 activity is also needed to restrict condensin I to the short arm of the bivalent. On monocentric chromosomes, Aurora B is enriched at the inner centromere and promotes homolog biorientation and sister coorientation. This zone of Aurora B activity is where centromeric cohesion must be protected in meiosis I. The role of Aurora B in condensin I and II targeting in meiosis in monocentric organisms is not known.

However, the role of Aurora B in the regulation of sister chromatid cohesin during meiosis I is different in monocentric and holocentric organisms. During meiosis I, sister chromatid cohesion is preserved at centromeres of monocentric chromosomes and at the long arm of holocentric chromosomes, whereas cohesion is released along chromatid arms of monocentric chromosomes and the short arm of holocentric bivalents. In monocentric organisms, Aurora B promotes preservation of cohesion at centromeres (Monje-Casas et al., 2007; Resnick et al., 2006; Yu and Koshland, 2007). By contrast, in worms, AIR-2/Aurora B functions to promote cohesion release at the short arm (Kaitna et al., 2002; Rogers et al., 2002).

In monocentric organisms, the activities that orient chromosomes and those that maintain connections between sisters during meiosis are located at the same place, the centromere. By contrast, in holocentric organisms these activities are located at opposite domains: chromosome orientation is achieved by activities along the short arms of bivalents, whereas preservation of connection between sisters is achieved along the long arm (de Carvalho et al., 2008; Martinez-Perez et al., 2008; Rogers et al., 2002). These spatial differences probably explain why Aurora B evolved different roles with respect to regulation of cohesion in these organisms. Given the similarities and differences in Aurora B functions in monocentric and holocentric organisms, it will be interesting to determine which aspects of condensin I regulation by Aurora B are conserved in meiosis in monocentric organisms.

Materials and Methods

C. elegans strains

All strains were maintained as described previously (Brenner, 1974) and grown at 20°C, unless indicated otherwise. For analysis of AIR-2 deficiency, air-2 (or207ts) L4 worms were shifted to 25°C for 24 hours. Strains include N2 Bristol strain (wild type), EKM28 unc-119(ed3) III; cldEx4 [Ppie-1::CAPG-1::GFP unc-119(+)], EU630 air-2(or207ts)I, VC666 rec-8(ok 978) IV/nT1[qIs51](IV; V), TY0420 dpy-27(y57)III, TY3837 dpy-28(s939)III/qC1, and TY4341 dpy-26(n199) unc-30(e191)/nT1(G)IV;V, EKM21 spo-11(ok79) IV/nT1(G) IV;V, EKM22 htp-1(gk174)IV/nT1(G) IV;V.

Antibodies

Primary antibodies were: rabbit anti-CAPG-1 antibody (Csankovszki et al., 2009); rabbit anti-KLE-2, anti-DPY-26 and anti-DPY-28 antibodies (Kirsten Hagstrom, University of Massachusetts, Worcester, MA) (Csankovszki et al., 2009), rabbit anti-HCP-6 antibody (Raymond Chan, University of Michigan, Ann Arbor, MI) (Chan et al., 2004), rabbit anti-AIR-2 antibody (Jill Schumacher, University of Texas, MD Anderson Cancer Center, Houston, TX) (Schumacher et al., 1998), mouse anti-H3S10-P (6G3; Cell Signaling Technology), mouse anti-NPC antibody (mab414; Abcam); and mouse anti-α-tubulin (DM1A; Sigma). Secondary antibodies were: FITC- or Cy3-conjugated donkey anti-rabbit-IgG and donkey anti-mouse-IgG antibodies (Jackson ImmunoResearch).

RNA interference

RNAi by feeding was performed as described previously (Kamath et al., 2003). To generate an RNAi construct for air-2, a genomic region was PCR amplified (using primers 5′-CATGCTCGAGTGGACATTTCCATGTAGCGA-3′ and 5′-GATCAAGCTTGGGGTTAGACGATTGGGAA-3′), digested with XhoI and HindIII cloned into the DT7 vector (Kamath et al., 2003). For lab-1 RNAi, bacterial cultures were grown at 37°C for 20 hours and induced with IPTG for 2 hours before plating; for air-2 RNAi, 50 ml bacterial cultures were grown at 37°C for 20 hours, induced with IPTG for 2 hours, pelleted and resuspended in 500 μl of fresh LB broth and plated as a concentrated bacterial lawn. RNAi was initiated at the L1 stage. L4 worms were transferred onto a fresh plate, and allowed to produce progeny (F1) for 24 hours. F1 worms were processed 24 hours post L4 for immunofluorescence. To deplete AIR-2 in meiosis, air-2(or207ts) hermaphrodites were grown on AIR-2 RNAi plates at 25°C from L1 to adulthood. Control experiments were performed using the same conditions as RNAi experiments.

Immunostaining

Adult worms were dissected in 1× sperm salts (50 mM Pipes pH 7, 25 mM KCl, 1 mM MgSO4, 45 mM NaCl and 2 mM CaCl2), fixed in 2% paraformaldehyde in 1× sperm salts for 5 minutes and frozen on dry-ice for 10 minutes. Slides were washed three times for 10 minutes each time in PBS with 0.1% Triton X-100 (PBST) before incubation with 30 μl of diluted primary antibody in a humid chamber, overnight at room temperature. Double labeling of samples was performed, with all primary antibodies simultaneously, during this overnight incubation. Slides were then washed three times with PBST, for 10 minutes each time, incubated for 1 hour with 30 μl diluted secondary antibody at 37°C, washed again twice for 10 minutes each with PBST, and once for 10 minutes with PBST plus DAPI. Slides were mounted with Vectashield (Vector Labs). For colocalization studies of CAPG-1 and AIR-2, or CAPG-1 and HCP-6, rabbit antibodies were directly labeled using the Zenon rabbit IgG labeling kit (Molecular Probes) according to manufacturer's instructions.

For detergent extraction, nucleoplasmic proteins were extracted from oocytes by dissecting adults in 1× sperm salts plus 1% Triton X-100 and processed as above. For methanol–acetone fixation (supplementary material Fig. S3A), adult hermaphrodites were dissected in 1× sperm salts and frozen on dry-ice for 10 minutes. The slides were fixed for, 1 minute each time, in methanol followed by acetone at −20°C. Slides were washed three times, for 10 minutes each time, in PBST, before incubation with primary antibody.

Embryos were obtained from hermaphrodites by bleaching, fixation with Finney fixative (2% paraformaldehyde, 18% methanol, 10 mM Pipes pH 7.5, 60 mM KCl, 8 mM NaCl, 2.6 mM EGTA, 0.4 mM spermidine, 0.16 mM spermine and 0.4% β-mercaptoethanol), before being frozen at −80°C for 20 minutes, thawed, fixed for 20 minutes at room temperature, and washed in PBST for 15 minutes. Samples were incubated with primary antibody overnight at room temperature. Embryos were washed three times (15 minutes each time) in PBST and incubated overnight with secondary antibody at room temperature. This incubation was followed by two PBST washes and a third wash in PBST plus DAPI. Embryos were mounted onto slides with Vectashield.

Images were captured using a Hamamatsu ORCA-ERGA CCD camera on an Olympus BX61 motorized X-drive microscope using a 60× PlanApo oil immersion objective with a NA of 1.42. Images were captured in Z-stacks with planes at 0.2 μm intervals and deconvolved and projected with 3i Slidebook software. Adobe Photoshop was used for image assembly.

Live imaging

Embryos were dissected into blastomere culture medium (Shelton and Bowerman, 1996) and mounted in a hanging drop to alleviate osmotic and mechanical pressures. Imaging was performed on a PerkinElmer spinning-disk confocal system with a Nipkow CSU10 scanner (Yokogawa), an EM-CCD camera (C9100-50, Hamamatsu Photonics), an inverted microscope (Axio Observer; Carl Zeiss) and a 63× Plan Apochromat 1.4 NA objective (Carl Zeiss). Images were acquired with Volocity acquisition software (PerkinElmer) and collected every 5 seconds with a 2-second exposure time and 2× binning. Images were processed with Gaussian blur to reduce noise, and level adjustments were made using ImageJ (Abramoff et al., 2004).

Acknowledgements

We thank Raymond Chan, Kentaro Nabeshima, Kirsten Hagstrom, Martha Snyder, Laura Custer and Michael Wells for comments on the manuscript, Uchita Patel and Emily Laughlin for technical help, Martha Snyder for assistance with some of the staining experiments and Cathy Collins for the use of spinning disk confocal system.

Footnotes

  • Funding

    This work was supported by the National Institutes of Health [grant number RO1 GM079533 to G.C.]; and by Predoctoral Training in Genetics, National Institutes of Health [grant number NIH T32 GM07544 to E. P. and K.C.]. Some nematode strains were provided by the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health National Center for Research Resources (NCRR). Deposited in PMC for release after 12 months.

  • Supplementary material available online at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.088336/-/DC1

  • Accepted June 14, 2011.

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Research Article
Different roles for Aurora B in condensin targeting during mitosis and meiosis
Karishma S. Collette, Emily L. Petty, Netta Golenberg, Joshua N. Bembenek, Gyorgyi Csankovszki
Journal of Cell Science 2011 124: 3684-3694; doi: 10.1242/jcs.088336
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