Correspondence to Francis J. McNally: fjmcnally{at}ucdavis.edu
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Introduction |
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Detailed studies of meiotic spindle movements in mouse (Maro et al., 1984, 1986; Verlhac et al., 2000) and Caenorhabditis elegans (Yang et al., 2003) have revealed a conserved series of movements that include translocation of the spindle to the cortex and rotation of the spindle from a parallel to a perpendicular orientation to allow chromosome segregation into a polar body. Movement and orientation of mitotic spindles in animals and fungi is thought to occur through astral microtubules that emanate from centriole-containing centrosomes or spindle pole bodies (Gonczy, 2002; Sheeman et al., 2003). However, the female meiotic spindles of humans (Sathananthan, 1997), cows (Navara et al., 1994), mice (Gueth-Hallonet et al., 1993), Drosophila melanogaster (Theurkauf and Hawley, 1992), and C. elegans (Albertson and Thomson, 1993) do not have centrioles or their associated astral microtubule arrays.
Mice and C. elegans have evolved different mechanisms for translocating their meiotic spindles to the oocyte cortex in the absence of astral microtubule arrays. Translocation of the mouse meiosis I spindle to the cortex is dependent on F-actin and c-mos but does not require microtubules (Verlhac et al., 2000). In contrast, we have previously shown that translocation of the C. elegans meiosis I spindle is dependent on microtubules and the microtubule-severing enzyme MEI-1 but is not dependent on F-actin (Yang et al., 2003). In both cases, the mechanism that polarizes cytoskeletal filaments toward the cortex and the mechanism of movement are unknown.
The microtubule dependence of C. elegans meiotic spindle translocation suggested that one or more microtubule motor proteins would be required either to establish bipolarity of spindle microtubules or to directly transport the spindle on the cytoplasmic microtubule array. To identify this motor (or motors), we initiated an RNA interference (RNAi) screen of the 23 microtubule motor subunits encoded in the C. elegans genome. We identified UNC-116, the kinesin-1 heavy chain (Patel et al., 1993), as essential for normal translocation of the meiotic spindle to the cortex. Because meiotic spindle structure appears normal in UNC-116depleted embryos, this result suggests that the spindle is translocated on the acentrosomal cytoplasmic microtubule array. Such directional transport on an acentrosomal microtubule array is also observed during mRNA localization in D. melanogaster oocytes (Cha et al., 2001) and vesicle transport in plant cells (Gunning and Steer, 1996).
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Results |
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Consistent with this hypothesis, the orientation of spindles moving toward the cortex was different between unc-116(RNAi) and wild-type spindles. In wild-type worms, 14/17 (82.4%) of the meiotic spindles approached the cortex in a sideways orientation (e.g., Fig. 1 A). After the prolonged stationary period, 10/12 (83.3%) unc-116(RNAi) spindles moved toward the cortex with one pole leading (e.g., Fig. 1 B). The velocity of late translocation in unc-116(RNAi) worms was also different than the velocity of early translocation in wild-type worms (Table I). Thus, unc-116(RNAi) worms appear to be completely defective in an early translocation mechanism but retain a distinct, late translocation mechanism.
The movement of unc-116(RNAi) spindles is mediated by a distinct, anaphase-promoting complex (APC)dependent mechanism
If movement of unc-116(RNAi) spindles to the cortex is indeed due to a discrete, late mechanism, this should be revealed by distinct genetic requirements for the early and late mechanisms. In a previous study (Yang et al., 2003), we showed that wild-type spindle translocation is followed sequentially by spindle shortening, spindle rotation, and anaphase chromosome segregation. Depletion of APC subunits had no effect on spindle translocation but blocked spindle shortening and rotation (Fig. 1 E; Yang et al., 2003). These results indicated that spindle shortening is APC dependent, whereas early translocation is APC independent. To test whether or not the late translocation observed in unc-116(RNAi) embryos is APC dependent, we compared the kinetics of spindle shortening and spindle translocation in wild-type worms with those of worms depleted of UNC-116, the APC subunit, MAT-2, or both.
In 19/19 wild-type worms, spindles initiated translocation while the zygote was in the spermatheca and arrived at the cortex 7.2 ± 1.7 min before spindle shortening initiated (Fig. 1 C). However, in 9/9 unc-116(RNAi) time-lapse sequences, where the spindle was oriented such that both translocation and shortening could be measured, translocation initiated at the same time that spindle shortening initiated and spindles arrived at the cortex 3.6 ± 1.1 min after shortening started (Fig. 1 D). As previously reported for worms depleted of the FZY-1 subunit of the APC (Yang et al., 2003), spindle shortening was blocked in mat-2 (ts) worms at nonpermissive temperature but spindle translocation occurred with wild-type kinetics. (Fig. 1 E, n = 5). In 5/5 of mat-2(ts); unc-116(RNAi) double mutant worms, spindle length did not change and spindle translocation to the cortex was completed blocked (Fig. 1 F). The complete block to spindle translocation in mat-2(ts); unc-116(RNAi) double mutant worms was confirmed by single time point analysis (Fig. 1 H) to eliminate any contribution by photodamage. These results demonstrated that the late spindle translocation observed in unc-116(RNAi) worms is APC dependent and kinesin-1 independent whereas the early translocation mechanism observed in wild-type worms is APC independent and kinesin-1 dependent.
UNC-116 is not required for other aspects of meiosis
We previously demonstrated that both tubulin and the katanin orthologue MEI-1 are required for meiotic spindle translocation (Yang et al., 2003). In both of these cases, however, spindle structure was severely perturbed, making it impossible to discern if MEI-1 is directly involved in spindle translocation or if normal spindle architecture is required for translocation. In contrast, unc-116(RNAi) meiotic spindles observed by GFP-tubulin fluorescence had a wild-type structure (Fig. 1, B and H). These spindles had wild-type length and width (Table I), exhibited normal anaphase chromosome segregation (Fig. 2 and Table I), and shortened with normal kinetics (Table I and Fig. 1 D). These results indicate that UNC-116 is primarily (if not exclusively) required in the early embryo for meiotic spindle translocation.
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UNC-116 is essential maternally because it is required for polar body formation
To determine the consequences of delayed spindle translocation, we filmed GFP-histone in unc-116(RNAi) worms to track chromosome movements during meiosis. In 5/5 cases where delayed spindle translocation was observed, anaphase chromosome segregation occurred normally with one set of chromosomes being pushed into the cortex during both anaphase I and II (Fig. 2). However, failures in polar body formation during meiosis I, meiosis II, or both were frequently observed. In the example shown in Fig. 2, chromosomes that segregated into the cortex at meiosis I (21 min) collapsed back into the embryo so that 12 rather than 6 chromosomes were present in the meiosis II spindle (24.3 min). In this example, meiosis II polar body formation was successful (36.3 min). In 60% of GFP-histone unc-116(RNAi) movies, one or both polar bodies failed to form, even though anaphase occurred at the cortex. In contrast, both polar bodies formed successfully in 100% of GFP-histone wild-type time-lapse sequences (n = 11). To eliminate any contribution from photodamage, polar body number was also scored by fixed time point analysis. A failure to form one or both polar bodies was observed in 67% of unc-116(RNAi) embryos compared with 5% of wild-type embryos (see Table III). A failure to form one or both polar bodies should result in embryos that are triploid or pentaploid. Whereas triploid worms can be viable (Madl and Herman, 1979), pentaploidy would be expected to cause embryonic lethality later in development. Indeed, 67% embryonic lethality was observed among embryos laid by unc-116(RNAi) worms (Table II). These results indicate that UNC-116 and the early spindle translocation pathway are required for meiotic cytokinesis, even though the spindles always achieve perpendicular cortical contact eventually.
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Consistent with the hypomorphic nature of the rh24sb79 allele, time-lapse imaging of GFP-tubulin unc-116(rh24sb79) worms revealed a block in preanaphase meiotic spindle translocation in only 7/14 worms (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200411132/DC1) versus 15/19 for unc-116(RNAi). Significantly, the combination of RNAi and this loss-of-function allele did not increase the level of embryonic lethality beyond that of unc-116(RNAi) alone (Table II). This finding strongly suggests that unc-116(RNAi) indeed represents the unc-116(null) embryonic phenotype. We conclude that both the spindle translocation defect and the maternal-effect embryonic lethality observed in unc-116(RNAi) worms is in fact due to a severe reduction (if not total elimination) of UNC-116 activity.
The kinesin light chains KLC-1 and -2 are required for early spindle translocation
Kinesin-1 purified from a variety of species, including C. elegans, consists of a tetramer with two heavy chain and two light chain subunits (Vale et al., 1985; Saxton et al., 1988; Signor et al., 1999). To test whether or not early meiotic spindle translocation depends on a kinesin-1 heavy chain/light chain complex, we acquired GFP-tubulin time-lapse sequences from worms treated with dsRNA corresponding to either of the C. elegans kinesin light chain homologues, KLC-1 or KLC-2. A block to early spindle translocation was observed in 3/6 klc-1(RNAi) worms, 2/7 klc-2(RNAi) worms, and 4/5 doubly treated klc-1(RNAi); klc-2(RNAi) worms (Fig. 3, A and B; and Table I). In addition, depletion of kinesin light chains in metaphase-arrested mat-2(ts) embryos resulted in spindles arrested far from the cortex (Fig. 3 C). Thus, the phenotype of klc-1(RNAi) or klc-2(RNAi) worms was qualitatively the same, but quantitatively weaker, than the phenotype of unc-116(RNAi) embryos. Western blots of klc-2(RNAi) worms probed with a KLC-2specific antibody revealed that considerable KLC-2 protein product remains in these worms (Fig. S1). Thus the weaker phenotypes observed were at least in part due to the incomplete effectiveness of the RNAi. Simultaneous treatment of worms with dsRNAs corresponding to both light chains (klc-1(RNAi); klc-2(RNAi)) resulted in embryonic lethality (Table II), polar body defects (Table III), and translocation defects that were quantitatively similar to unc-116(RNAi) worms. These results indicate that the two kinesin light chains act redundantly and that a complex of kinesin heavy and light chains is essential for the preanaphase translocation of the meiotic spindle.
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Discussion |
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KCA-1 as a novel cargo adaptor for the meiotic spindle
We showed that the previously uncharacterized gene product C10H11.10/KCA-1 can form a stochiometric protein complex with the kinesin light chain KLC-2b and the kinesin heavy chain UNC-116 in vitro. Furthermore, kca-1(RNAi) yields the same phenotype as depletion of heavy chain or depletion of both light chains. These results indicate that KCA-1 may be the cargo adaptor that allows UNC-116 to form transient attachments to the meiotic spindle. The Caenorhabditis briggsae predicted gene CBG03328(Wormbase) exhibits 40% amino acid identity over its entire length with KCA-1, but no homologues outside of nematodes or obvious sequence motifs are apparent. In the C. elegans global yeast two-hybrid analysis of Li et al. (2004), KCA-1 interacted only with the kinesin light chains KLC-1 and KLC-2 and with HPL-2. HPL-2 is one of two C. elegans orthologues of heterochromatin binding protein 1, a highly conserved component of pericentric heterochromatin (Couteau et al., 2002). If KCA-1 and HPL-2 interact in vivo, this interaction would complete a physical bridge between UNC-116 and the meiotic chromosomes. In this model, UNC-116 would transport meiotic chromosomes toward the cell cortex along cytoplasmic microtubules and the spindle would be dragged along with the chromosomes. This hypothetical mechanism would be consistent with the sideways orientation of spindles during wild-type spindle translocation.
Although our data support a model in which kinesin-1 transports the spindle along cytoplasmic microtubules, there is a 120-fold difference between the gliding velocity of purified C. elegans kinesin-1 (2 µm/s; Signor et al., 1999) and spindle translocation velocity (1 µm/min). The unexpectedly slow translocation might be explained if the UNC-116KLCKCA-1 complex actually acts indirectly, perhaps by organizing the cytoplasmic microtubule array. However, we favor a model in which translocation is slowed by opposing forces from kinesin-1 molecules that attempt to transport the spindle on microtubules with plus ends oriented away from the cortex.
Acentriolar spindles are positioned by unique mechanisms
Positioning of female meiotic spindles at the oocyte cortex is observed in all animal species, resulting in highly asymmetric divisions giving rise to small polar bodies and one large oocyte in most species. In some organisms such as Chaetopterus variopedatus (Lutz et al., 1988), Spisula solidissima (Palazzo et al., 1992), and starfish (Hamaguchi, 2001; Zhang et al., 2004), female meiotic spindles have robust astral microtubule arrays nucleated by centriole-containing centrosomes. In these species, it is likely that motor proteins associated with the cortex generate pulling forces on astral microtubules by the same mechanisms proposed for mitotic spindles in many species (Gonczy, 2002; Sheeman et al., 2003). In contrast, female meiotic spindles from organisms such as nematodes (Albertson and Thomson, 1993) and humans (Sathananthan, 1997) do not have centrioles and astral microtubule arrays are not apparent. It is reasonable to speculate that novel spindle positioning mechanisms have evolved to move acentriolar, anastral spindles to the oocyte cortex. Indeed, amphibian oocytes assemble a completely novel structure called the transient microtubule array that mediates movement of chromosomes to the cortex before a bipolar spindle is assembled (Becker et al., 2003). The genetic requirements for cortical translocation of preassembled, acentriolar meiotic spindles have been reported only for mouse (Verlhac et al., 2000) and C. elegans (Yang et al., 2003; this study), and these studies reveal apparently distinct requirements. Mouse meiotic chromosomes translocate to the cortex in the absence of microtubules using an actin-dependent mechanism, suggesting that the spindle is dragged along with the chromosomes (Verlhac et al., 2000). In contrast, movement of C. elegans meiotic chromosomes to the oocyte cortex is completely dependent on microtubules and apparently does not require F-actin (Yang et al., 2003). If the interaction between the C. elegans heterochromatin protein HPL-2 and the kinesin cargo adaptor KCA-1 occurs in vivo, then kinesin-1 may transport meiotic chromosomes, dragging the spindle along with the chromosomes. In this case, mice and worms would be using identical mechanisms mediated by different molecules.
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Materials and methods |
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In utero filming
Adult hermaphrodites were anesthetized with tricaine/tetramisole and mounted between a coverslip and a thin agarose pad as described previously (Yang et al., 2003). Stage temperature was 2224°C. Images were acquired at 15-s intervals on an upright microscope (model Microphot SA; Nikon) using a 60x PlanApo 1.4 objective and a Photometrics Quantix/KAF1400 camera (Roper Scientific). Illumination from an HBO100 light source was shuttered with a Uniblitz shutter. Time-lapse acquisition was controlled with IP Lab Spectrum software (Scanalytics). Quantitative analysis of translocation velocities was also performed with IP Lab Spectrum.
RNAi by soaking
The cDNA inserts of plasmid cDNA clones were amplified by PCR using one primer with a 5' T7 promoter extension and a second primer with a 5' T3 promoter extension. Linear PCR products were concentrated using spin columns (QIAGEN) and used as templates in separate T3 and T7 RNA polymerase transcription reactions (MegaScript T7 or T3 Kit; Ambion). After treatment with DNase I, RNAs were purified by LiCl precipitation and annealed. 4050 L4 worms were soaked in 10 µl of 1 mg/ml dsRNA dissolved in 66 mM K-PO4 and 10 mM K-citrate, pH 7.5, in a humidified chamber for 16 h at 25°C and then transferred to OP50-seeded plates to recover for 24 h before filming or lethality scoring. The following cDNA clones were used: KLC-1-yk1256g06, KLC-2-yk1323f03, C10H11.10-yk442h9, and UNC-116-yk255a4 (all obtained from Y. Kohara, National Institute of Genetics, Mishima, Japan). C10H11.10 was initially identified using RNAi by feeding with clone I-2I09 from the genomic RNAi feeding library (MRC Gene Service; Kamath et al., 2003).
Antibody generation
Rabbits were immunized with inclusion bodies composed of 6his-UNC-116 stalk tail or 6his-KLC-2b. UNC-116 or KLC-2specific antibodies were bound to 6his-UNC-116 or 6his-KLC-2b immobilized on nitrocellulose strips and eluted with 200 mM glycine, pH 2.4, before neutralization with Tris-Cl, pH 8.0.
Protein interaction experiments
Different fragments of KCA-1 were PCR amplified from the yk624e12 cDNA clone. In the amino acid numbering shown in Fig. 5, 1 corresponds to a methionine that is 43 aa downstream from the predicted start codon in Wormbase. These fragments were cloned into pET41a (Novagen), expressed in BL21(DE3) E. coli, and purified by Ni2+ chelate chromatography via the 6his tag on the GST encoded by pET41a. Full-length KLC-2b was PCR amplified from yk886b10 and cloned into pET28a (Novagen) for expression of 6his-KLC-2b and into pTYB12 (New England Biolabs, Inc.) for expression of CBD-KLC-2b. The COOH-terminal 442 aa of UNC-116 were PCR amplified from yk859d09 and cloned into pTYB12. Full-length MEI-1 was also PCR amplified and cloned into pTYB12. 6his-KLC-2b was purified by Ni2+ chelate chromatography after expression in E. coli. CBD-KLC-2b, CBD-UNC-116, and CBD-MEI-1 were expressed in E. coli, and high speed supernatants from microfluidized extracts were used directly in the binding assays without further purification after normalizing to equal concentrations of CBD fusion protein. 50 µl of glutathione Sepharose beads were incubated with an excess of GST-fusion protein for 1 h, and then washed extensively. Coated beads were incubated with 1 ml of E. coli lysate containing the appropriate CBD fusion and 25 mM Tris 8.0, 1 M NaCl, 0.1% Triton X-100, and 5% glycerol for 2 h at 22°C with constant rocking. After extensive washing with the same buffer, bound complexes were eluted with SDS-Laemmli buffer.
Isolation of unc-116(rh24sb79)
To identify a loss-of-function allele of unc-116, we reverted the gain-of-function properties of unc-116(rh24). These were isolated as intragenic revertants that blocked the temperature-sensitive enhancement between rh24/+ and mei-1(ct46ts)/+. Both unc-116(rh24) and mei-1(ct46ts)/+ result in small, misoriented first cleavage spindles. At 20°C, rh24/+ hermaphrodites segregate 12% dead embryos, 78% are lethal from mei-1(ct46ts)/+ animals, whereas 99.5% of the embryos from the double mutant rh24/+; mei-1(ct46ts)/+ fail to hatch. Replacing rh24 with a chromosomal deficiency of the region in nDf20/+; ct46ts/+ hermaphrodites produces 82% unhatched embryos, very close to the level seen with ct46ts)/+ alone (all reported values are corrected for the lethality stemming from nDf20 by itself). This finding indicates that unc-116(rh24) is a gain-of-function allele and that an unc-116(null)/+ revertant could be isolated in this background.
The strain mei-1(ct46ts) unc-29(e1072)/unc-13(e1091) daf-8(e1393) lin-11(n566) I; unc-116(rh24) dpy-17(e164)/qC1 III was mutagenized with EMS and grown at 15°C (where 85% of the embryos are dead). When the oldest F1 animals reached the last larval stage (L4), the plates were shifted to 20°C. A total of 7,200 F1 progeny were shifted, of which 1/4 (1,800) were the relevant rh24/+; mei-1(ct46ts)/+ genotype. Six plates produced an F2 generation, indicating the presence an F1 animal with a suppressor. One of these contained sb79, which is tightly linked to the original rh24 mutation. Three-factor mapping indicated that sb79 is <0.12 cM from rh24, consistent with an intragenic event (as confirmed by sequencing). The other five suppressors were linked to chromosome I and are likely alleles of mei-1 and/or mei-2, which mutate to dominant suppressors of ct46 at a high rate (Mains et al., 1990).
rh24sb79 behaves as a strong loss-of-function but not a null allele. unc-116(rh24sb79)/+; mei-1(ct46ts)/+ showed about the same level of lethality at 20°C as mei-1(ct46ts)/+ (70 vs. 78%), indicating that the enhancement of mei-1 was completely lost by rh24sb79. Similar results were seen for mutations of two other genes, mel-26(ct61) and zyg-9(b244), which are also strongly enhanced by rh24 but not by rh24sb79. Although the lethality of rh24/+ is semidominant, showing 26% unhatched embryos at 25°C, rh24sb79/+ segregated only 1.2% dead embryos. Together, the aforementioned data indicate that rh24sb79 is a strong loss-of-function mutation. However, it is not null because its behavior, when heterozygous with rh24, differs from that of a deficiency. Although the lethality of rh24/nDf20 at 25°C was 87%, the value was 48% for rh24/rh24sb79. This finding indicates that rh24sb79 has some unc-116(+) activity.
Online supplemental material
Video 1 corresponds to Fig. 1 A and Video 2 corresponds to Fig. 1 B. Fig. S1 shows antiUNC-116 and antiKLC-2 immunoblots of dsRNA-treated worms. Fig. S2 shows the spindle translocation defect in unc-116(rh24sb79). Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200411132/DC1.
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Acknowledgments |
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This work was supported by a grant from the University of California Cancer Research Coordinating Committee. P.E. Mains was supported by grants from the Canadian Institutes of Health Research and the Alberta Foundation for Medical Research.
Submitted: 22 November 2004
Accepted: 22 March 2005
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