Section of Molecular and Cellular Biology, University of California, Davis, CA 95616, USA
* Present address: Exelixis, South San Francisco, CA 94080, USA
Author for correspondence (e-mail: lsrose{at}ucdavis.edu)
Accepted 1 July 2002
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SUMMARY |
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Key words: Spindle orientation, Asymmetric division, Microtubules, Embryo, DEP domain, C. elegans
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INTRODUCTION |
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In C. elegans, the PAR proteins are required for cellular polarity and are asymmetrically localized at the cell periphery in response to the position of the sperm aster (Bowerman and Shelton, 1999; Goldstein and Hird, 1996
; Rose and Kemphues, 1998b
; Wallenfang and Seydoux, 2000
). PAR-3, PAR-6 and PKC-3 are localized to the anterior periphery of the one-cell embryo, whereas PAR-2 is restricted to the posterior periphery. PAR-3/PAR-6/PKC-3 and PAR-2 are interdependent for localization and are required for the posterior localization of PAR-1. PAR-1 is then necessary for the localization of MEX-5 and downstream cell fate determinants in anteroposterior (AP) domains (Bowerman and Shelton, 1999
; Gotta and Ahringer, 2001a
; Schubert et al., 2000
). The first mitotic spindle is aligned along the anteroposterior axis, resulting in the differential segregation of cell-fate determinants upon division; cleavage is also unequal, generating a larger anterior AB cell and a smaller posterior P1 cell. Anterior and posterior PAR domains are re-established in the P1 cell, which also divides asymmetrically.
To produce the asymmetric cell division described above, several polarized nuclear and spindle movements are required, including nuclear centration, rotation and asymmetric spindle positioning (Bowerman and Shelton, 1999; Gotta and Ahringer, 2001b
; Rose and Kemphues, 1998b
). In the one-cell embryo, the female and male pronuclei meet in the posterior and then move to the middle of the embryo in a process called centration. As the pronuclei move, the entire nuclear-centrosome complex undergoes a 90° rotation so that the spindle will form on the AP axis. The spindle also moves and elongates asymmetrically towards the posterior during anaphase resulting in unequal cleavage. Similar polarized nuclear and spindle movements occur in the P1 cell, which also undergoes asymmetric cell division. The nature of these polarized movements suggests that they are mediated by asymmetric forces, at least some of which are generated by interactions between astral microtubules and the cell cortex/periphery, and are PAR dependent (Cheng et al., 1995
; Grill et al., 2001
; Hyman, 1989
; Hyman and White, 1987
; Keating and White, 1998
; Waddle et al., 1994
). However, the mechanisms by which the asymmetric localizations of the PAR proteins are transduced into asymmetric forces on the centrosomes and the spindle remain to be elucidated. One potential target of the polarity pathway is the microtubule motor cytoplasmic dynein. Inhibition of the function of dynein or its associated dynactin complex (to levels that still allow formation of a spindle) blocks nuclear centration and rotation in one-cell embryos (Gönczy et al., 1999
). Some members of the dynein/dynactin complex appear enriched at the cell division remnant in two-cell embryos (Gönczy et al., 1999
; Skop and White, 1998
; Waddle et al., 1994
) and could thus provide an asymmetric cue for rotation. However, because dynein appears uniformly localized at the cortex of one-cell embryos, its presence alone appears insufficient to explain the asymmetric nature of nuclear centration and rotation at this stage.
The PAR proteins could asymmetrically regulate dynein or other cortical proteins directly. Alternatively, there could be intermediate proteins that transduce the polarity cues to the spindle orientation machinery, analogous to the MEX-5 intermediate that connects PAR asymmetry with the localization of cell fate determinants (Bowerman and Shelton, 1999; Gotta and Ahringer, 2001a
; Schubert et al., 2000
). There are three criteria for such intermediates that function downstream of the PAR proteins in spindle positioning. First, mutations in an intermediate gene should affect spindle position but not asymmetric localization of PAR proteins and other aspects of polarity. Second, an intermediate protein should directly or indirectly regulate the generation of forces on the spindle, and thus mutants should exhibit failures in some or all of the polarized nuclear and spindle movements described above. Third, at least one component of an intermediate pathway should be asymmetrically activated or localized in response to the PAR proteins.
Several genes have been described that fit the first two criteria for an intermediate. These include the trimeric G-protein subunit encoding genes gpb-1 (G protein ß-1), gpc-2 (G protein -2), goa-1 (G protein
1, class O) and gpa-16 (G protein
-16), as well as ric-8 (Gotta and Ahringer, 2001b
; Miller and Rand, 2000
; Zwaal et al., 1996
). Comparisons of the phenotypes of embryos depleted for GOA-1, GPA-16 and GPB-1 singly and in combinations suggest that the G
s function redundantly in asymmetric positioning of the first spindle, while Gß
functions in centrosome migration and nuclear rotation (Gotta and Ahringer, 2001b
). RIC-8 appears to play a positive role in GOA-1 signaling in the embryo (Miller and Rand, 2000). The G proteins are uniformly localized to the cortex and to microtubule asters and thus are not likely to depend on the PAR pathway for localization, but could be asymmetrically activated by the PARs.
Previous work on the let-99 gene shows that it also fits the first two criteria for an intermediate gene (Rose and Kemphues, 1998a). Recessive maternal effect lethal mutations in let-99 cause defects in nuclear rotation in the P lineage, while spindles in the AB lineage sometimes align ectopically on the anteroposterior axis; the localizations of PAR proteins and other polarity markers are normal. In addition, nuclear centration in one-cell mutant embryos is incomplete and the nuclear-centrosome complex exhibits a nuclear rocking phenotype. These phenotypes overlap with those of G-protein-depleted embryos (Gotta and Ahringer, 2001b
; Rose and Kemphues, 1998a
; Zwaal et al., 1996
) (Tsou and Rose, unpublished). Thus, the let-99 gene plays a crucial role in specifying spindle orientation after polarity is established, potentially as part of the G-protein signaling pathway.
We provide further data that let-99 is required for asymmetric forces on nuclei and spindles and new evidence that LET-99 fits the third criteria for an intermediate protein in the PAR pathway. The nuclear rocking exhibited by let-99 embryos is a hyperactive dynein-dependent movement and anaphase spindle pole movements are also abnormal, suggesting a role for LET-99 in regulating force generation. LET-99 is a novel DEP-domain containing protein that is enriched in a unique asymmetric pattern at the periphery of P cells in response to PAR polarity cues. These results indicate that LET-99 functions as an intermediate that transduces polarity information to the machinery that positions the mitotic spindle.
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MATERIALS AND METHODS |
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N2, wild type Bristol;
KK705, let-99(it141) unc-22 (e66)/nT1 [unc (n754) let];
KK805, let-99(s1201) unc-22 (s7) /nT1 [unc (n754) let];
RL19, let-99(or81) unc-22(e66) /nT1;
CB3843, fem-3(e1996)/dpy-20 (e1282) unc-24(e138);
KK302, unc-22(e66) dpy-4 (e1166);
NG2198, dpy-20(1282) ham-1(n1438) unc-31(e169);
KK747, par-2(lw32) unc-45(e286ts)/sC1 [dpy-1 (e1) let]; and
KK653, par-3(it71) unc-32(e189)/qC1.
Double mutants were as described previously (Rose and Kemphues, 1998a). Strains were provided by the C. elegans Genetics Center [N2, CB3843; the Garriga laboratory (NG2198), the Kemphues laboratory (KK strains)] or constructed during this study. The or81 allele used to construct RL19 was kindly provided by B. Bowerman (University of Oregon). All worms were grown at 20°C; filming was at 23-25°C. N2 was used for all wild-type controls.
Cloning and RNA analysis
The let-99 gene was mapped to the ham-1 unc-31 region using standard meiotic recombination; details are in Wormbase (Stein et al., 2001). Cosmids (from the C. elegans Sequence Consortium) and subfragments were co-injected with the pRF4 plasmid containing the dominant visible marker rol-6 (e187) (Mello and Fire, 1995
), into KK705 hermaphrodites. Heritable Roller lines were obtained and Roller let-99 segregants that gave rise to more than five adult progeny were scored as rescued. For mutant alleles, genomic DNA from let-99 hermaphrodites was amplified using Taq polymerase and primers flanking the let-99-coding region; PCR products were cloned into pGEMT Easy and three independent PCR reaction products were sequenced for each allele.
cDNAs were isolated from C. elegans libraries (gifts from B. Barstead and P. Okkema) and RNA isolation, northern blotting and hybridization were performed (see Watts et al., 2000). To determine the 5' end of the let-99 transcript, first strand cDNA was synthesized using a let-99-specific primer and polyA+ mRNA, then amplified with a 5' SL1 primer and a nested 3' let-99 primer; products were cloned into pGEMT Easy. cDNAs and genomic DNA were sequenced using ABI automated sequencers (Cornell University Sequencing, Davis Sequencing).
RNA interference
Antisense and sense RNAs were transcribed in vitro from linearized cDNA templates (Ambion MEGAscript) using a full-length let-99 cDNA, a lrg-1 cDNA encoding amino acids 25-350, and the dhc-1 cDNA yk161f11 (from Y. Kohara, National Institute of Genetics, Japan). Double-stranded RNA annealed as described elsewhere (Fire et al., 1998) was injected into adult hermaphrodites (1 mg/ml). Injection of single-stranded RNA was used for inhibition of dhc-1 (1.5 mg/ml). The progeny of injected worms were analyzed 24-50 hours post-injection.
Antibodies and Immunolocalization
A fragment of a let-99 cDNA, corresponding to amino acids 168-462, was cloned into the pMAL protein purification vector, expressed in bacteria, purified using amylose resin and injected into rabbits (Animal Resources Services, UC Davis). Antisera were purified using a GST:LET-99 fusion protein (pGEX) coupled to affigel. Western blotting was carried out as described previously (Basham and Rose, 2001), using dilutions of 1:3000 for LET-99 antibodies and 1:10,000 for tubulin DM1A (Sigma).
For in situ immunolocalization, worms were cut in egg buffer on poly-lysine coated slides, freeze-fractured, fixed with methanol and incubated with antibodies (anti-LET-99, 1:50; FITC-goat anti-rabbit, 1:200 in PBS) (see Miller and Shakes, 1995). Primary and secondary antibodies were pre-absorbed with acetone powders of GST-expressing bacteria and wild-type worms respectively. Single-section confocal images (mid-embryo focal plane) were analyzed using IP Images software (Scanalytic). Using the segmentation tool, the minimum pixel value displayed was increased until only the posterior band was labeled, thus defining the band, anterior and posterior domains. To quantify staining intensity, the line tool was used to mark the cortex, and the average pixel value of the marked region was measured. The unit of relative intensity in all tables is expressed as a ratio of peripheral staining to cytoplasmic staining (cytoplasmic values were obtained from the area beneath the cortex excluding the nuclei and asters). Embryos were staged by DAPI (4',6-diamidino-2-phenylindole dihydrochloride) staining of the nuclei.
Microscopy and analysis of living embryos
Embryos were mounted to avoid flattening the embryo and examined under DIC optics using time-lapse video microscopy (Rose and Kemphues, 1998a). Centrosome movements were quantified by measuring the angular velocity of the nuclear-centrosome complex, which was then converted to a linear velocity using the radius of the complex. Spindle length was determined by measuring the distance between the spindle poles at metaphase (just after nuclear envelope breakdown) and at cytokinesis onset (first ingression of the cleavage furrow). To generate embryos with lateral or posterior meiosis, N2 males were mated to fem-3 homozygous females as described elsewhere (Goldstein and Hird, 1996
). To produce spherical embryos, embryos were treated with 1% hypochlorite, 0.5% KOH for 2 minutes and rinsed twice in egg buffer. Embryos were then mounted in a drop of chitinase (Hyman and White, 1987
; Wolf et al., 1983
) and examined by time-lapse from pronuclear formation through second cleavage, during which time eggshell digestion and rounding of the embryo occurred. Embryos remained fixed to the coverslip, allowing accurate determination of the axis defined by the initial position of the sperm nucleus.
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RESULTS |
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The let-99 gene encodes a novel DEP domain-containing protein
We identified the let-99 gene using a combination of mapping, transformation rescue and RNA interference (Fig. 2A-C). Confirmation of the identity of the let-99 gene came from sequencing three mutant alleles, all of which are nonsense mutations (Fig. 2D). Previous genetic analysis (Rose and Kemphues, 1998a) and comparison of the phenotypes produced by these mutations to that produced by RNA interference (Table 2) indicates that all three mutations produce a strong or complete loss of function. Analysis of cDNA and genomic sequence confirmed the exon/intron structure predicted by The C. elegans Sequencing Consortium (The C. elegans Sequencing Consortium, 1998
) for open reading frame K08E7.3 (Fig. 2C) and indicated that the let-99 transcript can be SL1 spliced. The predicted 698 amino acid LET-99 polypeptide (Fig. 2D) contains an N-terminal DEP domain (domain in Disheveled, Egl-10 and Plekstrin) (Bateman et al., 1999
; Ponting and Bork, 1996
; Schultz et al., 2000
). Because many DEP-containing proteins function with trimeric or small G proteins (Ponting and Bork, 1996
; Schultz et al., 2000
), the presence of the DEP domain supports the hypothesis that LET-99 functions as part of the G-protein signaling pathway that controls spindle position (Gotta and Ahringer, 2001b
; Zwaal et al., 1996
). Although database searches (Altschul et al., 1997
) revealed no significant overall homology to proteins of known function, the C. elegans genome contains one let-99 related gene (lrg-1) that is highly similar at the nucleotide level to the entire let-99 transcribed region, but encodes a truncated protein (Fig. 2D). RNA interference experiments revealed no additional role for lrg-1 in the early embryo (Table 2), suggesting that there is no redundancy between lrg-1 and let-99 for spindle positioning.
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In mitotic-stage wild-type embryos, LET-99 was asymmetrically enriched at the cell periphery, beginning at pronuclear migration in the one-cell embryo (Fig. 3A-D, Table 3). The areas enriched for LET-99 encircled the posterior of the embryo but did not include the entire pole, and will be referred as posterior bands; one-cell embryos thus exhibited three distinct regions of LET-99 staining: the anterior domain, the posterior band and the posterior domain (Fig. 3L). The posterior bands were asymmetrically positioned at all stages; for example, in embryos at nuclear rotation stage, the posterior band extended from 51-74% egg length (Table 3, Fig. 3B). Quantification of average fluorescence intensity confirmed that the highest staining intensity was in the posterior band at all stages and that the intensity increased during the cell cycle (Table 3). In late anaphase embryos, a strong LET-99 band was still present, but the staining intensity of the posterior domain diminished (Fig. 3D, Table 3).
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Nuclear rotation fails in spherical par-3 1-cell embryos
If peripherally localized LET-99 functions as an intermediate to translate polarity cues into spindle orientation, then the pattern of LET-99 should correlate with nuclear rotation in par embryos as in wild type. In par-2 mutants, the mislocalization of LET-99 correlates with defects in nuclear rotation. In approximately half of par-2 one-cell embryos, the first spindle does not align on the AP axis before anaphase, and in virtually all par-2 two-cell embryos there is no nuclear rotation in P1 (data not shown) (Cheng et al., 1995). However, no defects in nuclear rotation have been reported for par-3 one-cell embryos (Cheng et al., 1995
; Kirby et al., 1990
), where LET-99 is symmetrically distributed around the periphery during prophase. We re-examined par-3 1-cell embryos and also observed rotation of the nuclear-centrosome complex onto the AP axis during prophase (n=13 1-cells); however, as previously noted (Kirby et al., 1990
), the pronuclei meet more centrally in par-3 embryos and thus centration is not comparable with wild type.
Although no intrinsic asymmetries appear to be present in par-3 embryos (Bowerman and Shelton, 1999; Rose and Kemphues, 1998b
), one obvious extrinsic asymmetry is the oblong shape of the egg itself. To test the hypothesis that nuclear rotation in par-3 embryos is due to egg shape rather than the normal rotation mechanism, we examined embryos in which the eggshell was removed by chitinase digestion. As reported previously (Hyman and White, 1987
), nuclear rotation still occurred in wild-type embryos that rounded up completely before rotation began (n=3; Fig. 6); this indicates that the spindle oriented with respect to the intrinsically polarized axis defined by the sperms position. By contrast, in spherical par-3 embryos, no nuclear rotation occurred and the spindle set up on a transverse axis (n=6; Fig. 6). In par-3 embryos in which the embryo rounded up during nuclear rotation, rotation stopped and the spindle set up on an oblique axis (n=5). Interestingly, in two additional cases in which the sperm was positioned laterally and the embryo was still oblong, the spindle oriented with respect to the long axis, rather than with the axis defined by sperm position. In addition, in all of these spherical par-3 embryos, both spindle poles oscillated during anaphase, and at the two-cell stage the spindles in both cells aligned towards the cell contact region, as in untreated par-3 embryos. We conclude that in par-3 one-cell embryos, the extrinsic asymmetry of the oblong egg results in nuclear rotation. The par-3 phenotype is thus consistent with the hypothesis that the LET-99 band plays a role in normal nuclear rotation. Furthermore, these results suggest that in par-3 embryos there is no remaining polarity at the one-cell stage (e.g. dictated by oocyte polarity or sperm position) that specifies anaphase spindle pole oscillations or two-cell spindle alignment, because these movements occurred regardless of first cleavage orientation. The cue for spindle alignment in two-cell par-3 embryos is likely to be the cell contact region itself (Skop and White, 1998
; Waddle et al., 1994
).
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LET-99 is required for ectopic anaphase spindle pole oscillations in par-3 embryos
We also examined whether LET-99 asymmetry correlates with anaphase spindle pole movements in par-3 mutants. In par-3 embryos, the central band of LET-99 seen at anaphase correlates with the oscillation of both spindle poles and symmetric spindle pole separation (Cheng et al., 1995). To test whether LET-99 is required for spindle pole oscillations in par-3 embryos, we examined par-3 let-99 embryos. Just as in let-99 embryos, nuclear and metaphase rocking was observed, but stopped abruptly during anaphase; no spindle pole oscillations were observed, and spindle pole separation was reduced (n=8, Table 1). This data, together with the let-99 single mutant phenotype, suggests that the LET-99 band plays a role in spindle pole oscillations.
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DISCUSSION |
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A model for LET-99 function in the 1-cell embryo
The current model for nuclear and spindle positioning in C. elegans embryos is that the asymmetric PAR protein domains lead to asymmetric cortical forces that control nuclear and spindle movements (Gotta and Ahringer, 2001a; Rose and Kemphues, 1998b
). However, it appears that the net forces acting on the centrosomes during nuclear centration/rotation (anteriorly directed) are opposite to those acting during anaphase spindle positioning (posteriorly directed). Thus, it is unclear how the same PAR domains could result in oppositely oriented forces.
The unique enrichment of LET-99 in a posterior band provides a model that simplifies this apparent paradox. The hyperactive dynein-dependent movement of the nuclear-centrosome complex in let-99 mutants suggests that the ultimate effect of LET-99 activity is a net inhibition of force generation between the cortex and astral microtubules (through several possible mechanisms). We thus propose that in wild type, the net force on the astral microtubules is lowest at regions enriched for LET-99 (Fig. 7A). A key feature of our model is that because of the geometry of centrosome position relative to the LET-99 band, the net forces produced on the centrosomes are different during centration/rotation and anaphase. Specifically, after pronuclear meeting the centrosomes are oriented transverse to the long axis of the embryo, parallel to the LET-99 band (Fig. 7A). Our model proposes that any small stochastic shift in centrosome position that places one centrosome more anterior (and thus more astral microtubules outside of the LET-99 band) would result in a net anterior force on that centrosome (Fig. 6A, red arrows). The band would similarly result in net posterior force on the other centrosome and thus create rotational torque. By contrast, during anaphase the centrosomes are aligned perpendicular to the LET-99 band. In early anaphase, the majority of anterior spindle pole astral microtubules are outside of the LET-99 band, which would result in radially uniform force on the spindle pole and the absence of anteriorly directed pole movements. By contrast, the posterior pole astral microtubules are partially in the LET-99 band; the inhibition of laterally directed forces in the band would produce a greater net posterior force on the spindle pole, causing posterior movement and lateral oscillations.
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In wild-type two-cell embryos, the asymmetric enrichment of LET-99 could function in a similar way to facilitate nuclear rotation and anaphase spindle positioning in P1. In par-3 mutants, the banded pattern of LET-99 per se does not appear to be required for nuclear rotation in AB and P1. The cell contact/cell division remnant probably provides the asymmetric cue for nuclear rotation in par-3 embryos. This region exhibits an enrichment of dynein/dynactin complex components and has been shown to be required for nuclear rotation in wild-type embryos (Gönczy et al., 1999; Skop and White, 1998
; Waddle et al., 1994
). Nonetheless, higher levels of LET-99 correlate with rotation in par-3 two-cell embryos, while lower levels or the absence of LET-99 correlates with lack of rotation in par-2 and let-99 mutants. Thus, we speculate that there is a threshold level of LET-99 that is required to inhibit net forces enough to allow nuclear rotation. In wild-type embryos at the two-cell stage, the banded pattern of LET-99 is predicted to facilitate rotation in conjunction with the cue from the cell contact region/cell division remnant.
LET-99 may regulate forces on centrosomes as part of a G protein signaling pathway
At the molecular level, LET-99 could function to inhibit cortical forces through several microtubule-based processes that are not mutually exclusive. There are no gross abnormalities in microtubule organization in let-99 mutant embryos (Rose and Kemphues, 1998) (data not shown). Nonetheless, LET-99 could inhibit dynein activity at the cortex, modify interactions between astral microtubules and the cortex, or cause changes in microtubule dynamics that are not detectable by conventional immunolocalization of tubulin. We found that reducing dynein activity suppressed the hyperactive rocking phenotype of let-99 embryos. The genetic interpretation of this result is that in wild type, LET-99 inhibits dynein directly or indirectly, consistent with the model in which LET-99 downregulates dynein activity and thus force at the cortex. However, the suppression could also result from changes in microtubule dynamics or microtubule organization caused by lowered dynein activity (Gönczy et al., 1999) and thus is also consistent with LET-99 acting through dynein-independent mechanisms. Only a few other microtubule-associated proteins that influence spindle orientation have been identified (Gönczy et al., 2001
; Matthews et al., 1998
). These or other microtubule-associated proteins could be targets of LET-99 activity instead of or in addition to dynein, and the targets in theory could be different for anaphase versus nuclear rotation.
LET-99 has no recognizable domains for interacting directly with the cytoskeleton but does contain a DEP domain, a motif implicated in recruitment to the cell periphery and found in components of G protein signaling pathways (Axelrod et al., 1998; Schultz et al., 2000
; Wong et al., 2000
). Therefore, we postulate that LET-99 plays a regulatory role, potentially as part of the G protein signaling pathway described in C. elegans embryos (Gotta and Ahringer, 2001b
; Zwaal et al., 1996
), rather than having a direct interaction with cytoskeletal proteins. The G proteins appear uniformly distributed; the distribution of LET-99 in response to PAR proteins could thus provide for asymmetric activation of the G proteins or asymmetric localization of effectors.
It has been found that trimeric G proteins and PAR-3 and its binding partners also play a role in asymmetric division in Drosophila. In that system, the G proteins function in localizing cell fate determinants in addition to orienting the spindle (reviewed by Doe and Bowerman, 2001; Knust, 2001
). In Drosophila, the Inscuteable protein serves as the link between the polarity cues and the G proteins, as we have postulated for LET-99 in C. elegans. LET-99 and Inscuteable have no sequence similarity or shared domains, but could be functioning similarly as adaptor proteins to organize protein complexes. Drosophila does not appear to have an ortholog for LET-99, even in terms of domain organization, nor does C. elegans have a clear Inscuteable ortholog. This lack of conservation could in part be due to differences in embryonic development. In C. elegans, as in many other organisms, early divisions take place in large cells that require long astral microtubules to reach the cortex. In Drosophila, early divisions occur first in cytoplasmic islands and then in small membrane domains within the syncitial blastoderm; similarly, the asymmetric divisions that require Inscuteable occur in small cells (Doe and Bowerman, 2001
; Knust, 2001
). The strict maternal requirement for LET-99 (Rose and Kemphues, 1998a
) suggests it is specialized for functioning in large embryonic cells. Both the mouse and human genomes encode several proteins with a similar domain organization as LET-99. It will be interesting to learn whether these DEP proteins function in any aspects of spindle positioning during the early development of these organisms.
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ACKNOWLEDGMENTS |
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