Article |
Address correspondence to Fumio Matsuzaki, Laboratory for Cell Asymmetry, Center for Developmental Biology, RIKEN, 2-2-3 Minatojima-Minamimachi, Chuou-ku, Kobe 650-0047, Japan. Tel.: 81-78-306-3217. Fax: 81-78-306-3215. email: fumio{at}cdb.riken.go.jp
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Abstract |
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Key Words: epithelium; cell polarity; heterotrimeric G protein; spindle orientation; Drosophila melanogaster
Abbreviations used in this paper: Baz, Bazooka; DaPKC, Drosophila atypical PKC; DmPar-6, Drosophila Par-6; GMC, ganglion mother cell; Insc, Inscuteable; NB, neuroblast; Pins, Partner of Inscuteable.
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Introduction |
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These asymmetric features of NB division are controlled by an apically localized multiprotein complex, which includes members of two signaling pathways that are distributed in different epithelial membrane compartments before NB delamination. One is an evolutionarily conserved signaling cassette consisting of Par-3 (called Bazooka [Baz]; Kuchinke et al., 1998; Schober et al., 1999; Wodarz et al., 1999), Drosophila Par-6 (DmPar-6; Petronczki and Knoblich, 2001), and Drosophila atypical PKC (DaPKC; Wodarz et al., 2000), which localizes to the subapical region of the adherens junction in neuroepithelial cells (Knust and Bossinger, 2002). The other includes the subunit (G
i) of heterotrimeric G protein (Schaefer et al., 2001) and its guanine nucleotide dissociation inhibitor called Partner of Inscuteable (Pins; Parmentier et al., 2000; Schaefer et al., 2000; Yu et al., 2000), which distributes laterally in epithelia. At delamination, NBs begin to express the founding member of the apical complex, Inscuteable (Insc), which integrates these two signaling groups into the apical cortex by associating with both Pins and Baz (Kraut and Campos-Ortega, 1996; Kraut et al., 1996).
Mutations in the known apical component genes more or less affect both spindle orientation and the localization of the determinants at NB division. In contrast, cell-size asymmetry is not severely affected in mutants for any single apical component. This difference in the effects of apical mutations turned out to be due to redundant functions of the two apical signals in promoting daughter cell size asymmetry (Cai et al., 2003). The BazDaPKCDmPar-6Insc complex and the PinsGi complex can independently localize to create spindle asymmetry.
Recent findings revealed that the Gß subunit (Gß13F) of heterotrimeric G protein, which uniformly distributes along the NB cortex, also participates in the formation of unequal-sized daughters (Fuse et al., 2003; Yu et al., 2003). Its elimination results in a large symmetric spindle in random orientations causing division into nearly equal-sized cells, but the cell-fate determinants localize over one spindle pole to segregate into the GMC, indicating unique roles of Gß13F signaling in asymmetric NB division. Although defective localization of the apical components has been described for Gß13F mutants (Fuse et al., 2003; Yu et al., 2003), the relationship between the apical complex and Gß13F signaling is not understood well enough to explain their phenotypic differences. In addition, these findings raise a fundamental question of whether the two apical signaling pathways have differential or equivalent roles in basal protein localization and spindle orientation; this question has not been answered definitively.
In this paper, we document the first mutant for the G1 gene encoding one of two Drosophila G
subunits of heterotrimeric G protein (Ray and Ganguly, 1992, 1994; Schulz et al., 1999). This G
1N159 mutant, which produces a truncated G
1, fails to localize Gß13F to the cortex and shows essentially the same phenotype as the loss of function mutant of Gß13F. G
1 is required for localizing Gß13F to the NB cortex, presumably as its binding partner, indicating the essential role of cortical Gß13FG
1 signaling in the induction of asymmetry in daughter cell size. We also found that, in both G
1 and Gß13F mutants, NBs retain asymmetric localization of the BazDaPKCDmPar-6Insc complex but not of PinsG
i. This localized Baz activity is responsible for Miranda localization and the residual asymmetry in NB daughter cell size in these mutants. These observations led us to reexamine the roles of the apical components, and we found evidence indicating distinct requirements for apical components in the two important processes for asymmetric NB division. BazDaPKCDmPar-6 are crucial for asymmetric localization of the cell-fate determinants, and PinsG
i mainly control spindle orientation. We suggest that the two apical signaling pathways play overlapping but distinct roles in the asymmetric division of NBs.
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Results |
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N159 is a mutation of the G1 gene
N159 turned out to include a nonsense mutation in the G1 gene (Ray and Ganguly, 1992, 1994). The mutant G
1 gene produces a truncated protein lacking the COOH-terminal isoprenylation site that acts in membrane anchoring (Fig. 3 A; see Materials and methods). N159 indeed fails to complement a P-insertion allele of G
1 (K08017). Expression of a wild-type G
1 transgene rescues the N159 mutant phenotypes with respect to gastrulation (unpublished data), daughter cell size (Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200309162/DC1), and spindle orientation (Fig. S1 C), as well as in the localization of the apical components (Fig. S1 B; see Fig. 4), indicating that the N159 characteristics originate from the mutated G
1 gene; we call this allele G
1N159. In situ hybridization revealed a high maternal contribution in early embryos and subsequent uniform expression of G
1 (Fig. 3 B). The other G
gene, G
30A, is not expressed in early embryos (unpublished data).
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We also performed immunoprecipitation of extracts from wild-type embryos expressing FLAG-tagged G1 with anti-FLAG antibodies and found that Gß13F coimmunoprecipitated with G
1 (Fig. 3 D, lane 3) but it did not for control embryos (Fig. 3 D, lanes 2 and 6). This result, together with the essential role of G
1 in recruiting Gß13F to the membrane, suggests that G
1 is an in vivo binding partner of Gß13F. We note that the association of Gß13F with G
1 does not appear to require the isoprenylation site of G
1 because both the G
1C67S (Fig. 3 D, lane 4) and G
1N159 (Fig. 3 D, lane 5) proteins coimmunoprecipitated with Gß13F. We could not determine the distribution of G
1 because of a lack of suitable antibodies. However, G
1 and Gß13F most likely colocalize in the cell cortex, because Gß13F localization depends completely on the ability of G
1 to bind to membranes.
Differential effects of G1Gß13F on the asymmetric localization of PinsG
i and BazDaPKCDmPar-6Insc
The asymmetric localization of Miranda and the residual difference in daughter cell sizes in the G1N159 mutant NBs indicate that some cell polarity remains in these NBs. The null Gß13F mutant shows similar residual cell asymmetry, which depends on baz activity (Table I; Fuse et al., 2003; Yu et al., 2003). We examined whether the residual asymmetry in the G
1N159 mutant depends on baz activity and found that elimination of baz activity (by RNAi) in G
1N159 results in complete loss of cell-size asymmetry and in uniform Miranda distribution (Fig. 2 A, D). These polar effects of Baz suggest asymmetric distribution of some apical components in the G
1N159 and Gß13F mutants, which prompted us to carefully reexamine the distribution of the apical components.
In wild-type NBs, apical components localize in the apical cortical crescent (PFig. 4, A, D, G, J, M, and P). In G1N159 NBs, Pins is distributed uniformly throughout the cell cortex and in the cytoplasm of all metaphase NBs (100%, n = 27; Fig. 4 B). G
i becomes undetectable at early embryonic stages (Fig. 4 E). The degradation of G
i was confirmed by Western blotting of G
1N159 mutant embryos (Fig. 3 E), suggesting that stability of G
i depends on the presence of the cortical Gß13FG
1 complex. Other apical components, Insc, Baz, DmPar-6, and DaPKC, also no longer distribute normally in G
1N159 NBs. Their staining intensity is decreased. However, we found that their distribution remains asymmetric in the majority of metaphase NBs; Insc distributes diffusely but still asymmetrically in the cytoplasm and cell cortex (Fig. 4 H; 75% of NBs localizing Miranda, n = 60). Baz (Fig. 4 K), DmPar-6 (Fig. 4 N), and DaPKC (Fig. 4 Q; 84% of NBs localizing Miranda, n = 56) form cortical crescents. The polarized distribution of DmPar-6 and DaPKC is more evident at the end of cytokinesis, even in divisions producing two equal-sized daughters (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200309162/DC1). DaPKC (87% of telophase NBs, n = 30) and DmPar-6 (unpublished data) remain in the daughter NB but are excluded from the sibling GMC, as also observed in wild-type NB divisions (100%, n = 30; Fig. S2, A and B). Interestingly, unlike DaPKC and DmPar-6, Insc is observed on both the NB and GMC sides in most telophase NBs in G
1N159 mutants (Fig. S2 E). Elimination of Gß13F has the same effects on localization of the various apical components as the G
1N159 mutation does (Fig. 4; Fig. S2, C and F). Thus, Gß
signaling in NBs is necessary for asymmetric localization of G
i and Pins but not of Insc, Baz, DmPar-6, or DaPKC. This remaining BazDaPKC pathway is responsible for asymmetric localization of the determinants and residual cell-size asymmetry in the absence of the Gß
signal, because elimination of baz activity in the G
1N159 background completely disrupts them (Fig. 2, C and D).
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PinsGi pathway predominantly regulates spindle orientation
Finally, we investigated the regulation of spindle orientation. In pins and Gi mutants, NBs show defects in spindle orientation (Schaefer et al., 2000; Yu et al., 2000, 2003). We found that epithelial cells also show defects in spindle orientation in these mutants. In wild-type embryos, epithelial cells divide parallel to the embryo's surface (Fig. 6, A, E, and F). In contrast, they often divide perpendicular (Fig. 6 B) or at an oblique angle (Fig. 6 C) to the embryo's surface in pins mutants (Fig. 6 Q). These observations suggest that the axis of division in mitotic epithelial cells is randomly oriented in pins mutants. In mitotic domain 9 of the procephalic neurogenic region, wild-type cells divide perpendicular to the surface, producing the smaller GMCs on the inner side (Fig. 6, I and M). It was reported that these cells divide parallel to the surface in pins mutants (Yu et al., 2000). However, our analysis indicates that the pins mutant cells in mitotic domain 9 divide in directions not only parallel but also perpendicular and often at an oblique angle to the embryonic surface (Fig. 6, J and N). Defects in the orientation of division are similarly observed in G
i mutants (Fig. 6, D, K, O, and Q). These results indicate that Pins and G
i are required for proper spindle orientation in both epithelial cells and mitotic domain 9 cells, as also observed in NBs (Yu et al., 2000).
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Discussion |
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Differential Gß signaling along the apicalbasal axis in NBs
Because the Gß13FG1 complex, which distributes uniformly in the cortex, functions in asymmetric organization of the spindle, differential activation or inactivation of Gß
signaling must occur in the apicalbasal direction. A recent work revealed that two apical signaling pathways are implicated in the apicalbasal difference in spindle development in a redundant fashion (Cai et al., 2003). What is the relationship between the apical signals and the Gß
signal? Spindle size is reduced by an increase in the amount of Gß
, but a lack of Gß
results in formation of a large, symmetric spindle (Fig. 1; Fuse et al., 2003). These findings raise the possibility that spindle development is suppressed by the Gß
signal, which is repressed by the presence of an apical complex on the apical side in the wild-type cells, resulting in a large apical and small basal spindle. This model suggests that the apical complex acts upstream of the Gß
signal. On the other hand, elimination of Gß13F affects the localization of the apical components: Pins becomes uniformly distributed and G
i becomes undetectable. In addition, G
1N159 and Gß13F mutations appear to destabilize the localization of the components in the BazDaPKC pathway, as judged by the reduced staining by their antibodies (although this may be an indirect consequence of the mislocalization of PinsG
i). The Gß
signal is thus required for normal distribution of the components of both apical pathways, consistent with the idea that the apical pathways acts downstream of the Gß
signal in regulating spindle asymmetry (Yu et al., 2003). Tests for epistasis between the apical pathways and the Gß
signal are needed to clarify their relationship in the regulation of spindle organization.
Dosage effects of apical components and the role of Insc
The effects of G1N159 and Gß13F mutations (this paper; Fuse et al., 2003) on cell-size asymmetry are remarkable but different from those in double mutants in which both apical pathways are disrupted simultaneously, where daughter cell sizes are completely equal (Cai et al., 2003; Yu et al., 2003). The cell-size ratio of GMCs to their sibling NBs shows a broad distribution: from 0.6 to 1 in the G
1 (and Gß13F) mutants (Fig. 2 A). This residual asymmetry in daughter cell size is due to BazDaPKC activity (Fig. 2, Fuse et al., 2003; Yu et al., 2003). Here, we showed that the components of this pathway indeed distribute asymmetrically in G
1 (and Gß13F) mutant NBs in which PinsG
i activity is no longer asymmetric (Pins is uniformly distributed and G
i is absent).
Why does this polarized BazDaPKC activity cause less asymmetry in daughter cell size in spite of the redundant function of the BazDaPKC pathway and PinsGi? Antibody staining for Baz, DaPKC, and DmPar-6 suggests that their levels and their polarized distribution are weakened in G
1 (and Gß13F) mutants. A possible explanation is that low levels of polarized BazDaPKC activity confer only low levels of asymmetry to the daughter cell size in the absence of polarized PinsG
i. Thus, the degree of cell-size asymmetry resulting from NB divisions may depend on the dosage of the components of one apical pathway when the other is absent or uniformly distributed. In contrast, Miranda localization does not appear to be severely impaired in G
1N159 and Gß13F mutants until late embryonic stages, indicating that the polarized BazDaPKC activity in these mutants is sufficient to localize Miranda. Therefore, full asymmetry in daughter cell size may require relatively higher levels of BazDaPKC activity than polarized distribution of cell-fate determinants does.
In G1N159 and Gß13F mutants, Insc has a different distribution than the other components of the BazDaPKC pathway. In most of these mutant NBs, Insc distributes broadly to both the cytoplasm and the cortex in a slightly asymmetric way, but Baz, DaPKC, and DmPar-6 localize asymmetrically in the cortex. The cytoplasmic distribution of Insc is also slightly asymmetric in pins mutant NBs (Fig. 5). It is not known whether cytoplasmic Insc is functional. Interestingly, Insc distribution often appears to correlate better with the asymmetry in daughter cell size than do the other components of the BazDaPKC pathway in G
1N159 and Gß13F mutants: in most telophase NBs that are cleaving into two equal daughters, DaPKC and DmPar-6 are excluded from the daughter GMC, but Insc tends to distribute evenly to both daughter cells (Fig. S2). This occurs also in pins mutants, in which
15% of NBs divide equally but most NBs divide unequally (Cai et al., 2003). In pins NBs cleaving equally, Insc is found equally in the cytoplasm of both daughter cells, but DaPKC and DmPar-6 remain in the newly forming NB; in unequally dividing NBs, all three components are found preferably on the NB side (unpublished data). These observations raise the intriguing possibility that Insc has more important roles in the generation of spindle asymmetry than do the other components of the BazDaPKC pathway. Because the absence of Baz results in mislocalization of Insc and vice versa, it is technically difficult to discriminate Insc-specific from Baz-specific functions. It may be Insc or some unknown Insc-associating effectors, rather than Baz, that functions in parallel with PinsG
i in the establishment of cell-size asymmetry.
PinsGi and BazDaPKC pathways have both overlapping and distinct functions
The question of whether the two apical pathways have redundant functions in aspects of NB division other than cell-size asymmetry has been elusive. In this paper, examination of G1N159 and Gß13F mutant NBs, as well as those overexpressing baz, suggest that the asymmetric localization of Miranda depends solely on polarized Baz activity and not on PinsG
i function. Miranda always distributes on the cortical side opposite to Baz in these mutants and in the wild-type. This also occurs for sensory precursor cells in the peripheral nervous system: in sensory precursory cell division Insc is not expressed, and Pins and Baz distribute on cortical sides opposite to each other, unlike in NBs; however, both Miranda and Numb localize to the cortex opposite Baz, as seen in NBs (unpublished data; Bellaiche et al., 2001).
A previous work showed that phosphorylation of the Lethal (2) giant larvae protein by DaPKC directs the localization of cell-fate determinants to the basal cell cortex (Betschinger et al., 2003). When baz is overexpressed in NBs, ectopically distributed Baz excludes Miranda from the Baz region and DaPKC colocalizes with the ectopic Baz, as shown in this work (Fig. 5). In contrast, a decrease in Baz activity in the wild-type results in cytoplasmic localization of DaPKC and uniform cortical distribution of Miranda. All these findings suggest that the Baz-directed localization of DaPKC excludes Miranda from the apical cortex via Lethal (2) giant larvae phosphorylation. In the absence of Baz, Miranda is eventually concentrated to the budding GMC during telophase by unknown mechanisms, a phenomenon called "telophase rescue" (Schober et al., 1999). We observed that this phenomenon did not occur by depleting both baz activity and Gß signaling (Fig. 2 D; Fuse et al., 2003), suggesting that telophase rescue involves Gß
signaling or asymmetric PinsG
i localization (Yu et al., 2003).
The absence of any single component of the apical complex has the same effect on spindle orientation during NB division, which is normally perpendicular to the apicalbasal axis (Kraut et al., 1996; Kuchinke et al., 1998; Wodarz et al., 1999, 2000; Schaefer et al., 2000; Yu et al., 2000, 2003; Petronczki and Knoblich, 2001). Thus, proper orientation of the spindle has been thought to require all the apical components. However, our observations on epithelial cells and mitotic domain 9 cells (summarized in Fig. 7 A) indicated that the spindle always points to the location of Pins when Pins is localized in the cell (Fig. 6). This alignment of the spindle toward Pins occurs irrespective of the localization of the Baz-pathway components. For instance, wild-type epithelial cells divide parallel (Pins direction) but not perpendicular to the apicalbasal axis (Baz direction); so do most epithelial cells and mitotic domain 9 cells in Gß13F and G1 mutants. Therefore, the PinsG
i pathway, rather than the BazDaPKC complex, is likely to play a dominant role in controlling spindle orientation.
In most NBs in pins, Gß13F, and G1 mutants, the spindle is oriented in the direction of Baz localization and therefore follows the localization of the cell-fate determinants. This coincidence results in the determinants' virtually normal segregation to one daughter cell despite the random orientation of division. Thus, only when PinsG
i are absent or uniformly distributed in NBs, polar Baz activity appears to be capable of directing spindle orientation. Alternatively, the mitotic spindle may position the BazDaPKC complex over one spindle pole.
In the NB in which the BazDaPKC pathway is depleted, PinsGi can still localize asymmetrically and orient the spindle (Yu et al., 2000, 2003). Interestingly, the Pins crescent forms in random orientations in this situation, leading to random spindle orientation. This fact suggests that the BazDaPKC complex or its combination with PinsG
i is necessary to orient the PinsG
i crescent in the apical direction of the NB, raising an intriguing possibility that there are unknown mechanisms by which formation of the apical complex occurs on the apical side. This postulated mechanism may involve interactions with neighboring epithelial cells.
What is the molecular mechanism by which PinsGi orient the spindle? It is interesting to assume that Pins has the ability to attract the spindle pole. This idea is consistent with previous evidence (Yu et al., 2000; Schaefer et al., 2001); although epithelial cells do not normally express Insc, its ectopic expression in these cells recruits PinsG
i to the apical cortex and reorients the mitotic spindle in the apicalbasal direction. The C. elegans homologues of Pins, GPR-1/GPR-2, interact with G
i/G
o and a coiled-coil protein, LIN-5, which is required for GPR-1/GPR-2 localization (Gotta et al., 2003; Srinivasan et al., 2003). All these molecules are indeed involved in the regulation of forces attracting spindles during early cleavages. Although Lin-5 has no obvious homologue in other species, functional homologues may regulate Pins localization and/or the connection between the spindle pole and Pins in Drosophila. Furthermore, the C. elegans gene ric-8, which interacts genetically with a G
o gene, is also required for embryonic spindle positioning (Miller and Rand, 2000). Its homologue in mammals acts as a guanine nucleotide exchange factor for G
o, G
q, and G
i (Tall et al., 2003). An analysis of the Drosophila RIC-8 homologue may give insight into the mechanisms by which PinsG
i regulate spindle orientation.
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Materials and methods |
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Transgenic flies and RNA interference
pUAST vectors (Brand and Perrimon, 1993) containing MFLAG-G1, MFLAG-G
1C67S, and MFLAG-G
1N159 were constructed, and fly strains carrying these constructs were established. G
1C67S, which encodes a single cysteine to serine change at amino acid 67, was made by PCR-based site-directed mutagenesis. For phenotype-rescue experiments, UAS-MFLAG-G
1, UAS-MFLAG-G
1C67S, and UAS-MFLAG-G
1N159 were driven by maternal Gal4 V32 (a gift from D. St. Johnston, Wellcome/CRC Institute, Cambridge, UK) in G
1N159 germline clone embryos. The same driver was used to overexpress UASbaz (a gift from A. Wodarz, Institute for Genetics, Heinrich-Heine University Dusseldorf, Dusseldorf, Germany). For RNA interference experiments (Kennerdell and Carthew, 1998), embryos were injected with baz dsRNA and were allowed to develop until the appropriate stages.
Immunohistochemistry
Embryos were fixed in 4% PFA for 20 min. For -tubulin staining, embryos were fixed in 38% formaldehyde for 1 min (Fuse et al., 2003). The following antibodies were used: anti-Miranda (Ikeshima-Kataoka et al., 1997), anti-Gß13F (Fuse et al., 2003), anti-Baz (Ohshiro et al., 2000), anti-PKC
C20 (Santa Cruz Biotechnology, Inc.), antiDmPar-6 (rabbit IgG against COOH-terminal peptide TIMASDVKDGVLHL), anti-Insc (a gift from W. Chia, MRC Center for Developmental Neurobiology, King's College, London, UK), anti-Cen190 BX63 (a gift from D.M. Glover, University of Cambridge, Cambridge, UK), anti-Pins (a gift from W. Chia), anti-G
i (a gift from X. Yang, Institute of Molecular and Cell Biology, Singapore), antineurotactin BP106 (Developmental Studies Hybridoma Bank), antiphosphohistone H3 (Upstate Biotechnology), antiß galactosidase (Cappel and Promega), anti
-tubulin (DM1A; Sigma-Aldrich), and anti-FLAG (Sigma-Aldrich). Cy3- or Alexa Fluor 488conjugated secondary antibodies were obtained from Jackson Laboratories or Molecular Probes, respectively. DNA was stained with TOTO-3 (Molecular Probes). A confocal microscope (BioRad Radiance 2000) was used to acquire images which were processed with Adobe PhotoShop.
In situ hybridization
In situ hybridization was done as described previously (Tautz and Pfeifle, 1989) by using a digoxigenin-labeled DNA probe containing the coding region of the G1 transcript.
Coimmunoprecipitation and Western blotting
Fly embryos overproducing MFLAG-G1 and MFLAG-Miranda (Ikeshima-Kataoka et al., 1997) under control of the maternal GAL4 V32, as well as wild-type embryos, were mixed with a fivefold volume of lysis buffer (50 mM Tris HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and protease inhibitor cocktail), purchased from Sigma-Aldrich, for 30 min at 4°C. The embryo lysates were centrifuged at maximum speed in a microcentrifuge for 30 min. The supernatants were immunoprecipitated with anti-FLAG antibodies and protein G beads (Amersham Biosciences). Beads were washed five times in the lysis buffer. Bound proteins were analyzed by Western blots with anti-Gß13F.
To compare the amount of Gi and Gß13F protein between wild-type and G
1 mutant embryos, the extracts from wild-type and G
1N159 germline clone embryos (016 h after egg laying) were analyzed by Western blots with anti-G
i and anti-Gß13F. Anti
-tubulin was used as a control to normalize these extracts.
Online supplemental material
Fig. S1 shows that the expression of the G1 transgene in G
1N159 mutants restores the asymmetric cell division of NBs and the cortical localization of Gß13F. Fig. S2 shows the distribution of DaPKC and Insc in telophase NBs in G
1N159 and Gß13F mutants. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200309162/DC1.
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Acknowledgments |
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This work was supported by grants-in-aid for Science Research from the Ministry of Education, Science, Sports, and Culture of Japan and by Core Research for Evolution Science and Technology for the Japan Science and Technology Corporation.
Submitted: 26 September 2003
Accepted: 20 January 2004
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