Distinct roles of Gi and G?13F subunits of the heterotrimeric G protei

杨柳

1 Institute of Molecular and Cell Biology, Singapore 117609
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( v* s/ E2 l/ }2 MRC Centre for Developmental Neurobiology, London SE1 1UL, UK/ d* z/ m) K$ T9 X

9 R. u5 O0 F2 |- `1 E: K7 y: `2 m! TAddress correspondence to William Chia, MRC Centre for Developmental Neurobiology, 4th Fl., New Hunts House, Guy's Campus, King's College London, London SE1 1UL, UK. Tel.: 44-207-8486544. Fax: 44-207-8486550. email: william.chia@kcl.ac.uk; or Xiaohang Yang, Institute of Molecular and Cell Biology, 30 Medical Dr., Singapore 117609. Tel.: 65-687-47848. Fax: 65-677-91117. email: mcbyangn@imcb.nus.edu.sg
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Abstract
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The asymmetric division of Drosophila neuroblasts involves the basal localization of cell fate determinants and the generation of an asymmetric, apicobasally oriented mitotic spindle that leads to the formation of two daughter cells of unequal size. These features are thought to be controlled by an apically localized protein complex comprising of two signaling pathways: Bazooka/Drosophila atypical PKC/Inscuteable/DmPar6 and Partner of inscuteable (Pins)/Gi; in addition, G?13F is also required. However, the role of Gi and the hierarchical relationship between the G protein subunits and apical components are not well defined. Here we describe the isolation of Gi mutants and show that Gi and G?13F play distinct roles. Gi is required for Pins to localize to the cortex, and the effects of loss of Gi or pins are highly similar, supporting the idea that Pins/Gi act together to mediate various aspects of neuroblast asymmetric division. In contrast, G?13F appears to regulate the asymmetric localization/stability of all apical components, and G?13F loss of function exhibits phenotypes resembling those seen when both apical pathways have been compromised, suggesting that it acts upstream of the apical pathways. Importantly, our results have also revealed a novel aspect of apical complex function, that is, the two apical pathways act redundantly to suppress the formation of basal astral microtubules in neuroblasts./ V+ Z6 e7 N6 {- G8 r+ c

- _  |, d/ u+ E7 ?9 EKey Words: neuroblast; asymmetric division; astral microtubules; heterotrimeric G proteins; Drosophila
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F. Yu's present address is Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore 117604.5 a# R. q2 \$ V5 d$ j2 u3 g' R9 n

) }/ e5 i2 }/ i3 J6 ?6 tAbbreviations used in this paper: baz, bazooka; CNN, centrosomin; DaPKC, Drosophila atypical PKC; insc, inscuteable; mira, miranda; NB, neuroblast; pins, partner of inscuteable; pon, partner of numb; pros, prospera; wt, wild type.
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Introduction8 b1 \6 V0 w9 q1 n. R

/ _: H+ n1 v: v+ [$ n7 [The Drosophila embryonic central nervous system is derived largely from neural progenitors called neuroblasts (NBs). NBs divide asymmetrically to generate two unequal size daughter cells: the larger apical daughter remains as a NB and continues to divide asymmetrically, and the smaller basal/lateral daughter (ganglion mother cell) divides terminally to generate two neurons/glial cells (Campos-Ortega, 1995). Three well-characterized features of the NB asymmetric division (Jan and Jan, 2001; Chia and Yang, 2002) are: (a) basal localization and asymmetric segregation of cell fate determinants and their associated proteins such as Numb/Partner of numb (Pon), Prospera (Pros)/Miranda (Mira), and pros RNA/Staufen; (b) reorientation of the mitotic spindle along the apical/basal axis at metaphase; (c) generation of an apically biased asymmetric mitotic spindle (Kaltschmidt et al., 2000) and the displacement of the spindle toward the basal cortex during ana/telophase, which leads to the formation of NB daughter cells that differ in size. An additional feature, which has not been extensively studied, is that late in NB mitosis an extensive astral microtubule network emanates from the apical but not the basal centrosome (Giansanti et al., 2001).0 Y% `2 h$ J7 O7 M' D9 X) E

1 W6 L9 b( f* E2 TThe well-characterized features of the NB asymmetric division are controlled by a complex of proteins that are apically localized in dividing NBs, which include the Drosophila homologues of the conserved Par3 (Bazooka )/Par6 (DmPar6)/aPKC (Drosophila atypical ) (Kuchinke et al., 1998; Schober et al., 1999; Wodarz et al., 1999, 2000; Petronczki and Knoblich, 2001) protein cassette first described in Caenorhabditis elegans (Kemphues, 2000; Matsuzaki, 2000; for review see Doe and Bowerman, 2001; Knoblich, 2001; Wodarz, 2002), the novel protein Inscuteable  (Kraut and Campos-Ortega, 1996; Kraut et al., 1996), and an  subunit of the heterotrimeric G protein complex (Gi) (Schaefer et al., 2001) and an evolutionarily conserved molecule, Partner of inscuteable (Pins) (Parmentier et al., 2000; Schaefer et al., 2000; Yu et al., 2000) that acts as a guanine nucleotide dissociation inhibitor for Gi. Since Insc can directly interact with both Baz and Pins in vitro, this apical complex of proteins can be viewed as comprising of two conserved protein cassettes, Baz/DmPar6/DaPKC and Pins/Gi, that are held together by Insc. Loss of function mutations exist for all members of the NB apical complex genes except Gi. Loss of single members of the apical complex, such as baz, insc, and pins, results in defective basal protein localization and spindle misorientation in mitotic NBs up to metaphase, although these defects can be partially corrected late in mitosis, a phenomenon called telophase rescue (Ohshiro et al., 2000; Peng et al., 2000; Cai et al., 2001). However, unlike basal protein localization and spindle orientation, the generation of an asymmetry spindle and its displacement toward the basal cortex are largely unaffected, and NBs lacking one component of the apical complex usually produce two unequal size daughter cells like wild-type (wt) NBs.& E. m1 K) q1 \+ {
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Recent findings indicate that the apical proteins are also involved in daughter cell size determination and can be further subdivided into two redundant pathways that control mitotic spindle geometry and displacement late in NB divisions (Cai et al., 2003). Baz, DaPKC, Insc, and probably DmPar6 belong to one pathway and Pins and (probably) Gi belong to the other. Members of each pathway can asymmetrically localize when members of the other pathway are mutated, suggesting that localized spindle extension signals derived from either one of these two pathways are sufficient to generate asymmetric spindle geometry and spindle displacement, resulting in unequal size daughter cells. Simultaneous disruption of both pathways destroys the localized spindle extension and displacement signals. Consequently, the two half spindle arms remain identical in length and mutant NBs produce two daughter cells with equal size.
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Heterotrimeric G protein signaling has been shown to be involved in controlling distinct microtubule-dependent processes in C. elegans P0 embryos (Gotta and Ahringer, 2001). G? is important for correct centrosome migration around the nucleus and spindle orientation. G is required for asymmetric spindle positioning in the one-cell embryos. In Drosophila, G protein signaling is also involved in microtubule-dependent processes such as the formation of an asymmetric spindle. When Gi is overexpressed (Schaefer et al., 2001) or when G?13F function is abolished (Schaefer et al., 2001), the ability to generate an asymmetric spindle is disrupted and NBs frequently divide to produce two daughter cells with equal size (Fuse et al., 2003). However, it has not been possible to assess the relative roles of G?13F and Gi in NB asymmetric divisions not only because Gi mutants are not available but also because in G?13F mutants Gi is undetectable in all cell types (Schaefer et al., 2001).
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In this study, we report the isolation and analysis of loss of function mutations in Gi and assessing the role of the apical complex components on NB astral microtubules and mitotic spindle geometry. Our findings indicate distinct roles for Gi and G?13F in NB asymmetric divisions. Loss of Gi releases Pins from the apical cortex into the cytosol and exhibits a similar array of phenotypes seen in pins mutant NBs. Mutations in Gi and one of the genes in Baz/DaPKC/Insc pathway cause NB to generate symmetric spindles and two equal size daughter cells, suggesting that Gi and Pins act in same pathway with respect to mediating mitotic spindle geometry. Formally, G?13F functions upstream of both Baz/DaPKC/Par6/Insc and Pins/Gi pathways and is required, at least in part, for the asymmetric localization and/or stability of all apical complex members. Mutation in G?13F can disrupt the asymmetric localization of members of both apical pathways in NBs and results in the formation of symmetric spindles and equal size daughter cells. Strikingly, our analyses has also revealed that the two apical pathways act downstream of G?13F to redundantly suppress the formation of basal astral microtubules during NB divisions.; a2 D# z9 O  i5 ~1 S) _" K& `
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Results* u5 P/ ?- v+ W2 m$ @$ v+ J
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Generation of antigen-minus alleles of Gi
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7 ?% G# v6 x# L# M" |0 |It has been shown that Gi is apically localized in mitotic NBs and its apical localization requires Pins. Gi interacts directly with the GoLoco motifs (Siderovski et al., 1999) in the COOH-terminal region of Pins, a region required for Pins to target to the NB cortex (Yu et al., 2002). In the absence of pins, Gi is localized uniformly to the cortex of dividing NBs. To ascertain the functions which are specific to Gi during asymmetric NB divisions, we generated Gi mutant alleles by imprecise excision of the P element (KG01907) inserted in the 5' flanking region of the Gi gene. Three revertants, GiP8, GiP29, and GiP20, associated with flanking deletions were isolated and mapped (Fig. 1). GiP20 is an embryonic lethal allele. Deletion in GiP20 removes not only the complete coding region of the Gi gene but also the putative gene CG10063. The precise 3' breakpoint of GiP20 has not been determined. GiP29 contains a deletion uncovering the first exon that includes the codon for translation initiation, whereas GiP8 carries a deletion that removes the first two exons. There is an EST sequence LD18889 with no obvious ORF in the first intron of the Gi gene that is deleted in GiP8 and GiP20. Similar to animals lacking zygotic pins function, homozygous GiP8 and GiP29 flies lacking zygotic Gi are viable, show locomotion defects, but nevertheless can lay fertilized eggs. The majority of the embryos derived from these homozygous animals lacking both maternal and zygotic components die as larvae. Western blot analysis and immunostaining with an anti-Gi antibody raised against the extreme COOH-terminal region, aa 327–355, of Gi (Schaefer et al., 2001) indicated that these GiP8 and GiP29 embryos are antigen minus (Fig. 1, B and C). Since these embryos exhibit NB phenotypes which are indistinguishable from germ line clone embryos derived from GiP20 (a complete deletion of the gene), they are likely to be null alleles. In the following experiments, unless otherwise specified, Gi mutant refers to GaiP8 embryos lacking both maternal and zygotic Gi function.4 b- }& F. y2 {
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Figure 1. Characterization of Gi deletion alleles. (A) Schematic representation of three Gi deletion alleles. The extent of the deletions are indicated by the parentheses. The Gi locus is deleted partially or fully in all three alleles. (B) Western blot analysis using a Gi COOH-terminal antibody indicates that embryos derived from homozygotes of either GiP8 or GiP29 are antigen minus (arrow). Gi signal is also undetectable in GaiP8 (C) and GiP29 (not depicted) NBs using immunofluorescence. Pins crescent (green) and Insc crescent (not depicted) reappears in GiP8 NBs (D) with ectopic Gi expression using a relatively mild sca-gal4 driver. The mitotic NBs are identified using DNA staining (cyan, C and D). Apical is up. Cell boundary is outlined with white dots. Bar: (C and D) 10 μm.: E( Q0 J& e! [" O; R. x
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Loss of maternal and zygotic Gi causes Pins to localize to the cytosol and produce phenotypic defects similar to those seen in pins NBs* G# U# f- V8 `6 s. K7 h9 C! |! M

. r- F5 \/ f7 k0 o* [Both Pins and Insc, which normally form apical crescents in wt NBs (Fig. 2, A, C, and E), are cytoplasmic in dividing Gi NBs (Fig. 2, B, D, and F). The apical localization of DaPKC (68%, n = 50) and Baz (unpublished data) remain largely unchanged although the intensity of the staining is reduced, sometimes dramatically (Fig. 2 H). Localization of the basal proteins are also affected. Basal proteins Mira/Pros (Fig. 2, I and J) and Pon/Numb (unpublished data) are often mislocalized in mitotic NBs up to metaphase; however, telophase rescue occurs normally, and basal proteins subsequently segregate primarily to just one daughter during telophase (Fig. 2 L). In Gi mutant NBs, G?13F remains uniformly cortical as in wt NBs (PFig. 2, O and P). The RP2sib to RP2 cell fate change is also observed in Gi embryos (Fig. 2 R), which serves as a good indication of defective ganglion mother cell asymmetric divisions. Anti-Eve staining shows that RP2sib adopts RP2 cell fate in 10% (n = 248) of mutant hemisegments. In addition, the RP2 missing phenotype is also observed (11%, n = 248). Mitotic spindle reorientation is also affected in Gi mutants. In mitotic domain 9, mitotic spindles fail to undergo 90～ reorientation, and these cells divide parallel to the embryonic surface (Fig. 2 N), whereas their wt counterparts reorientate and divide perpendicular to the surface (Fig. 2 M). These defects are similar to those observed for NBs lacking pins function (Yu et al., 2000).
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: O! R$ L( `4 y7 O4 f7 mFigure 2. Gi function is required for correct asymmetric NB divisions. In wt dividing NBs, Pins (green, A and C), Insc (green, E) and DaPKC (red, G) always localize to the apical cortex. Pins (green, B and D) and Insc (green, F) are cytoplasmic in all Gi mutant NBs (100%, n = 80); DaPKC localization is largely unchanged in the majority of NBs (H); however, its levels can be drastically reduced (see Results). Mira (red, I–L) can be mislocalized in Gi NBs at metaphase (compare wt I and mutant J) but, nevertheless, is redistributed to only one of the daughters at telophase (compare wt, K, and mutant, L). Anti– ?-tubulin staining (green) indicates that spindle reorientation in cells of mitotic domain 9 does not occur in Gi embryos (N). Spindle axis in wt domain 9 cells is perpendicular to the surface; hence, only the more apical spindle pole can be seen from the surface (M), whereas spindles of mitotic domain 9 cells are aligned parallel to the surface in Gai embryos so both spindle poles can be seen (N). G?13F (red) cortical localization is independent of Gi (wt, O, and mutant, P). Motoneuron RP2 (arrow) can be duplicated or missing in Gi embryos (R) as indicated by the anti-Eve staining. For NB panels, apical is up. DNA staining is in cyan. For panels Q and R, anterior is toward left. Bars: (A–P) 10 μm; (Q and R) 50 μm.
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Several observations further support the view that the above described defects are caused by the loss of Gai function. Introduction of the nested gene LD18889 into GaiP8 does not rescue the defects in asymmetric NB division. Furthermore, the small deletion GiP29, which contains intact LD18889, exhibits the same phenotypes seen in GiP8. Moreover, low level expression of a UAS-Gai using the sca-gal4 driver in Gi mutant background can partially restore apical localization of Pins (81%, n = 52; Fig. 1 D) and Insc (unpublished data) in mitotic NBs, suggesting that defects in NB divisions are due to loss of Gi function.
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# N9 \' G( n! t+ pGi and Pins act in the same pathway to regulate asymmetric spindle geometry and unequal cell size divisions% B. p. p- J, M
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Gi has been implicated previously in the generation of spindle asymmetry from overexpression and RNAi experiments (Schaefer et al., 2001; Cai et al., 2003). The availability of Gi loss of function alleles enables us to more definitively assess the role of Gi in NB spindle geometry and the generation of daughters of unequal cell size. In wt NBs, the mitotic spindle is symmetric until metaphase. Starting from anaphase, the differential extension of the apical half spindle arm results in an apically biased asymmetric spindle (Kaltschmidt et al., 2000): the distance from the midspindle to the apical centrosome is larger than that to the basal centrosome. In addition, the spindle is displaced basally: the apical centrosome is located away from the NB apical cortex, whereas the basal centrosome lies close to the basal cortex (Cai et al., 2003). Consequently, the future cleavage plane is located toward the basal side of the NBs. Similar to pins, the majority of Gi mutant NBs generate an asymmetric spindle and produce two daughter cells with different cell sizes; however, similar to pins NBs, 21% (n = 86) of Gi NBs produce a symmetric spindle and give rise to equal size daughters (Fig. 3 B).$ K- Z# U! Q" j6 A! s( H/ v9 a/ Y
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Figure 3. Gi and pins form part of the same apical pathway for regulating NB mitotic spindle geometry. Confocal images of triple labeled telophase NBs (BP106, a membrane marker, red; DNA, cyan; Asense, a NB marker, cytosolic green in A–D or CNN, a centrosome marker, green in E–F) showing unequal size divisions in wt (A and E) and equal size divisions in various mutant combinations. 21% of Gi mutant NBs generate two approximately equal size daughter cells (B); further removal of baz function in Gi NBs (C) greatly increases the frequency of equal size divisions; similarly, Gi/insc NBs (D) also show high frequency of equal size divisions (100%, see Results). In wt NBs, the mitotic spindle, deduced from positions of the centrosomes, is asymmetric and displaced toward the basal cortex (E). In equal size NB divisions (e.g., Gi/insc NBs), the mitotic spindle is symmetric and the two centrosomes both lie in close vicinity of the cell cortex (F). Apical is up. Bar, 10 μm.
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To ascertain how Gi acts in the context of our two pathway models for the control of mitotic spindle geometry in NBs, we analyzed spindle geometry and daughter cell size in various combinations of double mutants with Gi. A high frequency of equal size divisions (Gai/baz RNAi, 100%, n = 39 ; Gai/insc, 100%, n = 66 ) is observed only when Gi and one of the components of Baz/DaPKC/Insc pathway are simultaneously disrupted. In contrast to wt NBs (Fig. 3 E), in these double mutants, for example, in Gi/insc NBs, the spindle geometry revealed with anticentrosomin (CNN) staining remains symmetric even at telophase with the cleavage plane being equidistant to both centrosomes (Fig. 3 F). Furthermore, the spindle is positioned symmetrically with both centrosomes lying in close proximity to the cell cortex (Fig. 3 F). In contrast, the frequency of equal size divisions in the Gi/pins double ablation NBs is low, comparable to frequencies seen in Gi or pins single mutants (Cai et al., 2003). These data indicate that Gi and Pins belong to the same pathway with respect to regulating asymmetric spindle geometry. Like pins, Gi loss of function in combination with mutation in baz, DaPKC, or Insc will disrupt both pathways which control spindle asymmetry and displacement in mitotic NBs, leading to the formation of a symmetric spindle and equal size daughters.
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Apical functions are necessary to suppress basal astral microtubule formation
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/ x7 w& ^( \8 u" U2 B0 x6 k/ H0 gOne striking observation seen with anti–-tubulin staining of mitotic NBs that had not been noted before is the influence of the apical functions on the asymmetric nature of the astral microtubules associated with the two centrosomes. In wt NBs, astral microtubules are nucleated at the apical centrosome, and the intensity of this staining increases markedly during the later stages of mitosis from metaphase onwards (Fig. 4, A–C), resulting in the formation of a prominent astral microtubule cap structure associated with the apical centrosome. In contrast, little astral microtubules can be seen near the basal centrosome. Although this preferential formation and association of astral microtubules with only the apical centrosome is not affected in single mutants of apical complex genes or double mutants affecting components of the same apical pathway (unpublished data), a dramatic change is observed in double mutants which affects both the Pins/Gi and Baz/DaPKC/Insc pathways. In these double mutant NBs, both centrosomes are associated with astral microtubules, with a cap structure forming over each centrosome from metaphase onwards (Fig. 4, J–L, N, and O). In addition, overexpression of Gi, which can lead to the uniform cortical localization of all apical components, and the loss of G?13F (see next section), also result in the production of prominent astral microtubules over both centrosomes (Fig. 4, G–I). This symmetric astral microtubule association with both centrosomes is similar to the astral microtubule structure seen in dividing epithelial cells (Fig. 4, D–F). These observations suggest that the presence of either of the asymmetric apical pathways is sufficient to suppress the formation of basal astral microtubules in NBs (see Discussion).6 ]5 W' Z. D8 F. s
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Figure 4. Apical complex functions regulate the asymmetric formation of astral microtubules in dividing NBs. Confocal images of triple labeled NBs showing microtubule structures (-tubulin, red; Mira, green; and DNA, cyan) in dividing NBs. In wt NBs (A–C), astral microtubules are weak or undetectable before metaphase; from metaphase onwards, astral microtubules associated with the apical centrosome grow out robustly and form a prominent, cap-like structure (arrow). In contrast, few astral microtubules associate with the basal centrosome during mitosis. Similar astral microtubule cap structures can be seen in dividing epithelial cells (arrow, D–F). In epithelial cells of the epidermis from metaphase onwards, astral microtubules form two cap-like structures; each associates with one of the centrosomes. Overexpression of Gi in wt embryos changes the astral microtubule structures in dividing NBs (G–I). In addition to the formation of a symmetric spindle, two astral microtubules cap-like structures (arrows) are formed, associated with each centrosome, similar to that seen in epithelial cells. Similar astral microtubule behavior (arrow) can be observed in NBs in which the Pins/Gi and Baz/DaPKC/Par6/Insc pathways are simultaneously compromised: Gai/insc NBs (J–L), Gi (M), insc/pins (N), baz/pins (O), and baz/Gi (unpublished data). Mira is distributed uniformly around the cell cortex in both baz/Gi and baz/pins NBs, suggesting the possible involvement of Baz in "telophase rescue" of basal proteins. For NB panels, apical is up. (D–F) Surface view of epithelial cells. Cell boundary is outlined with white dots. Bar, 10 μm.
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! K" ~+ P! b7 C9 N! gG?13F function is required for the asymmetric localization of apical components
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, w$ ?- @( s9 h, a3 y7 pTo compare and contrast the roles of Gi and G? in NB divisions, we analyzed G?13F mutant NBs. In contrast to Gi, G?13F, which has been shown previously to have a role in NB asymmetric divisions, is evenly distributed to the cortex of mitotic NBs. It has been reported (Schaefer et al., 2001) and we have confirmed that in G?13F mutants Gi is progressively degraded during embryonic development and becomes undetectable at stage 10 with anti-Gi staining (unpublished data), presumably due to the instability of Gi in the absence of G?13F. In G?13F mutant NBs, Insc is cytoplasmic (Fig. 5 A) and Pins levels are also strongly reduced and it appears to be distributed throughout the cell cortex and in the cytoplasm of all NBs (100%, n = 21 ). Hence, in all G?13F mutant NBs, both the stability and the asymmetric localization of Pins are drastically affected. In addition, in agreement with the findings of Fuse et al. (2003), we observed that spindle asymmetry is lost in the majority (65%, n = 110 ) of the G?13F NBs, and a similar proportion of NBs divide to produce two equal size daughter cells (Fig. 5 E).* X" N& e0 H" g1 Q- h2 T, }% a
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Figure 5. Loss of G?13F disrupts both apical pathways. In embryos lacking both maternal and zygotic G?13F, localization of apical proteins are disrupted (A–D): Insc becomes cytoplasmic (green, A); Pins is strongly reduced, and the residual Pins is either cortical or cytosolic (green, B); DaPKC is delocalized in the majority of NBs (71%) (green, D), and in 35% of NBs, DaPKC remains asymmetric but the crescent could be mislocalized (green, C). About 65% of the G?13F NBs undergo equal size divisions (E, Asense, red; BP106, green), suggesting that asymmetric spindle geometry and spindle displacement are defective. Further attenuation of Baz functions with RNAi treatment in G?13F germline clone embryos drastically increases the frequency of equal sized NB division (94%, n = 45, F). Anti–-tubulin staining shows that in G?13F NBs that undergo equal size divisions spindle is symmetric and two astral microtubule caps (arrows) are formed, each associated to one centrosome (red, G–I). In addition, spindle displacement is defective (I). Nevertheless, the Mira is asymmetrically segregated into only one of the daughter cells (green, G–I). Apical is up. DNA staining is in cyan. Cell boundaries are outlined (white dots). Bars: (A–I) 10 μm.
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- A6 E/ Q" [# ]7 i( SFigure 6. Depletion of free G? by overexpression of Gi or Go results in equal size NB divisions. (A) Western blot analysis of expression levels of Gi driven by maternal gal4 driver (mata), sca-gal4 driver, and in wt embryos. Gi levels based on densitometry are about fivefold (in mata-gal4 embryos) and twofold (in sca-gal4 embryos) higher than that in wt. Immunofluorescence data also show that mata-gal4 drives higher levels of Gi expression than sca-gal4 in NBs derived from stage 10 embryos. (B) Frequencies of equal size NB divisions induced by ectopic expression of Gi and Go and in G?13F germline clone embryos with and without attenuation of baz function. (C) Western blot showing coimmunoprecipitation of Go47A with G?13F when Gao is overexpressed using a maternal driver. Anti-G? antibody was used for immunoprecipitation. PI is a preimmune serum. Bar, 10 μm.* c6 N# w' m" Y- a

6 k* y, D5 S6 ]+ D  k7 O0 cSince we have previously shown that the loss or the uniform cortical localization of both Pins/Gi and Baz/DaPKC pathway members can abolish spindle asymmetry and result in equal size NB divisions, we wondered whether the equal size divisions seen in the G?13F NBs can be rationalized according to our model. If G?13F functions upstream of the apical complex members to regulate their asymmetric localization, stability, or function, we would expect Baz/DaPKC asymmetric localization/function to also be affected in G?13F mutant NBs. Indeed the anti-Baz and anti-DaPKC immunostainings show that Baz (unpublished data) and DaPKC asymmetric localization is lost or undetectable in 71% (n = 45) of G?13F NBs. In the rest of NBs, Baz (unpublished data) and DaPKC (Fig. 5 C) form cortical crescents. Further removal of Baz through RNAi in G?13F germline clones leads to equal size divisions (Fig. 5 F) in 94% of NBs (n = 45) (Fig. 6 B), suggesting that the function of the Baz/aPKC pathway is disrupted only in 71%, whereas the function of the Pins/Gai pathway is compromised in all of the NBs in G?13F embryos. Astral microtubules can be seen associated with both centrosomes in G?13F NBs undergoing equal size divisions (Fig. 5, G–I).
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These data suggest that G?13F (presumably in association with G) can function upstream of both apical pathways and act to promote the asymmetric localization/stability of the Baz/DaPKC and Pins/Gi pathway members. In the absence of G?13F, the functions of both apical pathways are compromised in the majority of NBs; they fail to generate an asymmetric mitotic spindle and consequently undergo equal size divisions. In the remainder of mutant NBs, although the function of the Pins/Gi pathway is compromised, Baz/DaPKC remain asymmetrically localized and functional; consequently asymmetric spindles and daughter cells of unequal size are produced. These findings support and extend on our earlier two pathway model (Cai et al., 2003) for the generation of an asymmetric mitotic spindle.
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Dosage-dependent effects of Gi overexpression on equal size NB divisions/ u% ~! w1 ?% H) b+ k
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Our previous study (Cai et al., 2003) showed that the equal size NB divisions caused by overexpression of Gi driven by sca-gal4 was dependent on pins function. Our interpretation of these results was that both proteins need to be present in a complex in order for a signal to be generated. However the observations that overrepression of Gi, but not constitutively activated form of Gi, in NBs disrupted asymmetric divisions and produced two equal size daughter cells (Schaefer et al., 2001) suggest that it is the depletion of free G? (caused by an excess of GDP-Gi) that might be the cause for the equal size NB divisions; the equal size NB divisions seen in the G?13F embryos provide further support for this view. If this were the case, then one would expect that under conditions in which Gi was in excess (with respect to all other molecules it can complex with like Pins and G?) free G? should be depleted whether Pins was present or not. How can these seemingly contradictory observations be reconciled?
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One possible explanation is that under conditions that we used previously (sca-gal4 driving UAS-Gi) Gi is not overexpressed to excess. Under these conditions, the phenotypic effects produced are caused by uniform Pins/Gi signaling from the cortex, and not by the sequestration of G? due to excess Gi, and therefore are Pins dependent. To test whether the equal size division phenotype is dependent on Pins under circumstances in which Gi is overexpressed to higher levels, we used a stronger driver (mata-gal4 VP16 V32). This driver increases Gi levels by about fivefold (compared with wt) compared with a twofold increase by sca-gal4 as judged by Western blot analysis of embryonic extracts (Fig. 6 A). In immunofluorescence experiments using identical conditions, mata-gal4 VP16 V32 also drives a higher level of expression than sca-gal4 in NBs (Fig. 6 A). The increased levels of Gi overexpression leads to a high frequency of equal size NB divisions (83%, n = 55 ) which is largely independent of Pins, since overexpression in the absence of Pins only marginally reduce the frequency of equal size NB divisions (62%, n = 62 ).
8 ^, h7 o* U" ~0 X* X% K- Y( z& a2 l1 y7 E
Our interpretation of these observations is that overexpression of Gi can cause NBs to undergo equal size divisions via two different mechanisms. With the levels of overexpression obtained with sca-gal4, Gi binds primarily to Pins and recruits Pins uniformly to the NB cortex (Cai et al., 2003). The cortical Pins/Gi can, presumably through a signaling function, disrupt the Baz/DaPKC apical localization, resulting in equal size NB divisions. In the absence of Pins, although both endogenous and ectopic Gi molecules are uniformly cortical, Gi alone cannot or is less able to interfere with Baz/DaPKC asymmetric localization. With higher levels of ectopic Gi (mata-gal4 VP16 V32 driver), not only are Pins/Gi uniformly cortical but the excess Gi can also bind to and deplete free G?. With limiting levels of free G?, both apical pathways can be disrupted as seen in the G?13F mutants. In the presence of higher levels of Gi, Pins is not required for the majority of the equal size NB divisions since its absence would not affect the ability of Gi to sequester free G?.# M9 @6 W& w# u

8 F6 B6 h- \/ K) [, |( dOverexpression of Go causes equal size NB divisions
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8 U! d8 M. P; L( IIf the depletion of free G? can disrupt asymmetric NB divisions, we might expect that other G molecules that can interact with G? may also be able to reproduce the Gi overexpression phenotypes when ectopically expressed in NBs. One such molecule, Go47A, which shares high homology with Gi, is able to bind/complex G?13F in vivo as indicated by the observation that it coimmunoprecipitates with G?13F when it is overexpressed (Fig. 6 C). Anti-Go47A staining shows a weak cortical localization of the protein in NBs (unpublished data; Schaefer et al., 2001). However, removal of both maternal and zygotic Go47A does not affect any aspect of NB asymmetric division, indicating that Go47A is not normally required in wt NBs. When Go47A is overexpressed, we observe a high frequency of NB equal size divisions (85%, n = 41 ), similar to that seen with Gi overexpression (Fig. 4 I). In metaphase NBs overexpressing Go, it shows a strong uniform cortical signal (Fig. 7 A); Gi levels are reduced dramatically (100%, n = 76 ); Pins is cortical (Fig. 7 C); Insc is delocalized (100%, n = 23 ); DaPKC becomes uniformly cortical or undetectable (100%, n = 36 ); and spindle geometry late in mitosis remains symmetric (Fig. 7, I and K), suggesting the disruption of both apical pathways. In addition, Mira is delocalized and can segregate into both daughter cells (75%, n = 40 ).
2 B2 F/ X0 X* ]7 V8 i8 }- `8 ?- o7 Y5 G, f
Figure 7. Overexpression of Go mimics G? mutant phenotypes. In mitotic NBs ectopically expressing Go, double label confocal images (A and B; C and D) show that Go (red, A) gives a strong uniformly cortical signal whereas Gi (green, B) is weak or undetectable; Pins (green, C) becomes uniformly cortical and Insc (red, D) shows punctuated, delocalized staining. DaPKC is no longer apical as seen in wt (green, E) but weak and uniformly cortical in most of NBs (green, F). Mira basal localization is also disrupted in the presence of ectopic Go in the majority of the NBs: Mira is delocalized at metaphase (red, G), and telophase rescue often does not occur late in mitosis (red, H). The majority of NBs (85%, n = 41) undergo equal sized divisions when Gao is ectopically expressed (I and K); two microtubule caps (red, I) are prominent (arrow); each is associated with one centrosome and mitotic spindle geometry is symmetric (CNN, green, K). Induction of similar mutant phenotype cannot be achieved when the constitutively active form of GoQ205L that should mimic the GTP-bound form of Go is overexpressed in the embryos (J and L); only one prominent microtubule cap is formed (arrow, J), and the mitotic spindle is asymmetric (red, J; CNN, green, L) like in wt NBs. Apical is up. DNA staining is in cyan. Mira is green in I and J. Cell boundaries are outlined (white dots) or marked by BP106 (K and L). Bars: (A–L) 10 μm.
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1 ~& ?5 F; [4 F' b8 l- MOverexpression of a putative constitutively active GoQ205L in NBs does not show any defects in spindle geometry (Fig. 7, J and L), suggesting that it is the GDP-bound Go which is responsible for the defect in size asymmetry in the overexpression experiments. Our results therefore suggest that depletion of free G? either by mutation or by greatly increasing the levels of G subunits can compromise the function of both apical pathways. These data are consistent with the view that G?13F (G?) can act genetically upstream of apical complex members to mediate their asymmetric localization., c' V5 `& z- m5 j$ m5 L7 r
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Discussion& O+ c: A( D6 L

: r  y* G% F" o' y; ]' E) ZHere we report the isolation and analysis of loss of function mutations in Gi and show that the loss of Gi and G?13F have distinct effects on NB asymmetric cell divisions. Gi is required for Pins cortical association and asymmetric localization; loss of Gi causes Pins to localize to the cytosol, and mutant NBs exhibit phenotypes which are highly similar to those seen in pins mutants. Analyses of double mutant combinations confirm Gi RNAi results showing that Pins/Gi and Baz/DaPKC/Insc act in an redundant fashion to mediate the formations of an asymmetric mitotic spindle and the generation of NB daughters of unequal size. Importantly, our analyses also revealed a new aspect of apical complex function: that the two apical pathways also act redundantly to suppress the formation of astral microtubules from the basal centrosome of NBs. In contrast, G?13F appears to act upstream of the apical components and is required for their asymmetric localization/stability. The defects associated with NBs lacking G?13F function are highly similar to those seen when the function of both apical pathways have been compromised. In addition, we show that high level overexpression of two different G subunits which can bind/complex to G?13F result in similar phenotypes seen in G?13F mutant NBs, suggesting that it is the depletion of free G?13F, which is responsible for the mutant phenotypes.& b( {1 y5 N% e

* v' ^- R# w* d/ Q6 W1 ?Gi is required to target Pins to the NB cortex% ^* H8 ~8 k: Y7 v% b6 T& m
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Our results indicate that Pins and Gi apical localization are mutually dependent. In pins NBs, Gi is evenly distributed to the NB cortex, and in Gi mutant NBs, Pins localizes to the cytosol. We have provided evidence previously that Pins asymmetric localization to the apical cortex of the NBs is a two-step process (Yu et al., 2002): Pins need to be targeted to the cortex first, which requires the COOH-terminal Goloco motifs that can bind Gi before it can be recruited to the apical cortex in a process which requires its NH2-terminal TPR that can interact with Insc. Our current results therefore suggest that Pins cortical targeting is most likely mediated by Gi, which cannot only bind Pins but is also able to localize to the plasma membrane through lipid modifications (Casey, 1994).
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% j# H# f: D( [8 T! bHowever, in G?13F mutant NBs, although the levels of Pins are drastically reduced, the residual Pins is localized both to the cytosol and to the cell cortex. This poses a problem since in the G?13F mutant NBs not only is G?13F absent but Gi also is undetectable with an anti-Gi antibody. One possible explanation is that although Gi is undetectable, there is still some Gi remaining in the G?13F NBs which may account for the low level residual uniform cortical distribution of Pins. Alternatively, we cannot formally rule out the possibility that the cortical Pins in G?13F NBs is due to some unknown molecule that can recruit Pins to cortex in the absence of both Gi and G?13F.
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  Y/ B3 R8 @9 v9 P' P) `G?13F acts upstream of the apical components to mediate their asymmetric localization5 n6 z" }. I& C$ h: @; Q
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The analysis of G?13F function is complicated by the fact that in the G?13F mutant NBs, Gi levels are also down-regulated presumably due to the instability of the protein in the absence of G?13F. Although loss of either Gi or G?13F causes aberrations in localization of the basal components and orientation of the mitotic spindle, it is clear that at least some of the defects associated with the loss of G?13F cannot be attributable solely to the depletion of Gi. In the great majority of Gi mutant NBs, DaPKC and Baz still localize asymmetrically to a subset of the cell cortex. And consistent with our proposal that spindle geometry and the size asymmetry of the NB daughters are mediated by two redundant apical pathways, Pins/Gi and Baz/DaPKC, the great majority (79%) of the Gi mutant NBs generate an asymmetric mitotic spindle and divide to produce unequal size daughters. In contrast, in G?13F NBs not only do Pins/Gi always fail to become asymmetrically localized but the majority of mutant NBs (71%) also fail to asymmetrically localize Baz/DaPKC; consequently 65% of NBs fail to generate an asymmetric mitotic spindle and divide to produce equal size daughters. Therefore, at least formally, G?13F acts upstream of the two apical pathways (Fig. 8 A).4 Y' |- |) y  x4 R; b

' B8 Q) J3 d1 }6 WFigure 8. Models. (A) A schematic diagram depicting our proposed hierarchical relationship between G?13F and the apical pathways. (B) We propose that an as yet unidentified adaptor protein(s) (blue) acts to promote/stabilize the formation of astral microtubules. In wt NBs or when at least one of the apical pathways is intact, this adaptor protein is asymmetrically localized to a specific region of the cortex and promotes asymmetric formation of astral microtubules (and cap) only in association with the proximal centrosome. However, when both apical pathways are compromised this protein becomes delocalized and consequently astral microtubules form over both centrosomes. See Discussion.1 |/ T. O/ W& p4 g
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We believe that the major reason for the phenotypes associated with loss of G?13F function is due to the disruption of G? signaling. We show, as previously reported (Schaefer et al., 2001; Cai et al., 2003), that overexpression of Gi will cause a high frequency of equal size divisions. In addition, we show here that the overexpression of Go, a G subunit that interacts with G?13F but is not itself required for asymmetric divisions in wt NBs, will also mimic the G?13F loss of function phenotype. For both overexpression of Gi and Go, the frequency of equal size divisions is significantly higher than that seen in G?13F loss of function (80 versus 65%). This difference may be due to the existence of other G? subunits which might also function in NB asymmetric divisions. Three G? genes have been identified by the Drosophila genome project, and although one of these genes, concertina, appears not to be involved in the process (Schaefer et al., 2001), it is possible that overexpression of G molecules may deplete not only G?13F but also G?76C. This possibility could be addressed by the analysis of double mutants of G? genes. Nevertheless, these observations are consistent with the view that the depletion of free G?, and not Gi, is the major cause for the symmetric divisions seen in G?13F mutant NBs (Fuse et al., 2003). Hence, although previous analysis of G?13F loss of function did not report any effects on NB daughter size, our data are in agreement with those of Fuse et al. (2003) and consistent with the notion that G?13F plays a major role in mediating the distinct size of NB daughter cells.
. |1 M# w" F/ B% C, I6 |  L& X
8 i7 N* a0 }/ f# F8 }7 {7 Q1 d- {" x6 HApical pathways act redundantly to prevent basal astral microtubule formation
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4 z9 w! C8 Z" o/ g1 ]0 A3 I3 BThe apical centrosome associates with prominent astral microtubules, whereas the basal centrosome connects to few if any astral microtubules in wt NBs and in mutants in which one of the two apical pathways is compromised. In contrast, in NBs that lack both apical pathways a symmetric mitotic apparatus is established that features extensive arrays of astral microtubules at both centrosomes. Therefore, either of the two apical pathways appears sufficient to prevent formation of basal astral microtubules. It is not clear how this might be accomplished at a mechanistic level. However, one might speculate that there exists an asymmetrically localized molecule, which can act to promote the formation of astral microtubules. When either of the apical pathways is functional, this molecule is asymmetrically localized and promotes the formation of astral microtubules only over the centrosome it overlies. However, when both apical pathways are mutated, or when G?13F is mutated or when all apical components become uniformly cortical, e.g., when Gi is overexpressed, then the hypothetical molecule becomes uniformly cortical and can promote the formation of astral microtubules over both centrosomes (Fig. 8 B). This type of model can readily explain why either loss or uniform cortical localization of both apical pathways leads to symmetric astral microtubule formation over both centrosomes.% s; n+ o: b0 y
1 Y% @2 c, H1 V" T
In summary, our results demonstrate that for NB asymmetric divisions Gi and G?13F play distinct roles. Gi and Pins are members of one of the two apical pathways and Baz/DaPKC/Insc forms the other. Loss of Gi function results in defects in NB asymmetry that are essentially indistinguishable from those seen in pins mutants. G?13F (G?) functions upstream of both Pins/Gi and Baz/DaPKC/Insc pathways to mediate their stability and/or asymmetric localization (and function). Without G?13F, the function of both apical pathways are attenuated; Gi levels are dramatically reduced and Pins/Gi pathway is defective; in addition, the asymmetric localization of members of the Baz/DaPKC/Insc pathway is often defective. Consequently, loss of G?13F function yields phenotypes which are similar to those seen when both apical pathways are disrupted by mutations. A schematic summary depicting the hierarchical relationship between G?13F and the apical pathways and our speculative model of how the apical pathways might act to "suppress" the formation of basal astral microtubules are depicted in Fig. 8.: u1 G% H, ^" u. B% |) a# d

: H" l4 F% |, N& N/ DMaterials and methods0 T, G- a" \6 k: }

# G8 g; O# _3 K! t  v% gFlies" q' Q& G% m( l& n. G( O; Q
% w: p% V0 t, l0 e9 D1 ~2 \
insc (insc22), pins (pins62, pins89), scabrous-gal4 (sca-gal4), and UAS-Gai were described earlier (Yu et al., 2000; Cai et al., 2003). KG01907 was a gift from H. Bellen (Baylor College of Medicine, Houston, TX). UAS-Go and bkh 007, an allele of Go, were a gift from M. Semeriva (LGPD, Centre National de la Recherche Scientifique, Marseille, France). FRT101-G?13F was provided by J.A. Knoblich (Research Institute of Molecular Pathology , Vienna, Austria).
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Mobilization of P element
2 n! i$ z) v) }; k  e, Z7 m' B/ a# e( K2 T) b  `- X; b
KG01907 carrying a P element derivative that contains the white gene is inserted near the 5' end of the Gi transcription unit at cytological location 65D6. The P element in this stock was mobilized using P(ry 2–3)(99B) as a transposase source. 300 independent w- revertant lines were established. These were analyzed on Southern blots using various portions of the Gai cDNA as hybridization probes. Several small deletion events which resulted in deletions that removed some or all of the Gai coding region were recovered.1 o5 j4 D/ T9 S0 [5 @& C

6 H# V6 G2 B) v, o; AGermline transformation, overexpression studies, and RNAi experiments
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+ V; k& j3 ]' b1 N& a/ Q& bTransgenes were expressed in NBs using either the maternal GAL4 driver V32 (obtained from D. St. Johnston, Wellcome/CRC Institute, Cambridge, UK) or scabrous-gal4 (Brand and Perrimon, 1993). UAS-Gao and UAS-Gao Q205L were created by cloning the full-length Gao cDNA (Fremion et al., 1999) or a mutant version in which glutamine 205 had been replaced with leucine into pUAST (Brand and Perrimon, 1993). Rescue experiments were performed by driving the expression of the UAS-Gai transgene with a sca-gal4 driver in Gi mutant background.
* u, \% B: O1 C  S2 R! a0 G* u2 g0 K- p' ^! h3 s* w
A 0.8-kb PstI fragment of baz cDNA (from Andreas Wodarz, University of Duesseldorf, Duesseldorf, Germany) was used as a template for RNAi experiments and subcloned into a modified pBluescript vector (pKS-ds-T7) (Cai et al., 2001) for double strand RNA synthesis.
9 {- _; ~0 K3 b1 K8 D9 K. O+ X  X' \
Immunocytochemistry and confocal microscopy
  @; w: S! r9 a$ e9 X& y
# a: g. [( d. P# S5 rEmbryos were collected and fixed according to Yu et al. (2000); for -tubulin and ?-tubulin stainings, embryos were fixed with 38% formaldehyde for exactly 1 min. Rabbit anti-Asense (provided by Y.-N. Jan, University of California, San Francisco, San Francisco, CA), rabbit anti-Baz (provided by F. Matsuzaki, Center for Developmental Biology, RIKEN, Kobe, Japan), mouse anti-Eve (Kai Zinn, Caltech, Pasadena, CA), rabbit anti-Insc, rabbit and rat anti-Pins, rabbit anti-Gi (aa 327–355; provided by J.A. Knoblich, IMP), guinea pig anti-Go (provided by M. Forte, Oregon Health Sciences University, Portland, OR), rabbit anti-PKC C20 (Santa Cruz Biotechnology, Inc.), rabbit anti-G?13F (provided by J.A. Knoblich), rabbit anti-Mira (provided by F. Matsuzaki), rabbit anti-Pon (provided by Y.-N. Jan), rabbit anti-Numb (provided by Y.-N. Jan), mouse anti- tubulin (DM1A; Sigma-Aldrich), rabbit anti–-tubulin (provided by D. Glover, University of Cambridge, Cambridge, UK), rabbit anti-CNN (provided by T.C. Kaufman, Indiana University, Bloomington, IN), anti-Pros MR1A (provided by C.Q. Doe, University of Oregon, Eugene, OR), mouse anti-? gal (Chemicon), anti–?-tubulin E7 (Developmental Studies Hybridoma Bank ) and anti-Nrt BP106 (DSHB) were used in this study. Cy3- or FITC-conjugated secondary antibodies were obtained from Jackson Laboratories. Stained embryos were incubated with ToPro3 (Molecular Probes) for chromosome visualization and mounted in Vectashield (Vector Laboratories). Embryos were analyzed with laser scanning confocal microscopy (Bio-Rad Laboratories MRC 1024 and Zeiss LSM510 ). Images were processed with Adobe Photoshop?.+ T  n$ ]1 W; \. [, v

& [! B2 Y) Z/ F  _2 [/ FCoimmunoprecipitation and Western blot
! B, z3 G6 ^  ]% f3 S. o* h
) G+ @1 Q" {+ e  @% y8 a% B9 nEmbryos overexpressing Go using the maternal Gal4 driver V32 were ground in liquid nitrogen and mixed with fives times volume of the lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM PMSF, and protease inhibitor cocktail from Roche) for 30 min at 4°C. The embryo lysate was centrifuged at maximum speed in a microcentrifuge for 20 min. The supernatant (embryo extract) was used to immunoprecipitate with anti-G?13F antibody and the protein A/G beads (Amersham Biosciences). Beads were washed three times (10 min each) in lysis buffer. Bound proteins were analyzed by Western blots with anti-Go and anti-G?13F.
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' ?' i, I, m1 Q9 T6 s, X7 y. ]4 oAcknowledgments
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We thank our colleagues referred to in the Materials and methods section, DSHB (University of Iowa), and the Bloomington stock center for generously providing antibodies and fly stocks. We are grateful to F. Matsuzaki and N. Fuse (Center for Developmental Biology, RIKEN) for generously providing conditions for anti–-tubulin staining and exchanging and discussing data prior to publication. F. Yu would like to thank S Oliferenko for helpful discussion.
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- t2 e$ h8 ^4 c& Q0 s% o5 G. |$ RX. Yang is an adjunct staff, Department of Anatomy, National University of Singapore. W. Chia is a Wellcome Trust Principal Research fellow. This work was supported by A*STAR Singapore and the Wellcome Trust.+ r# a2 J" q+ M8 T# q

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红旗

干细胞库  

榴榴莲

哈哈 我支持你

bluesuns

免疫细胞治疗  

nauticus

人气还要再提高  

杏花

有才的不在少数啊  

MIYAGI

真是佩服得六体投地啊  

dypnr

看或者不看，贴子就在这里，不急不忙  

昕昕

回帖是种美德.  

foxok

小生对楼主之仰慕如滔滔江水连绵不绝,海枯石烂,天崩地裂,永不变心.