Cell fate diversity is generated in part by the unequal segregation of cell-fate determinants during asymmetric cell division. In the Drosophila bristle lineage, the sensory organ precursor (pI) cell is polarized along the anteroposterior (AP) axis by Frizzled (Fz) receptor signaling. We show here that Fz localizes at the posterior apical cortex of the pI cell prior to mitosis, whereas Strabismus (Stbm) and Prickle (Pk),which are also required for AP polarization of the pI cell, co-localize at the anterior apical cortex. Thus, asymmetric localization of Fz, Stbm and Pk define two opposite cortical domains prior to mitosis of the pI cell. At mitosis, Stbm forms an anterior crescent that overlaps with the distribution of Partner of Inscuteable (Pins) and Discs-large (Dlg), two components of the anterior Dlg-Pins-Gαi complex that regulates the localization of cell-fate determinants. At prophase, Stbm promotes the anterior localization of Pins. By contrast, Dishevelled (Dsh) acts antagonistically to Stbm by excluding Pins from the posterior cortex. We propose that the Stbm-dependent recruitment of Pins at the anterior cortex of the pI cell is a novel read-out of planar cell polarity.
During development, cell fate diversity is generated in part by asymmetric cell division, in which a mother cell divides to produce two daughter cells with distinct developmental potentials. Adoption of different fates often relies on the unequal segregation of cell-fate determinants at mitosis. To ensure unequal partitioning of cell-fate determinants at anaphase, the mitotic spindle must be lined up with the polarity axis revealed by the position of cell-fate determinants at the cortex. Polarization of the mother cell regulates both mitotic spindle positioning and asymmetric localization of cell-fate determinants.
Recent studies have shed light on the mechanisms that regulate cell polarization during asymmetric division in the Drosophila peripheral nervous system. Each bristle sensory organ is composed of four distinct cells that are generated from a single sensory organ precursor cell (pI for primary precursor) via a series of stereotyped asymmetric divisions(Fichelson and Gho, 2003; Gho et al., 1999; Reddy and Rodrigues, 1999). In the pupal notum, the pI cell divides asymmetrically within the plane of the single-layered epithelium, along the anteroposterior (AP) axis of the pupa, to generate an anterior pIIb cell and a posterior pIIa cell(Gho and Schweisguth, 1998). The cell-fate determinants Numb and Neuralized (Neur) localize at the anterior cortex of the pI cell during prophase (Le Borgne and Schweisguth, 2003; Rhyu et al., 1994). At prometaphase, the mitotic spindle rotates and lines up with these cell-fate determinants (Bellaiche et al.,2001a; Roegiers et al.,2001). Numb and Neur co-segregate into the anterior cell, where they both act to bias the Notch-mediated pIIa/pIIb fate decision(Berdnik et al., 2002; Guo et al., 1996; Le Borgne and Schweisguth,2003; Rhyu et al.,1994). The anterior localization of these two cell-fate determinants and the AP orientation of the mitotic spindle depend on the activity of the frizzled (fz) gene(Bellaiche et al., 2001a; Gho and Schweisguth, 1998; Roegiers et al., 2001). In fz mutant pupae, the division of the pI cell is oriented randomly relative to the AP axis, with Numb localizing asymmetrically at one pole of the mitotic spindle in most pI cells. Thus, fz activity is necessary to orient the planar cell polarity (PCP) of the pI cell along the AP axis but is not required for asymmetric localization of cell-fate determinants.
Fz encodes a seven-pass transmembrane receptor that signals to polarize cells within the plane of the epithelium(Adler, 2002). Recent studies have indicated that Fz signaling must be spatially restricted within the cell to ensure establishment of PCP (Strutt,2003; Strutt,2002). In pupal wing epidermal cells, which are polarized along the proximodistal axis of the wing, Fz becomes asymmetrically localized at the distal edge of the apical cortex of each epidermal cell(Strutt, 2001b). The asymmetric distribution of Fz is regulated by PCP proteins, three of which also localize asymmetrically. Dishevelled (Dsh) co-localizes with Fz at the distal-apical cortex (Axelrod,2001; Shimada et al.,2001), whereas Prickle (Pk), a LIM- and PET-domain protein, and Strabismus (Stbm; also know as Van Gogh), a four-pass transmembrane protein,form a complex and localize opposite to Fz(Bastock et al., 2003; Gubb et al., 1999; Jenny et al., 2003; Tree et al., 2002; Wolff and Rubin, 1998). The activity of each of these PCP proteins is required for each of the others to become localized asymmetrically. Thus, formation of two opposite domains at the apical cortex appears to be essential to spatially restrict Fz signaling to a specific domain at one pole of the apical cortex, which, in turn, is translated into the planar polarization of wing epidermal cells.
How Fz regulates the anterior localization of Numb and Neur as well as the AP orientation of the spindle is not well understood. Two signaling complexes localizing opposite to each other have, however, been implicated in this process. The first complex includes two PDZ-containing proteins, Bazooka (Baz;the fly homolog of Par3) and Par-6, together with the atypical Protein Kinase C (aPKC) (for a review, see Henrique and Schweisguth, 2003). This complex re-localizes from the apical cortex to the posterior lateral cortex at mitosis(Bellaiche et al., 2001b). The second complex includes the proteins Discs-large (Dlg), Partner of Inscuteable(Pins) and the Gαi subunit of heterotrimeric G proteins(Bellaiche et al., 2001b; Schaefer et al., 2001). This complex localizes at the anterior lateral cortex of the dividing pI cell. The Dlg protein contains three PDZ domains, one SH3 domain and a C-terminal GUK domain. In epithelial cells, Dlg localizes at septate junctions and regulates epithelial cell polarity (Bilder et al.,2000; Woods and Bryant,1991; Woods et al.,1996). In mitotic pI cells, Dlg interacts via its SH3 domain to Pins and regulates the accumulation of Pins at the anterior cortex(Bellaiche et al., 2001b). Pins is a modular protein with seven tetratricopeptide repeats (TPR) and three G protein regulatory (GPR) motifs (Schaefer et al., 2000; Yu et al.,2000). The GPR motif has the ability to bind the GDP-bound form of Gαi and has been proposed to promote Gβγ signaling by dissociating the Gβγ dimer from Gαi·GDP(De Vries et al., 2000; Schaefer et al., 2001). The downstream targets of the Pins-Gαi, Gβγ and aPKC signaling activities in the pI cell are not known.
We have investigated the mechanisms by which PCP signaling regulates the anterior localization of the Dlg-Pins-Gαi complex during the asymmetric pI cell division. Our data indicate that Fz localizes at the posterior cortex prior to mitosis, whereas Stbm and Pk co-localize at the anterior cortex. At mitosis, Stbm forms an anterior crescent that overlaps with the distribution of Pins and Dlg, and promotes Pins cortical localization at prophase. We propose that a read-out of PCP in the pI cell is the Stbm-dependent localization of the Dlg-Pins-Gαi complex at the anterior cortex.
Materials and methods
The null fzK21, fzKd4a, stbm6c, bazXJ106 alleles and the dsh1 allele are described in FlyBase(http://flybase.bio.indiana.edu/). pkpk-sple14 is a null allele (D. Gubb, personal communication).
Somatic clones were generated in: (1) Ubx-flp bazXJ106 FRT9-2/ubi-nlsGFP FRT9-2; (2) Ubx-flp dsh1 bazXJ106FRT9-2/ubi-nlsGFP FRT9-2; and (3) Ubx-flp bazXJ106 FRT9-2/nlsGFP FRT9-2; stbm6cpupae. The Ubx-flp stock was a kind gift of J. Knoblich.
The arm –Fz::GFP line is described elsewhere(Strutt, 2001b). The neurP72GAL4 (Bellaiche et al.,2001a) line was used to express Histone2B::YFP(Bellaiche et al., 2001a),Pon::GFP (Lu et al., 1999a),tau::GFP (Kaltschmidt et al.,2000), Pk (Gubb et al.,1999), GFP::Stbm, GFP::StbmΔPBM and Stbm using the UAS/GAL4 expression system (Brand and Perrimon,1993). The UAS-Stbm, UAS-GFP::Stbm, UAS-GFP::StbmΔPBM, arm-Stbm, arm-GFP::Stbm, arm-StbmΔPBM and arm-GFP::Stbm transgenic flies were generated by P-element transformation. In all GFP::Stbm constructs, the sequence of m6GFP (gift of A. Brand) was fused in frame to the first codon of Stbm (the stbm cDNA was a gift of T. Wolff). Cloning details for the UAS-GFP::Stbm,UAS-GFP::StbmΔPBM, arm-Stbm, arm-StbmΔPBM and arm-GFP::Stbm plasmids are available upon request.
Live GFP imaging was carried out as described(Bellaiche et al., 2001a). Orientation of the pI cell division was measured at telophase using tau::GFP or Pon::GFP in living pupae of the following genotypes: (1)neurP72GAL4/UAS-tau::GFP; (2) neurP72GAL4/UAS-Pon::GFP;(3) fzK21 UAS-tau::GFP/fzKd4aneurP72GAL4; (4) stbm6c;neurP72GAL4/UAS-Pon::GFP; (5) pkpk-sple14;neurP72GAL4/UAS-Pon::GFP. For all other genotypes, the orientation of the pI cell division was measured at telophase in fixed pupae described previously (Bellaiche et al.,2001a). Localization of Pon::GFP was analyzed in: (1) dsh1/Y; neurP72GAL4 UAS-Pon::GFP/+; (2) stbm6c; neurP72GAL4 UAS-Pon::GFP/+; (3) pkpk-sple14; neurP72GAL4 UAS-Pon::GFP/+; and(4) UAS-Pk/+; neurP72GAL4 UAS-Pon::GFP/UAS-Stbm pupae.
Fluorescence Recovery After Photo-bleaching (FRAP) analysis was performed on arm-Fz::GFP pupae. Photobleaching was achieved by scanning a region of interest using the 488 nm laser source at maximal intensity.
All images were acquired on a SP2 confocal microscope and assembled using NIH image and Photoshop software.
Rabbit polyclonal anti-Stbm antibodies were raised against a mixture of the two peptides CNVLAEEVVDPKSNKFV and MENESVKSEHSGRSRC.
Pupal nota were dissected and processed as previously described(Gho et al., 1999). Primary antibodies were: guinea pig anti-Senseless (Sens; gift from H. Bellen;1:3000); mouse anti-Cut (2B10 obtained from the DSHB; 1:500); rat anti-DE-Cadherin (gift from T. Uemura; 1:50); guinea pig anti-Dlg (gift from P. Bryant; 1:3000); rabbit anti-Baz (gift from A. Wodarz; 1:3000); rabbit anti-Pins (gift from J. Knoblich; 1:1000); rat anti-Pins (gift from P. Bryant;1:1000); rabbit anti-Pk (gift from J. Axelrod; 1:500); rabbit anti-Stbm(affinity-purified;1:400); and rabbit anti-GFP (Molecular Probes; 1:1000). Cy3- and Cy5-coupled secondary antibodies were from Jackson's Laboratories and Alexa-488-coupled secondary antibodies were from Molecular Probes.
Protein interaction assays
A cDNA fragment encompassing the intracellular domain of Stbm with(303-584) or without (303-581) the PBM was subcloned into pGEX2KG. A PCR product encompassing Stbm553-584 was subcloned into pGEX2KG to generate GST-PBM. Similarly, cDNA fragments encoding the full-length DlgA isoform(GST-Dlg) or the PDZ domains of Dlg (GST-PDZ1-3: amino acid 39-640) were subcloned into pGEX2KG. Met 35S radiolabelled Stbm and Dlg proteins were in vitro synthesized using the TNT kit (Promega). Stbm was synthesized in presence of Canine Pancreatic Microsomal Membranes (Promega). Pull-down assays were performed as previously described (beads were incubated in PBS, NP40 0.5%and 0.5 M NaCl in the final wash) (Brou et al., 1994).
Fz localizes at the posterior apical cortex of the pI cell
In the notum of wild-type pupae, pI cells divide within the plane of the epithelium and along the AP axis (Fig. 1A) (Gho et al.,1999; Gho and Schweisguth,1998). In fz mutant pupae, the mitotic spindle is randomly oriented relative to the AP axis(Fig. 1B). This PCP phenotype was fully rescued by a transgene directing the ubiquitous expression of a Fz::GFP fusion protein (Fig. 1C) (Strutt,2001b). Using this functional Fz::GFP protein, we have examined the localization of Fz in the developing notum at 16-17 hours APF, when pI cells divide. Fz::GFP co-localized with Baz and E-Cadherin (Cad) at the apical cortex of epidermal cells (not shown), and showed no obvious sign of planar asymmetry. In pI cells, however, Fz::GFP predominantly co-localized with Baz at the posterior cortex at prophase (Fig. 1D-D″). Posterior localization of Fz::GFP was confirmed using Fluorescence Recovery After Photo-bleaching(Fig. 1E-E″). The posterior cortex of the pI cell and the epidermal cells posterior to the pI cell were photo-bleached prior to division of the pI cell. If Fz::GFP accumulates at the anterior cortex of epidermal cells, then no significant fluorescence recovery should be observed at the contact region with the pI cell because the entire pool of Fz::GFP molecules of these posterior epidermal cells has been photo-bleached. By contrast, if Fz::GFP accumulates at the posterior cortex of the pI cell, then fluorescence recovery should be observed because of the redistribution of fluorescent Fz::GFP molecules within the pI cell. After photo-bleaching, a recovery of Fz::GFP was seen at the posterior cortex of the pI cell (Fig. 1E-E″). We therefore conclude that Fz::GFP accumulates at the posterior pole of the pI cell.
Time lapse-analysis revealed that Fz::GFP localized uniformly at the apical cortex of pI cells in dsh1, stbm6c and pkpk-sple14 mutant pupae(Fig. 1F-H; data not shown),indicating that posterior accumulation of Fz::GFP in pI cells required the activity of the dsh, stbm and pk genes. Thus, our results extend to the pI cell the observation first made in wing epidermal cells showing that unipolar accumulation of Fz::GFP depends on PCP signaling(Strutt, 2001b; Strutt, 2002).
Stbm and Pk co-localize at the apical anterior cortex of the pI cell
The stbm and pk genes not only regulate the posterior localization of Fz::GFP in pI cells but also polarize the pI cell along the AP axis. In stbm6c and pkpk-sple14 mutant pupae, pI cells divided with a random orientation(Fig. 2A,D) and Numb localized asymmetrically but at a random position relative to the AP axis (not shown). Thus, stbm and pk mutant pI cells exhibit planar polarity defects that are similar to the ones previously described for fz,dsh1 and fmi mutant pupae(Gho and Schweisguth, 1998; Lu et al., 1999b).
To study the distribution of Stbm, we generated antibodies that specifically detect Stbm on western blots and on fixed tissues (data not shown; see Fig. S1). In epidermal cells, Stbm co-localized with Cad, apical to Dlg, with no clear sign of planar asymmetry (Fig. 2F-G). In pI cells, however, Stbm localized asymmetrically at the anterior cortex prior to division, and formed a crescent opposite to Baz upon mitosis (Fig. 2H-J″).
We further studied the distribution of Stbm using a GFP::Stbm fusion protein. Uniform low-level expression of GFP::Stbm fully rescued the stbm6c mutant PCP phenotype(Fig. 2B), indicating that GFP::Stbm is functional (GFP::Stbm was expressed at a level similar to endogenous Stbm: see Fig. S1). Expression of GFP::Stbm (or Stbm) in pI cells did not modify the stereotyped orientation of the pI cell division (Fig. 2C; data not shown). GFP::Stbm localized at the apical anterior cortex of the pI cell prior to division(Fig. 3A) and formed an anterior crescent at mitosis (Fig. 3B,B′). Anterior accumulation of GFP::Stbm was also seen in epidermal cells (Fig. 3C). This suggests that endogenous Stbm may also localize asymmetrically in epidermal cells but that this asymmetry is difficult to visualize because cells in the notum do not form a regular hexagonal lattice.
We then investigated the localization of Pk using anti-Pk antibodies(Tree et al., 2002). Pk co-localized with Cad at the apical cortex of epidermal cells(Fig. 4A-A″). As observed above for Fz::GFP and Stbm, no sign of planar asymmetry was detected in the distribution of Pk in epidermal cells. However, the higher level of Pk accumulation in pI cells allowed us to detect an accumulation of Pk at the anterior cortex of pI cells, opposite to Baz(Fig. 4A-B). Consistent with the notion that Pk and Stbm directly interact(Bastock et al., 2003; Jenny et al., 2003), Pk co-localized with GFP::Stbm (Fig. 4C,C′). We conclude that Pk and Stbm co-localize at the anterior apical cortex of the pI cell, opposite to Fz, prior to mitosis.
The asymmetric accumulation of Pk and Stbm was dependent on the activity of fz and dsh. In fzKD4a/fzK21 and dsh1 mutant cells, Pk, Stbm and GFP::Stbm were uniformly distributed at the apical cortex (Fig. 2K-K″, Fig. 3D,E and Fig. 4F-F″; and data not shown). Similarly, Stbm and Stbm::GFP localized uniformly at the apical cortex of pkpk-sple14mutant pI cells (Fig. 3I,J and data not shown). By contrast, Pk was mostly cytoplasmic in stbm6c mutant cells, with only a small amount of Pk remaining at the cell cortex (Fig. 4D-E) (Bastock et al.,2003), indicating that Stbm is required for the cortical localization of Pk. Furthermore, we found that the intracellular tail of Stbm promotes dimerization in vitro and that Stbm forms oligomers in vivo (see Fig. S1). Thus, oligomerization of both Pk and Stbm may contribute to the clustering, hence asymmetric localization, of Pk- and Stbm-containing complexes at one pole of the pI cell (Bastock et al., 2003; Jenny et al., 2003).
Overlapping distribution of Stbm, Pins and Dlg in dividing pI cells
The anterior accumulation of Stbm suggests that Stbm may co-localize with Pins and Dlg at the anterior cortex in dividing pI cells(Bellaiche et al., 2001b; Schaefer et al., 2001). Cortical staining for Pins was first detected at prophase. Pins co-localized with Stbm at the anterior apical cortex at this stage, whereas Dlg predominantly accumulated at a more basal position, with only the apical-most fraction of Dlg overlapping with Pins and Stbm(Fig. 5A-B'''; arrowhead in B). This suggests that Pins is first recruited at prophase at the anterior apical cortex where Stbm and Dlg appear to overlap. At prometaphase, Stbm localization appeared to extend to a slightly more lateral position where it overlapped with the apical-most region of the Dlg-Pins crescent(Fig. 5C-C''' and E-E''';arrowhead in E; see Fig. S2in order to view the complete stack). At a more basal position, high levels of Pins and Dlg were detected anteriorly, whereas Stbm levels were very weak(Fig. 5D-E'''). Thus, Stbm overlapped with the apical-most part of the cortical domain containing the Dlg-Pins complex at the anterior pole of the dividing pI cell from prophase onwards.
The PBM of Stbm binds Dlg and regulates the re-localization of Stbm at mitosis
As the distribution of Stbm overlapped with the localization of Dlg during mitosis, we tested whether Stbm interacts with Dlg. The presence of a class I PDZ Binding Motif (PBM) at the C terminus of Stbm indeed suggests that Stbm may directly interact with the PDZ domains of Dlg that are known to bind class I PBM. GST-Dlg was found to directly interact with Stbm in vitro. This interaction required the PBM of Stbm (Fig. 6A). Conversely, the C-terminal intracellular tail of Stbm[GST-StbmIntra(303-584)] bound both Dlg and a N-terminal fragment of Dlg containing the PDZ domains in a PBM-dependent manner(Fig. 6B). Similarly, the PBM motif of Stbm was sufficient to bind Dlg or the PDZ domains of Dlg(Fig. 6B). These data indicate that Dlg may directly interact via its PDZ domains with the PBM of Stbm.
The function of the PBM of Stbm was tested using a phenotypic rescue assay. Uniform low-level expression of Stbm rescued the stbm6cmutant PCP phenotype in pI cells (division angle relative to the AP axis:–10±43°; to be compared with–30±29° in wild-type). A similar result was obtained with StbmΔPBM, a Stbm protein deleted of its last three amino acids (division angle relative to the AP axis:–25±28°). Thus, the PBM is dispensable for the PCP function of stbm in pI cells [see Bastock et al.(Bastock et al., 2003) for a similar result in wing epidermal cells]. Furthermore, anterior localization of Stbm at interphase does not require its PBM(Fig. 3D). At prometaphase,GFP::Stbm relocalized from the anterior apical cortex to a more lateral position in 72% of the pI cells at prometaphase (n=18; Fig. 3B,B′), while GFP::StbmΔPBM remained predominantly apical in 86% of the pI cells(n=14; Fig. 3E,E′). This suggests that a PDZ-containing protein,possibly Dlg, regulates localization of Stbm at mitosis in pI cells.
Stbm promotes Pins localization at the anterior cortex at prophase
We next investigated the role of Stbm in the anterior localization of Pins. In wild-type pupae, Pins localized at the anterior cortex of 92% of the pI cells at prophase (n=24, Fig. 7A). By contrast, Pins localized asymmetrically in only 42% of the pI cells in stbm6c mutant pupae at prophase(n=17). In the remaining 58% of the pI cells, Pins was not restricted to a single pole at the cortex and was also found in the cytoplasm(Fig. 7B). This initial defect in Pins localization appeared to be corrected during prometaphase as Pins localized asymmetrically (but at a random position) in stbm6c mutant pI cells at prometaphase(Fig. 7E,F). These results indicate that Stbm is required for the initial localization of Pins at the anterior cortex.
Conversely, co-expression of Stbm and Pk using the neurP72GAL4 driver disrupted PCP in pI cells (Fig. 2E) and resulted in a broadened accumulation of cortical Pins in 78% of the pI cells at prophase (n=9; Fig. 7D). Again, this initial defect in Pins localization was partly corrected because the Pins crescent appeared to be extended in only 30% of the pI cells at prometaphase(Fig. 7H; n=20). We conclude that Stbm acts together with Pk to promote the anterior localization of Pins in mitotic pI cells.
Dsh excludes Pins from the posterior cortex at prophase
In contrast to the Stbm-Pk complex shown above to promote Pins cortical localization, Dsh appears to antagonize it. In 63% of the dsh1 mutant pI cells, Pins localized in a broad cortical region at prophase (Fig. 7C, n=33). This defect was rescued during prometaphase because in 75% of the dsh1 mutant pI cells, Pins accumulated asymmetrically(but at a random position) at prometaphase (n=28; Fig. 7G) opposite to Baz (data not shown). This suggests that Dsh acts antagonistically to the Pk-Stbm complex at prophase. As Dsh acts downstream of Fz, we propose that activated Dsh at the posterior cortex prevents Pins from accumulating there.
Dsh and Pk-Stbm act antagonistically to organize the anterior cortex
The anterior cortex has been proposed to regulate mitotic spindle positioning. First, the anterior pole of the mitotic spindle is more tightly associated with the anterior cortex than the posterior pole is with the posterior cortex (Roegiers et al.,2001). Second, in the fz mutant pI cells in which Pon::GFP, a marker for Numb localization, is mis-partitioned into the two daughter cells, both poles of the spindle appeared to be tightly associated with the `anterior-like' Pon::GFP-positive cortex(Bellaiche et al., 2001a; Doe, 2001; Roegiers et al., 2001). It is thought that a broadening of the `anterior-like' cortex results in the capture of both spindle poles, hence leading to a mis-partitioning of Pon::GFP(Fig. 8A,B). To further test the role of Dsh, Stbm and Pk in organizing the anterior cortex, we have used the defective partitioning of Pon::GFP as a read-out for the broadening of the anterior domain. In wild-type pI cells, Pon::GFP was asymmetrically localized at the anterior cortex from prophase onwards and was unequally segregated into the anterior cell at anaphase (100%; n=54). By contrast, Pon::GFP was mis-partitioned in 15% of fz mutant pI cells (n=33)(Bellaiche et al., 2001a) and in 11% of the dsh1 mutant pI cells (n=114). Such a defect was not seen in pkpk-sple14 mutant pupae (0%; n=17) and was very rare in stbm6c mutant pupae(1%; n=84). We conclude that the loss of fz or dshbut not of pk or stbm activities leads to a broadening of the anterior domain of the pI cell. Conversely, overexpressing Pk and Stbm resulted in defective partitioning of Pon::GFP (16%; n=70; Fig. 8D-E′). Moreover, a transient extension of the Pon::GFP-positive cortical domain could also be seen in cells that divide unequally with Pon::GFP segregating in only one of the two cells (Fig. 8C-C″). Thus, our analysis of both Pins localization(Fig. 7A-D) and Pon::GFP mis-partitioning (Fig. 8)indicate that the Pk-Stbm complex promotes the `anteriorization' of the cortex and that Fz/Dsh signaling acts antagonistically to restrict this`anteriorization'.
Dsh and Stbm have distinct functions at prometaphase
Finally, we have studied the mechanism that acts during prometaphase in stbm6c and dsh1 mutant pI cells to correct the initial defects in Pins localization(Fig. 7A-D). This mechanism may possibly involve the Baz-Par6-aPKC complex that localizes opposite to Pins. We have therefore analyzed the localization of Pins in pI cells that are double mutant for dsh1 and bazXJ106 or for stbm6c and bazXJ106. Although Pins correctly localized at the anterior cortex of bazXJ106mutant pI cells (Fig. 7I)(Bellaiche et al., 2001b),asymmetric localization of Pins was strongly reduced and/or lost in 71% of the dsh1 bazXJ106 double mutant pI cells at prometaphase (n=24, Fig. 7K). This shows that Baz functions in the rescue mechanism operating in dsh1 mutant pupae, possibly by excluding Pins. It further indicates that Dsh acts redundantly with Baz to maintain Pins at the anterior cortex of the pI cell during prometaphase. By contrast, Pins was found to localize asymmetrically in 67% of the bazXJ106stbm6c double mutant pI cells at prometaphase (n=15, Fig. 7J). This indicates that Baz does not play an essential role in the rescue mechanism operating in stbm6c mutant pupae. These data also show that Stbm and Dsh have distinct functions at prometaphase in dividing pI cells, at least in the absence of baz activity. We therefore conclude that the asymmetric localization of Pins is maintained in a baz-dependent manner in dsh1 mutant pupae and in a baz-independent manner in stbm6c mutant pupae.
PCP proteins define two opposite cortical domains prior to mitosis
Asymmetric localization of Fz, Stbm and Pk at the apical cortex define two opposite domains in the pI cell prior to its division. Unipolar accumulation of Fz, Stbm and Pk depends on PCP signaling: Fz appears to exclude Stbm and Pk from the posterior cortex and, conversely, Stbm and Pk appears to exclude Fz from the anterior cortex. In addition, the cortical localization of Pk depends in part on Stbm. As Stbm is an integral membrane protein and as Pk directly binds Stbm (Bastock et al.,2003; Jenny et al.,2003), one function of Stbm is to tether Pk at the membrane.
Recent studies have shown that Fz, Dsh, Stbm and Pk become asymmetrically distributed during planar polarization of the wing epidermis(Axelrod, 2001; Bastock et al., 2003; Shimada et al., 2001; Strutt, 2001b; Tree et al., 2002). In this tissue, planar polarity is established along the proximodistal axis. Fz and Dsh preferentially accumulate at the distal vertex of wing epidermal cells,whereas Pk and Stbm localize at the opposite proximal pole. Planar polarization of the eye epithelium also involves the asymmetric distribution of Fz, Dsh, Stbm and Pk (Das et al.,2002; Rawls and Wolff,2003; Strutt et al.,2002; Strutt,2002). Thus, our analysis of the distribution of Fz, Stbm and Pk in the pI cell reinforces the idea that unipolar distribution of PCP proteins is a landmark of planar polarization.
Pins cortical localization as a novel read-out for PCP
Planar polarization of the pI cell occurs prior to division and is required, upon entry into mitosis, to direct the Dlg-Pins-Gαi and Baz-Par6-aPKC complexes at the anterior and posterior cortex, respectively(Bellaiche et al., 2001b). We have shown that the localization of Pins at the anterior cortex is regulated positively by the Stbm-Pk complex and negatively by Dsh. First, loss of stbm activity results in a delay in the cortical localization of Pins during prophase. Second, concomitant expression of Stbm and Pk leads to a broadening of the cortical crescent of Pins at prophase. Third, loss of dsh PCP activity similarly results in an extended Pins crescent at prophase. Moreover, our analysis of the defective partitioning of Pon::GFP suggests that the Stbm-Pk complex acts antagonistically to Dsh to localize at the anterior cortex a centrosome-attracting activity. We propose that the Stbm-Pk complex organizes the anterior cortex and recruits the Dlg-Pins-Gαi complex as well as molecules regulating spindle positioning.
Cortical localization of Pins is a novel read-out of PCP signaling in the pI cell that is distinct from the ones previously identified in wing and eye cells (Fig. 9). In wing epidermal cells, Fz promotes the formation of a polarized actin cytoskeleton via a pathway that possibly involves a direct interaction between Dsh and a Daam1-Rho complex (Habas et al.,2001) and a Rho Kinase-dependent phosphorylation of cytoplasmic myosin (Strutt, 2001a). Whether Dsh also regulates microfilament assembly in pI cells remains to be studied. In photoreceptor cells, the read-out for PCP signaling is the transcriptional regulation of the Delta gene in R3(Cooper and Bray, 1999; Das et al., 2002; Fanto and Mlodzik, 1999; Mlodzik, 1999; Strutt et al., 2002). Thus,the conserved core of PCP signaling molecules have different, cell-type specific read-outs.
How does Stbm direct the localization of Pins to the anterior cortex? One hypothesis is that Stbm directs the anterior localization of Pins via the regulated assembly of a Stbm-Dlg-Pins complex. We have previously shown that the anterior accumulation of Pins depends on its interaction with Dlg(Bellaiche et al., 2001b). We provide evidence that Stbm may bind Dlg. First, in vitro binding studies indicate that Stbm interacts with Dlg. We note, however, that PDZ-containing proteins other than Dlg may also bind Stbm in this assay. Second, the localization of Stbm overlaps with the distribution of Pins and Dlg in dividing pI cells. Third, the PBM motif of Stbm appears to regulate the re-localization of Stbm in pI cells. Our data are therefore consistent with a model in which, upon mitosis, the binding of Stbm to Dlg in turn promotes the binding of Pins to Dlg and, hence, localization of Pins at the anterior cortex where Stbm and Dlg accumulations overlap. This model predicts that the PBM of Stbm should be required for the anterior localization of Pins. We have found,however, that StbmΔPBM is fully functional and that Pins is properly recruited at the anterior cortex in stbm6c mutant pI cells expressing StbmΔPBM (F.S., unpublished). One interpretation of this result is that Stbm regulates the localization of Pins not only via the PBM-dependent assembly of the Dlg-Pins complex but also via a second PBM-independent mechanism. As Dsh acts redundantly with Baz to localize Pins asymmetrically, we suggest that this second mechanism may involve Dsh. Accordingly, in stbm6c mutant pI cells, uniformly distributed Dsh activity would prevent Pins cortical localization. By contrast, as the PCP function of stbm does not depend on its PBM, the activity of Dsh should be restricted to the posterior cortex in stbm6c mutant pI cells expressing StbmΔPBM. Dsh should therefore restrict Pins localization to the anterior cortex in this mutant background. Another interpretation of the correct localization of Pins in stbm6c mutant pI cells expressing StbmΔPBM is that Stbm recruits Pins via a mechanism that does not involve an interaction with Dlg (or any other PDZ-containing proteins). Future studies will address how the Stbm-Pk complex regulates the localization of Pins in the pI cell.
Maintenance of Pins anterior localization in the mitotic pI cell
Different mechanisms appear to cooperate to maintain Pins asymmetric localization. We have shown here that baz is required for the asymmetric localization of Pins in the absence of dsh PCP activity. This indicates that Baz can regulate the maintenance of Pins asymmetric localization at prometaphase. The loss of asymmetric localization of Pins in dsh baz mutant pI cells suggests that Dsh may also contribute to maintain Pins asymmetric localization at prometaphase. Dsh does not merely act by excluding Stbm, a positive regulator of Pins localization in prophase,because Pins localizes asymmetrically in baz stbm double mutant pI cells. The mechanisms by which Baz and Dsh regulates Pins localization are not known. However, as Pins regulates its own localization via a Gβ13F-dependent positive feedback loop(Fuse et al., 2003; Schaefer et al., 2001), one hypothesis is that Baz and/or Dsh negatively regulates Gβ13F signaling activity.
Implications for the read-out of PCP in vertebrates
One of the best examples of PCP in mammals is the stereotyped planar orientation of the stereociliary bundles that are located at the apical cortex of each mechanosensory hair cells within the cochlea. In these cells, the first sign of polarization is the stereotyped movement, at the luminal surface of the cell and along the neural-abneural axis, of the kinocilium, the single tubulin-based cilium, from the center towards the abneural pole of the cell(Montcouquiol et al., 2003). Recently, a mutation in a stbm homolog, Vangl2, has been shown to result in the defective orientation of the stereociliary bundles. This planar cell polarity defect appears to result from the randomly oriented center-to-periphery movement of the kinocilium(Montcouquiol et al., 2003). Because LGN, a mammalian homolog of Pins(Yu et al., 2003), is known to regulate microtubule stability (Du et al.,2002), it is tempting to speculate that Vangl2 may regulate via LGN a microtubule-dependent process regulating kinocilium movement along the neural-abneural axis. Future studies will reveal whether the regulation of Pins/LGN cortical localization is a conserved read-out of PCP.
Note added in proof
A recent study by Lee et al. (Lee et al., 2003) indicates that Stbm directly interacts with Dlg via its C-terminal PBM. Dlg and Stbm can be co-immunoprecipated from a Drosophila embryonic extract. This direct interaction was also observed with mammalian homologs of Stbm and Dlg. The authors propose that the Dlg-Stbm interaction is important for the formation of new plasma membrane during cellularisation of the Drosophila embryo.
Supplemental data available online
We thank J. Axelrod, H. Bellen, J.-P. Borg, A. Brand, P. Bryant, W. Chia,R. Finley, D. Gubb, Y.-N. Jan, J. Knoblich, D. Strutt, T. Uemura, A. Wodarz and T. Wolff for generously providing us with flies, yeasts, plasmids and antibodies. We also thank A. Brand for suggesting the FRAP experiment and P. Bryant and T. Wolff for sharing unpublished information. We thank A. Bardin,Z. Gomes, C. Goridis, R. Le Borgne and M. Wassef for critical reading. This work was supported by an ACI grant from the Ministère de la Recherche and by the Association pour la Recherche sur le Cancer (ARC 4512 to F.S. and ARC 4726 to Y.B.).