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First published online December 30, 2003
doi: 10.1242/10.1242/dev.00928

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The planar cell polarity protein Strabismus promotes Pins anterior localization during asymmetric division of sensory organ precursor cells in Drosophila

Yohanns Bellaïche^*, Olivia Beaudoin-Massiani, Isabella Stüttem and François Schweisguth

Ecole Normale Supérieure, UMR 8542, 46, rue d'Ulm, 75230 Paris, Cedex 05, France

View larger version (120K): [in a new window]   Fig. 1. Fz-GFP localizes to the posterior cortex. (A-C) Orientation of the pI cell division in neurP72GAL4 UAS-tau::GFP (wild type; A), fzK21 UAS-tau::GFP/fzKd4a neurP72GAL4 (B) and arm-Fz::GFP/+; fzK21 UAS-tau::GFP/fzKd4a neurP72GAL4 pupae (C). Expression of Fz::GFP rescued the fz PCP phenotype. Each red dot indicates the orientation of one division measured by live imaging as described elsewhere (Bellaiche et al., 2001a). Anterior is leftwards, midline is towards the top. Mean and standard deviation values are given for genotypes exhibiting a stereotyped orientation. (D-D'') Fz::GFP (anti-GFP; green in D,D') co-localized with Baz (blue in D,D'') at the apical posterior cortex of pI cells at early prophase. Sens (red in D) was used as a pI cell marker. (E-E'') FRAP analysis indicates that Fz::GFP localizes at the posterior cortex in the pI cell. Live GFP imaging of a 16.5 hours APF arm-Fz::GFP pupa showing a pI cell (circle) prior to photobleaching (E; t=–3 minutes), soon after photobleaching (E'; t=1 minute) and 17 minutes after photobleaching (E''). The photobleached region includes the posterior cortex of the pI cell (arrow) and the epidermal cells posterior to this pI cell. (F-H) Live GFP imaging of wild-type (F), dsh1 (G) and stbm6c (H) mutant pupae expressing arm-Fz::GFP. The arrow in F indicates the posterior accumulation of Fz::GFP in the pI cell prior to mitosis. Asymmetric Fz::GFP localization depends on dsh and stbm activities. pI cells (circle) were identified based on their higher level of Fz::GFP accumulation at 16.5 hours APF and on the stereotyped morphology of their daughter cells. In this and all other figures, anterior is towards the left. Scale bars: 5 µm.

View larger version (125K): [in a new window]   Fig. 2. Stbm localizes to the anterior apical cortex. (A-E) Orientation of the pI cell division in stbm6c (A), stbm6c; arm-GFP::Stbm (B), neurP72GAL4/UAS-GFP::Stbm (C), pkpk-sple14 pupae (D) and UAS-Pk/+; neurP72GAL4/UAS-Stbm (E). Low-level ubiquitous expression of GFP::Stbm rescued the stbm6c PCP phenotype, whereas overexpression of Pk and Stbm in the pI cell partially disrupted planar polarity. Orientation of cell division was measured at anaphase and telophase in fixed pupae stained for Sens and either Numb or Pins as described elsewhere (Bellaiche et al., 2001a). (F,G) Localization of Stbm (green), Cad (blue) and Dlg (red) in dorsal thorax epithelial cells at 16.5 hours APF. Stbm co-localized with Cad at the apical cortex (apical section in F-F''), apical to Dlg (basal section in G). (H) The distribution of Cad (blue), Dlg (red), Stbm (green) and Fz (yellow) in pI cells prior to division. (I-K'') Localization of Stbm (green) and Baz (red) in wild-type (I-I'',J-J'') and fzK21/fzKd4a mutant (K-K'') pI cells expressing Histone2B::YFP (blue) under the control of neurP72GAL4. Stbm localized opposite to Baz at early prophase (I-I''; note that Histone2B::YFP was not detected in this apical section) and prometaphase (J-J'') in wild-type cells. By contrast, Stbm localized rather uniformly in fz mutant pI cells (K-K''). Scale bar: 5 µm.

View larger version (117K): [in a new window]   Fig. 3. Distribution of GFP::Stbm and GFP::StbmPBM. Localization of GFP::Stbm (green in A-C and G-J) and GFP::StbmPBM (green in D-F) in living pI cells expressing Histone2B::YFP (yellow in A-H) under the control of neurP72GAL4. GFP::Stbm and GFP::StbmPBM similarly localized at the apical anterior cortex of both pI (A,D) and epidermal cells (C,F; a few isolated epidermal cells expressed low levels of GAL4 in the notum of neurP72GAL4 pupae). In mitotic pI cells, low levels of GFP::Stbm are seen at the apical cortex (B). By contrast, GFP-StbmPBM remained apical (E) and did not accumulate laterally as GFP::Stbm (B,B' and E,E' show two different confocal sections of the same cell). Anterior localization of GFP-Stbm in pI and epidermal cells was disrupted in dsh1 (G,H) and pkpk-sple14 (I,J) mutant pupae. Scale bars: 5 µm.

View larger version (156K): [in a new window]   Fig. 4. Pk colocalizes with Stbm at the anterior cortex. (A-A'') Localization of Pk (green), Cad (blue) and Dlg (red). Pk accumulated at a higher level in pI cells where it localized at the anterior pole. This slightly oblique section shows that Pk co-localized apically with Cad (arrowheads) apical to Dlg (arrow) in epidermal cells. (B) Pk (green) localized at the anterior apical cortex of the pI cell (Sens in blue), opposite to Baz (red) at prophase. (C,C') Pk (red) co-localized with GFP::Stbm (green) at the anterior cortex of stbm6c arm-GFP::Stbm pI cells. (D-E) Pk (green) accumulated in the cytoplasm of stbm6c mutant cells whereas Cad (blue) and Dlg (red) remained cortical. A faint, uniformly distributed cortical Pk staining was detected in pI cells (arrow in E). (F-F'') Pk (green) localized uniformly at the cortex and did not accumulate opposite to Baz (red) in fzK21/fzKd4a mutant pI cells (Sens in blue). Scale bar: 5 µm.

View larger version (168K): [in a new window]   Fig. 5. Stbm overlaps with Pins and Dlg at the anterior lateral cortex. Localization of Stbm (green), Pins (blue) and Dlg (red) in dividing pI cells. At prophase (A-B'''), Stbm (A',B') co-localized with Pins (A'',B'') at the anterior apical cortex whereas Dlg (A''',B''') was predominantly basal to Stbm and Pins. Although, in this particular cell, the rounding up of the posterior cortex resulted in a weak Dlg posterior staining, no asymmetry in Dlg accumulation is usually detected at this stage. At prometaphase (C-E'''), Stbm (C',D',E') localized at the anterior lateral cortex where it overlapped with Pins (C'',D'',E'') and Dlg (C''',D''',E'''). Only low levels of Stbm (C') were seen at a more basal position, where Pins (C'') and Dlg (C''') predominantly accumulated. A z-section view is shown in (E-E'''). z-section axes are indicated by arrowheads in A,C. pI cells were identified based on the anterior accumulation of Pins. Mitotic staging was deduced from cell morphology and accumulation of Pins. Scale bar: 5 µm.

View larger version (16K): [in a new window]   Fig. 6. Stbm interacts with Dlg. (A,B) GST pull-down assays showing that Stbm interacts in vitro with Dlg via its PBM. GST served as a negative control and input lanes correspond to 10% of the amount of radiolabelled proteins used for interaction. GST-Dlg interacts with Stbm but not with StbmPBM (A). GST-PBM and GST-Stbm, but not GST-StbmPBM, interact with Dlg or with the PDZ1-3 domain of Dlg (B).

View larger version (127K): [in a new window]   Fig. 7. Stbm promotes and Dsh inhibits Pins cortical localization at prophase. Localization of Pins (green) in wild-type (A,E), stbm6c (B,F), dsh1 (C,G), bazXJ106 (I), bazXJ106; stbm6c (J), bazXJ106 dsh1 (K) and UAS-Pk/+; neurP72GAL4/UAS-Stbm (D,H) pI cells (Sens in red) at late prophase (A-D; the onset of nuclear breakdown was detected by the weak cytoplasmic Sens staining) and prometaphase/metaphase (E-K). At prophase, reduced Pins cortical staining was observed in stbm6c pI cells (B), whereas increased cortical staining was seen in dsh1 (C) and UAS-Pk/+; neurP72GAL4/UAS-Stbm (D) pI cells. Despite this early defect, Pins localized asymmetrically at prometaphase/metaphase in all genotypes analyzed here (E-J) except in bazXJ106 dsh1 double mutant pI cells (K). Note that Pins localized randomly relative to AP axis in UAS-Pk/+; neurP72GAL4/UAS-Stbm, stbm6c and dsh1 mutant pupae. Scale bar: 5 µm.

View larger version (103K): [in a new window]   Fig. 8. Stbm and Dsh have opposite activities in organizing the anterior cortex. (A,B) Schematic interpretation of the Pon::GFP mis-partitioning phenotype. In wild-type pI cells (A), centrosomes (red) are randomly oriented at late prophase and rotate to line up with the AP polarity axis during prometaphase (Bellaiche et al., 2001a; Roegiers et al., 2001). An anterior `centrosome-attracting activity' (blue) has been suggested to attract and anchor the anterior centrosome, thereby regulating spindle rotation (Doe, 2001). Alignment of the spindle with Pon::GFP (Pon::GFP co-localizes with the blue dots of the `centrosome-attracting activity') results in the unequal segregation of Pon-GFP at anaphase (A). We suggest that this `centrosome-attracting activity' is distributed over a larger cortical region in fzK21/fzKd4a, dsh1 and UAS-Pk/+; neurP72GAL4/UAS-Stbm pI cells such that both centrosomes can be simultaneously anchored to this cortical domain. This would therefore lead to a mis-partitioning of Pon::GFP at anaphase (B). (C-E) Co-expression of Pk and Stbm in pI cells led to a broadening of the Pon::GFP domain. In 84% of the pI cells, the crescent of Pon::GFP was progressively restricted to a single pole during prometaphase and was unequally inherited by only one daughter cell (C-C''). Arrows indicate the extent of the Pon::GFP crescent at late prophase (C) and metaphase (C''). In the remaining 16% of the cells, Pon::GFP accumulated along a broad cortical domain (D) or in a bipolar manner (E) and was mis-partitioned in the two daughter cells at anaphase (D,E'). Scale bar: 5 µm.

View larger version (18K): [in a new window]   Fig. 9. Different read-outs for PCP in Drosophila. In pI cells of the notum (top), Fz localizes opposite to the Stbm-Pk complex prior to mitosis. Upon division, PCP signaling localizes the Dlg-Pins-Gi and Baz-Par6-aPKC complexes along the AP axis. The Sbm-Pk complex recruits the Dlg-Pins-Gi complex at the anterior apical cortex. Conversely, Dsh restricts the localization of Pins at the cortex, at least partly via the regulation of the localization of Stbm as well as via Baz. In wing epidermal cells (center), Fz signals at the distal vertex to promote the formation of an actin-based structure, the epidermal hair, via a pathway that may involve direct interactions between Dsh, the fly homolog of Daam1 and Rho. Localized activation of Rho would control the spatial regulation of myosin activity via the Rho Kinase and the Myosin Light Chain Kinase (Strutt, 2001a). In the eye (bottom), higher levels of fz activity in the equatorial cell leads to an up-regulation of the expression of the Delta gene (Cooper and Bray, 1999; Fanto and Mlodzik, 1999; Mlodzik, 1999; Strutt et al., 2002). This in turn leads to the activation of Notch in the polar cell that, therefore, becomes R4.

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