Article. The Conserved Discs-large Binding Partner Banderuola Regulates Asymmetric Cell Division in Drosophila

Current Biology 24, 1811 1825, August 18, 2014 ª2014 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2014.06.059 The Conserved Discs-large Binding Partner Banderuola Regulates Asymmetric Cell Division in Drosophila Article Federico Mauri, 1,2 Ilka Reichardt, 1 Jennifer L. Mummery-Widmer, 1 Masakazu Yamazaki, 1,3 and Juergen A. Knoblich 1, * 1 Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna 1030, Austria Summary Background: Asymmetric cell division (ACD) is a key process that allows different cell types to be generated at precisely defined times and positions. In Drosophila, neural precursor cells rely heavily on ACD to generate the different cell types in the nervous system. A conserved protein machinery that regulates ACD has been identified in Drosophila, but how this machinery acts to allow the establishment of differential cell fates is not entirely understood. Results: To identify additional proteins required for ACD, we have carried out an in vivo live imaging RNAi screen for genes affecting the asymmetric segregation of Numb in Drosophila sensory organ precursor cells. We identify Banderuola (Bnd), an essential regulator of cell polarization, spindle orientation, and asymmetric protein localization in Drosophila neural precursor cells. Genetic and biochemical experiments show that Bnd acts together with the membrane-associated tumor suppressor Discs-large (Dlg) to establish antagonistic cortical domains during ACD. Inhibiting Bnd strongly enhances the dlg phenotype, causing massive brain tumors upon knockdown of both genes. Because the mammalian homologs of Bnd and Dlg are interacting as well, Bnd function might be conserved in vertebrates, and it might also regulate cell polarity in higher organisms. Conclusions: Bnd is a novel regulator of ACD in different types of cells. Our data place Bnd at the top of the hierarchy of the factors involved in ACD, suggesting that its main function is to mediate the localization and function of the Dlg tumor suppressor. Bnd has an antioncogenic function that is redundant with Dlg, and the physical interaction between the two proteins is conserved in evolution. Introduction Although most cell divisions are symmetric, some cells can divide asymmetrically into two daughter cells that assume different fates [1 3]. During development, asymmetric cell division (ACD) allows specific cell types to be generated at precise locations relative to surrounding tissues. To achieve this, the axis of ACD needs to be coordinated with the architecture and polarity of the developing organism. Over the past years, a conserved protein machinery for ACD has been identified, but how this machinery connects to the organism architecture is less clear. 2 Present address: IRIBHM, Universitè Libre de Bruxelles, Brussels 1070, Belgium 3 Present address: Research Center for Biosignal, Akita University, Akita 010-8543, Japan *Correspondence: juergen.knoblich@imba.oeaw.ac.at The fruit fly Drosophila melanogaster is one of the bestunderstood model systems for ACD. In particular, the development of the Drosophila CNS and peripheral nervous system relies heavily on ACD and has contributed much to our current understanding of this process. In the peripheral nervous system, external sensory (ES) organs are formed by two outer cells (hair and socket) and two inner cells (neuron and sheath). The four cell types arise from a single sensory organ precursor (SOP) cell, which divides asymmetrically into an anterior piib cell and a posterior piia cell. In a second round of ACD, piia and piib generate the outer or inner cells of the ES organ, respectively [4]. The difference between piia and piib cells arises from different levels of Notch signaling in the two daughter cells. This difference is established by the asymmetric segregation of the Notch inhibitor Numb into the piib cell. Numb is known to regulate endocytosis, but how it inhibits Notch signaling is not precisely understood [5, 6]. In SOP cells, the polarity axis is coordinated with the anterior-posterior planar polarity axis of the overlying epithelium [7, 8]. Planar polarity involves the localization of mutually inhibitory components of a well-characterized machinery to the anterior or posterior plasma membrane [8, 9]. In SOP cells, the planar polarity protein Strabismus (Stbm) localizes to the anterior cortex and initiates the reorganization of plasma membrane domains to establish the axis of ACD [9]. One of the most upstream events of this process is the recruitment of the membrane-associated guanylate kinase (MAGUK) Discs-large (Dlg) to the anterior cortex [10]. This may involve a direct interaction of Dlg with the planar polarity protein Stbm [11]. Dlg was originally identified as a tumor suppressor involved in the regulation of epithelial cell polarity [12, 13] and later shown to play a role in ACD [14, 15] and synaptogenesis [16, 17]. Despite its widespread functions, the biochemical pathways regulated by Dlg in those various cell types are not entirely understood. In SOP cells, Dlg associates with the adaptor protein Pins to direct the protein Bazooka (Baz) to the basal-posterior side of the dividing SOP cell [10]. Together with Par-6 and apkc, Baz forms the so-called Par protein complex that plays a pivotal role during ACD in many different cell types [18]. Eventually, the kinase apkc phosphorylates Numb, mediating its release from the posterior plasma membrane and thereby causing its accumulation to the anterior side [19 21]. To ensure the asymmetric segregation of Numb to the anterior piib cell, the mitotic spindle has to be oriented along the polarity axis [22]. This function is mediated by Pins through the binding of the microtubule binding protein Mushroom body defect (Mud), which forms a cortical attachment site for astral microtubules, aligning the spindle into the correct orientation [23 25]. The binding to Pins requires the heterotrimeric G protein Gai, which associates with Pins to mediate its recruitment to the anterior plasma membrane and switches it to an open conformation in which Pins can bind Mud [26, 27]. The same protein machinery directs ACD in neuroblasts, the stem cell-like progenitors of the Drosophila CNS. Neuroblasts divide asymmetrically into self-renewing daughter neuroblasts and smaller ganglion mother cells (GMCs) that generate two differentiating neurons through a terminal symmetric division

Current Biology Vol 24 No 16 1812 [28]. The asymmetric segregation of the cell fate determinants Numb, Prospero (Pros), and Brat into the GMC is required for proper differentiation. The asymmetric partitioning of Pros and Brat is mediated by the adaptor protein Miranda [29 32], and the asymmetric localization of both Miranda and Numb depends on phosphorylation by apkc [21, 33]. Mutations in any of the three segregating determinants lead to the generation of excessive numbers of neuroblasts and ultimately cause the formation of lethal, transplantable brain tumors [34]. As in SOP cells, Pins, Dlg, and Baz are required for ACD in neuroblasts, but they act in a characteristically different manner. First, neuroblast divisions are oriented along the apical-basal axis and not the planar polarity axis. Second, Pins, Dlg, and Baz colocalize apically in neuroblasts while they occupy opposite domains in SOP cells [35]. In part, those differences can be explained by the recruitment of the adaptor protein Inscuteable (Insc) in the apical complex [36]. Insc is not expressed in SOP cells, but in neuroblasts, it coordinates cortical polarity and spindle alignment by connecting Pins to Baz, ensuring the correct segregation of cell fate determinants in the differentiating daughter cell [37 40]. In addition, Dlg has a neuroblast-specific role in mediating spindle orientation, acting downstream of Pins to align the spindle pole through the interaction with the kinesin motor Khc-73 [41]. Pins, Dlg, and Khc-73 also regulate a pathway called telophase rescue that corrects ACD defects during late mitotic stages [42]. This pathway realigns cortical polarity along the spindle axis independently of the Par complex through a Dlg cortical clustering mechanism to ensure that determinants eventually segregate asymmetrically and daughter cell fates are correctly specified. How Dlg performs those seemingly divergent roles in SOPs and neuroblasts is currently unclear. Because our knowledge about ACD is evidently incomplete, we performed several RNAi screens to identify additional players required for the correct establishment of daughter cell fates [43 45]. Here, we used the results from one of those screens [43] to identify Banderuola (Bnd), a new key regulator of ACD that acts both in neuroblasts and in SOP cells. We demonstrate that Baz, Pins, and Dlg are all mislocalized in bnd mutant SOP cells, placing Bnd at the top of the hierarchy for ACD. In bnd mutant neuroblasts, the asymmetric segregation of cell fate determinants is disrupted because apkc and Dlg fail to accumulate apically. Importantly, Bnd interacts physically and genetically with Dlg, suggesting that it supports Dlg in performing its divergent functions in various cell types. Because Bnd is conserved in evolution, our data identify a new member of the universal machinery for ACD that might direct cell polarity in vertebrates as well. Results A Screen for GFP-Pon Localization Identifies banderuola A genome-wide RNAi screen has identified 134 genes that are potentially involved in ACD because they cause defects in the SOP lineage [43]. To identify genes regulating Numb localization, we used an enhanced green fluorescent protein (EGFP) fusion of the Numb binding partner Pon expressed in SOP cells and their descendants from the phyllopod promoter [43, 46]. This construct allowed live imaging of asymmetric protein segregation while simultaneously expressing upstream activation sequence (UAS)-RNAi lines using the pnr-gal4 driver (Figure 1A). Three RNAi lines caused defects in EGFP::Pon.LD localization and targeted genes not previously implicated in ACD. TID38988 targets APC4 (CG32707), a subunit of the anaphase promoting complex; TID31742 targets san (separation anxiety, CG12352), an acetyltransferase required for sister chromatid cohesion; and TID27759 targets CG45058, a gene not previously characterized in flies (Figure 1B). Because only CG45058 RNAi causes defects in the establishment of polarity, whereas the other two lines appear to be required for maintaining polarity (data not shown), we focused on this line for further analysis. Lineage staining shows that RNAi of CG45058 causes the formation of ES organs that contain two neurons and two sheath cells, indicating a cell fate transformation of piia into piib (Figure 1C). CG45058 encodes a 180 kda protein containing a low complexity region, two Ankyrin repeat-containing domains (ANK), a fibronectin type III domain (FN3), and a C-terminal Ras association (RA) domain (Figure 1D). CG45058 is conserved throughout most eukaryotes, but not in fungi. The predicted human ortholog (InParanoid, [47]) is ANKFN1-201 (Ensembl gene ID ENSG00000153930). The most conserved portion of the protein includes the ANK domains, the FN3 domain and a large sequence following those domains that we defined as the CG45058 Motif (Figure 1D). The RA domain is not conserved in mammalian orthologs. We named the gene banderuola (bnd; the Italian word for weathercock, indicating that crescent formation is variable like the blowing wind) to describe the randomized crescent formation in the mutants. Bnd Is Required for Asymmetric Cell Division To understand the role of Bnd in asymmetric cell division, we analyzed and quantified the EGFP::Pon.LD localization defects. Before anaphase onset, EGFP::Pon.LD was completely symmetric in 11% of the mitotic SOPs and weakly asymmetric in 55% of the mitotic SOPs. In 6%, EGFP::Pon.LD localization was severely delayed (Figures 1B and S1 available online), and only 28% of the SOPs normally localized the protein to the anterior cell cortex at the onset of metaphase. Remarkably, most of these defects were corrected later in mitosis, and, in anaphase/telophase, 45% of the SOP cells showed no phenotype. In 33% of those late mitotic cells, EGFP::Pon was asymmetric, but the crescent was not aligned with the mitotic spindle (Figure 1B, arrowhead), whereas 11% of those late mitotic cells showed split or nonanterior crescents (Figure S1). In only 11%, EGFP::Pon.LD remained symmetric throughout mitosis and segregated equally into both daughter cells (n = 30 for control; n = 18 for bnd RNAi). Thus, Bnd is required for initiating asymmetric protein localization in prometaphase but seems to be less important during later mitotic stages. To verify the RNAi phenotype, we generated a bnd loss-offunction mutant by Flp-mediated recombination of two P elements that carry flippase recognition target (FRT) sites flanking the gene (Figure S2) [48]. The resulting recombinants were expected to be complete loss-of-function alleles because the entire bnd coding region was removed. Homozygous mutants were pupal lethal, with only a few adult escapers (<1% of the progeny) that displayed severe locomotion defects and died within hours after eclosion (data not shown). In mutant SOP cells, Numb was not asymmetric or was only very weakly asymmetric (27%, 7 of n = 26), or the Numb crescent was not aligned with the mitotic spindle (15.4%, 5 of n = 26; Figures 2A 2D). Again, those defects were less obvious during anaphase and telophase (data not shown). Live imaging of RFP::Pon.LD together with the mitotic spindle marker Zeus::GFP [49] (Figures 2E and 2F) showed that the asymmetric accumulation of RFP::Pon.LD was generally delayed

Banderuola Regulates Asymmetric Cell Division 1813 A B C D Figure 1. Identification of CG45058 through a Live Imaging Assay (A) Setup of the live imaging assay. The notum of living pupae was exposed and imaged at the confocal microscope (top). The Numb localization reporter EGFP::Pon.LD was fused to the SOP-specific phyl promoter while expressing the RNAi lines in the notum with pnr-gal4. This allowed us to score for Numb localization defects in mitotic SOP cells upon gene knockdown (bottom, frame from a live movie). (B) Identification of novel regulators of ACD through live imaging. Anterior is up. In the first row, images from a control movie showing EGFP::Pon.LD localization to the anterior side are shown. In the second and third rows, images from APC4 and san RNAi movies showing defects in the maintenance of polarity are shown: the asymmetric localization of the reporter is lost by anaphase onset. In the last row, images of CG45058 RNAi displaying defects in the establishment of polarity and its coordination with spindle alignment (arrowhead) are shown. Time is expressed in min:s and counted considering nuclear envelope breakdown (NEBD) as reference point (t = 0:00). Scale bars represent 5 mm. n R 18 mitotic SOP cells from 4 5 pupae for each RNAi line, recording mitosis in its full length. (C) Lineage staining showing cell fate transformations caused by CG45058 knockdown (asterisk): only inner cells (two neuron and two sheath cells) are detectable. Su(H) (green) marks the socket cell. Pros (blue) marks the sheath cell. Cut (red) marks all cell types. (D) Structure and evolutionary conservation of CG45058. The CG45058 protein consists of two ankyrin repeat-containing domains (ANK), a FN3 domain, and a RA domain. The conservation of the protein is shown as percentage of sequence identity and similarity, calculated with pairwise alignment among the depicted species on the whole sequence and on the most conserved portion of the protein (ANK domains, FN3 domain, and CG45058 motif). and less striking, and the mitotic spindle displayed abnormal rotation movements during the alignment to the planar cell polarity (PCP) axis (Figures 2E 2G). In particular, whereas the crescent of PON was clearly detectable at prometaphase (t = 0:00) in wild-type SOP cells, in the bnd loss-of-function (LOF) background, it was either undetected or weak, and it became clear only at later time points (Figure 2H). In telophase, however, the segregation of PON::RFP into the piib cell was almost always correct (data not shown). In the bnd heterozygous background, the spindle became aligned with the planar polarity axis early in mitosis (3 4 min before the metaphaseanaphase transition) and displayed limited rotation movements (Figures 2E and 2G). In the bnd null background, however, there were extensive rotation movements carrying on

Current Biology Vol 24 No 16 1814 E F D G H Figure 2. Numb Is Mislocalized, and Spindle Positioning Is Affected upon bnd LOF (A C 0 ) Numb localization is affected upon bnd LOF. In bnd heterozygous flies (A), Numb (green) is normally segregated to the anterior side during SOP cells division. Upon bnd LOF (B and C), about half of the cells display defects in Numb localization, either due to misalignment of the Numb crescent and the spindle (yellow arrow in B 0 ) or due to very weak or absent polarization of Numb (C). Sanpodo (Spdo, red) marks SOP cells, ph3 (white) marks mitotic cells, Centrosomin staining (Cnn, green) shows the position of the spindle, and DAPI marks DNA. Anterior is left. The schematics to the right of the figure describe the corresponding Numb localization phenotypes. Scale bars represent 5 mm. (D) Summary of Numb localization phenotypes. Heterozygous bnd has been used as control (bnd D/+ n = 18; bnd D/D n = 26). (E and F) Time-lapse imaging of Numb localization and spindle alignment in dividing SOP cells in the bnd deletion background. SOP cells were identified based on morphology, position, timing of division, and asymmetric segregation of PON::RFP at cytokinesis, as done in [9]. The microtubule marker Zeus::GFP is expressed ubiquitously and used to image the mitotic spindle, whereas the expression of the Numb localization reporter RFP::Pon.LD is driven in the notum by the pnr-gal4 driver. Anterior is up. t = 0:00 corresponds to the formation of the mitotic spindle (prometaphase). Metaphase-anaphase transition takes place at t = 7:50 in the heterozygous and t = 8:30 in the homozygous mutant. In the heterozygous background (bnd D/+; E), RFP::Pon.LD asymmetry is established already at prometaphase (t = 0:00), whereas in the bnd LOF background (bnd D/D; F), its accumulation is much weaker, and just at the metaphase-anaphase transition (t = 8:30), the reporter is clearly segregated to the anterior side. Also, the spindle displays abnormal rotation movements (arrows) that are more intense and prolonged in the homozygous mutant, continuing throughout metaphase. Scale bars represent 5 mm. Time is expressed in min:s. (G) Quantification of the spindle rotation movements measured between prometaphase and anaphase. Each bar corresponds to the division of one SOP cell. The extent of rotation movements is expressed in degrees and quantified in a cumulative way over time. The blue part of the bar shows the angle value of the early rotation movements that are happening between t = 0:00 and midmetaphase, which is defined as the midpoint between the formation of the mitotic spindle and the metaphase-anaphase transition. The red part of the bar gives the value of late rotation movements, which are happening between midmetaphase and the metaphase-anaphase transition. In the bnd D/D background, the spindle rotation movements are more intense, especially the later ones. (H) Quantification of RFP::Pon.LD asymmetric accumulation over time. In the bnd homozygous mutants, PON crescent is formed later during mitosis compared to the heterozygous counterpart. Each point corresponds to the division of one SOP cell (sample Homozygous 5 has been excluded because it never formed a PON crescent). throughout metaphase (Figures 2F and 2G). A similar phenotype has already been described for other polarity proteins such as Dlg [10]. Thus, Bnd is required for both spindle positioning and asymmetric protein localization in mitosis, indicating that it might be involved in setting up the polarity axis. Because the asymmetric localization of Numb in SOP cells requires the Par protein complex [10, 20, 50, 51], we tested the localization of Baz and apkc. While both proteins normally accumulated on the posterior side of the SOP cells in heterozygous control animals, either they were not asymmetrically localized, or their accumulation was not aligned with the mitotic spindle in about half of the SOP cells in homozygous bnd mutant animals (Figures 3A 3F). Baz asymmetry is established by the Dlg/Pins complex that connects the axis of ACD to planar polarity [10] and also

Banderuola Regulates Asymmetric Cell Division 1815 mediates the alignment of the mitotic spindle with the polarity axis through the interaction with Gai and Mud [26, 27, 52]. We therefore assessed the localization of Pins, Gai, and Mud upon loss of bnd. All three proteins accumulated asymmetrically at the anterior cortex in heterozygous control animals (Figures 3G, 3J, and 3M). In bnd null mutants, however, Pins, Gai, and Mud showed localization defects similar to those observed for the Par proteins and Numb (Figures 3H, 3K, and 3N). Crescents were weak or not aligned with the polarity axis in more than half of the mutant cells (Figures 3I, 3L, and 3O). The anterior enrichment of Dlg [10] was also affected in a similar way (Figures 3P 3R). Because Dlg is the most upstream component for establishing polarity in SOP cells and because we did not observe planar polarity defects [43] (Figure S3 and data not shown), we conclude that Bnd acts between the planar polarity machinery and Dlg to establish cortical polarity and align it with the mitotic spindle in SOP cells. Bnd Localizes to the Centrosomes and the Cell Cortex To further characterize the role of Bnd, we analyzed its subcellular localization in SOP cells by live-cell imaging and immunofluorescence. Multiple attempts to generate a specific antibody that reliably detects the endogenous protein failed. Therefore, we expressed a GFP-tagged version of the protein in SOP cells together with Histone::RFP using the neuralized- Gal4 driver. Bnd overexpression does not affect polarity and spindle orientation in dividing SOP cells (data not shown). The GFP-tagged protein is functional because its expression using the insc-gal4 driver can rescue the viability of bnd mutants (Figures S4A and S4B) and the loss-of-neuroblast phenotype (see below) we observe in bnd mutants (Figures S4C and S4D). During interphase, Bnd::GFP is localized diffusely in the cytoplasm and enriched at the apical cortex (Figure S5A; Movie S2). At the onset of mitosis, the protein accumulates at centrosomes (Figure 4A; Figure S5B; Movies S1 and S2). During metaphase and anaphase, it is enriched on the mitotic spindle as well, and, upon cytokinesis, it is found concentrated on the central spindle (Figures 4A and S5; Movies S1 and S2). In addition, the protein shows transient accumulation on various domains along the cell cortex throughout mitosis, although those domains are not always correlated with the known anterior or posterior polarity proteins (Figure 4A; Movie S1). The Bnd localization pattern was confirmed by coexpressing a CFP-tagged version of the protein (Bnd::CFP) with Zeus::GFP [49] using pnr-gal4 (Figures 4B 4E). Bnd::CFP colocalizes with the spindle microtubules and centrosomes during mitosis (Figures 4B and 4C) and also recapitulates the transient cortical enrichment observed with the GFP fusion (Figures 4B 4D). Centrosomal and cortical enrichment were also confirmed by anti-gfp staining of SOP cells expressing Bnd::GFP from the neur-gal4 driver (Figure 4F). Thus, the subcellular localization of Bnd is consistent with a role in establishing cortical polarity and orienting the mitotic spindle. bnd Is Required for Self-Renewal and Asymmetric Cell Division in Neuroblasts Because most regulators of ACD are conserved between SOP cells and neuroblasts, we analyzed the bnd mutant phenotype in the CNS. Staining for the neuroblast markers Miranda and Deadpan and the neuronal marker Prospero revealed a strong loss of neuroblasts in bnd mutant brains, which was becoming more severe during larval development (Figures 5A, 5B, 5H, and 5I). Consistently, there was a strong decrease in the number of phospho-histone H3 (ph3)-positive mitotic cells (Figures 5D, 5E, and 5J) in bnd mutant larval brains, whereas we did not detect any increase in the levels of activated proapoptotic Caspase 3 (Figures 5F and 5G). Because this is a fairly nonspecific defect that could have been caused by a second site mutation as well, we confirmed the specificity by rescuing the phenotype with a bacmid carrying the bnd locus (Figures 5C and 5H) and with a UAS-bnd::GFP transgene under the control of the insc-gal4 driver (Figures S4C and S4D). Interestingly, the number of optic lobe neuroblasts was unaffected, and the overall structure of the brain was normal, although homozygous mutant brains were slightly smaller on average. Thus, we conclude that the loss of bnd causes premature differentiation of larval neuroblasts. To test whether the loss of neuroblasts correlates with defects in asymmetric cell division, we analyzed the establishment of cortical domains. The remaining neuroblasts in bnd mutants were smaller than their wild-type counterparts. The basal localization of both the adaptor protein Miranda (Figures 6A 6I) and the cell fate determinant Numb (Figures 6E, 6F, and 6L) was deficient, and the apical localization of the Par complex member apkc and of Inscuteable (Figures 6A 6D, 6J, and 6K) was also abnormal in bnd mutant neuroblasts. In particular, apkc did not form apical crescents and often displayed a uniform cortical localization (Figure 6B). In addition, the apical enrichment of Dlg that has been described in neuroblasts was also essentially absent (Figures 6G, 6H, and 6M). We observed similar defects for all the polarity proteins we analyzed, such as Pins, Baz, and Mud (Figure S6 and data not shown). Similar to what we observed in SOP cells, the localization of the basal cell fate determinants was often rescued during later mitotic stages, even in the absence of the apical complex (Figures S6G and S6H), presumably by the action of the so-called telophase rescue pathway [42]. Notably, in neuroblasts, we did not observe any defect in the coordination between the polarity axis and the mitotic spindle. Thus, Bnd is required to ensure neuroblast selfrenewal, and it exerts its function in the early stages of polarity establishment in multiple types of asymmetrically dividing cells. Banderuola Interacts with Discs-large To define the possible mechanism of action of Bnd, we performed coimmunoprecipitation assays of FLAG-tagged Bnd in transfected Drosophila S2 cells with proteins involved in the establishment of cortical polarity and the alignment of the mitotic spindle. Among other proteins, we focused on Dlg because dlg mutant alleles display similar polarity and spindle rotation phenotypes [10 12]. We could coimmunoprecipitate Bnd::FLAG with Dlg::GFP in S2 cells (Figure 7A), and the biochemical interaction between Bnd and Dlg was confirmed in vivo using Drosophila embryos that express a Bnd::FLAG transgene under the control of a heat shock inducible promoter. In these embryos, Dlg coimmunoprecipitated with Bnd::FLAG (Figure 7B), and, similarly, Bnd::FLAG was detected upon immunoprecipitation of Dlg (data not shown). Moreover, Dlg and Bnd showed transient colocalization in dividing neuroblasts and SOP cells (Figure S7), further suggesting that there is a functional interaction between the two proteins. To test whether this interaction is conserved in evolution, we expressed the mammalian homologs of Bnd and Dlg (ANKFN1 and Dlg1, respectively) in 293T cells. Indeed, immunoprecipitation of GFP-tagged ANKFN1 coprecipitated MYCtagged Dlg1 from those cells (Figure 7C). Bnd is therefore an interaction partner of Dlg, and because this interaction is

Current Biology Vol 24 No 16 1816 C F I L O R Figure 3. Loss of bnd Affects Polarity Establishment and Spindle Alignment in SOP Cells (A F) The Par complex proteins apkc and Baz are mislocalized in bnd LOF. In the bnd heterozygous control, apkc (A, green) and Baz (D, green) accumulate to the posterior side of dividing SOP cells. In the bnd null background, both apkc (B, green) and Baz (E, green) display localization defects. In more than half of mitotic SOP cells (summarized in C and F), crescent formation is weak or absent, the crescent is not aligned with the spindle poles (misaligned crescent), or the crescent is correctly polarized, but the spindle is not aligned along the anterior-posterior axis (PCP defects). (G O) Proteins involved in the spindle alignment are mislocalized in bnd LOF. The localization of Pins (G and H, green; summarized in I), Gai (J and K, green; summarized in L), and Mud (M and N, green; summarized in O) is abnormal in 60% 70% of the mitotic SOP cells in the bnd LOF background, while in the heterozygous background they are correctly segregated to the anterior side. PCP defects describe cases in which the protein is polarized to the correct side, but the spindle is not aligned with the polarity of the tissue. (legend continued on next page)

Banderuola Regulates Asymmetric Cell Division 1817 conserved in evolution, we assume that it is important for Bnd function. To verify the close functional connection between Dlg and Bnd, we analyzed their genetic interactions. For this purpose, we expressed bnd and dlg RNAi lines in all neuroblasts using insc-gal4 [29] (Figures 7D 7G). Knockdown of dlg alone does not cause an obvious change in neuroblast numbers (Figures 7E and 7M), but it does induce defects in the basal segregation of Miranda, consistent with what has been reported previously [14, 15, 53] (Figure 7I). Knockdown of bnd alone also causes defects in Miranda localization but no changes in neuroblast number (Figures 7F, 7J, and 7M), presumably because RNAi results in a partial loss-of-function phenotype. Strikingly, however, the simultaneous knockdown of both dlg and bnd causes a massive overproliferation of neuroblasts that is accompanied by increased mitotic activity (Figure 7G). To confirm this data, we performed genetic interaction experiments using dlg sw and dlg HF321 hypomorphic alleles [54, 55] (Figures 7K and 7L and data not shown). Although dlg sw mutants per se did not display neuroblast overproliferation phenotypes (Figures 7K and 7M), the combination with bnd LOF caused the onset of massive brain tumors (Figure 7L). The same results were obtained with the dlg HF321 allele (data not shown). These data establish a strong genetic interaction between dlg and bnd, and this, together with their physical interaction, supports the model that the genes are functionally connected. In addition, these data suggest that Bnd might have additional functions that go beyond simply localizing Dlg because the combined knockdown phenotype is even stronger than the dlg null mutant phenotype. We conclude that Bnd acts together with Dlg to establish polarity in both asymmetrically dividing neuroblasts and SOP cells. In addition, our data establish that Bnd has antioncogenic functions, acting redundantly with Dlg to prevent the formation of brain tumors in Drosophila. Discussion Our results establish Bnd as a new component of the machinery for asymmetric cell division. We show that bnd RNAi or loss-of-function mutations cause defects in the establishment of polarity and the positioning of the mitotic spindle in mitotic SOP cells. We also demonstrate that bnd is required for ACD and continued self-renewal activity in Drosophila larval neuroblasts. Because Bnd interacts both biochemically and genetically with the tumor suppressor protein Dlg, we propose that it exerts its function during ACD by regulating the function of Dlg. Moreover, the spindle rotation phenotype we observed in mitotic SOP cells in bnd mutants is very similar to that of dlg sw mutants [10], further strengthening the possibility that the two proteins are functionally connected. Because the mammalian homologs of these two proteins also interact, this function might be conserved in higher organisms as well. Banderuola Regulates Asymmetric Cell Division The process of ACD involves the establishment of a polarity axis, the orientation of the mitotic spindle, the polarized distribution of cell fate determinants, and, ultimately, the establishment of different daughter cell fates. In SOP cells, the axis of polarity is established when Dlg and Pins interact with components of the planar polarity pathway to concentrate anteriorly [9]. Because Bnd binds to Dlg and is required for Pins and Dlg localization, but not for planar polarity (Figures 3 and S3), our data indicate that it acts at the very top of this hierarchy. Because Dlg is also mislocalized in bnd mutant neuroblasts, the role of Bnd in this tissue appears to be similar. Nevertheless, because the defect in asymmetry establishment is not completely penetrant, it is plausible that bnd function is partially redundant. Alternatively, it might also be that the residual protein derived from maternal contribution is sufficient to maintain, at least partially, the asymmetric partitioning of determinants. Further experiments will be needed to address these issues and clarify the instructive role of Bnd in establishing cell asymmetry. How could Bnd perform its function on a molecular level? Bnd::GFP localizes at the centrosomes, on the spindle, and, transiently, at the cell cortex. Because Bnd contains both Ankyrin repeats and an FN3 domain, it could mediate proteinprotein interactions leading to the anterior localization of Dlg downstream of the PCP pathway. The localization of Dlg and Pins to the anterior side of dividing SOP cells is regulated by Strabismus (Stbm) and Dishevelled (Dsh) [9]. It is thought that Dsh excludes Dlg/Pins from the posterior side, whereas Stbm binds Dlg at the anterior cortex, promoting the association with Pins. This hypothesis is reinforced by the fact that Dlg interacts directly with the PDZ binding motif (PBM) of Stbm in Drosophila embryos [11]. However, Pins is localized to the anterior cortex in stbm mutant SOP cells expressing a Stbm protein lacking the PBM domain [9]. Hence, the localization of Dlg/Pins can be regulated independently of a direct binding to Stbm. It is tempting to speculate that Bnd could be a mediator between the PCP pathway and the establishment of the asymmetry axis in mitotic SOP cells. Alternatively, however, Bnd could also affect the function of Dlg and other cortical proteins through its RA domain. RA domains mediate binding to small GTPases and regulate their activity. Small GTPases are involved in the modification of the actomyosin network, and the establishment of polarity is influenced by myosin activity and by the contractility of the actomyosin mesh [2, 56, 57]. In particular, Cdc42, a small GTPase of the Rho family, plays a central role in the establishment of polarity in a wide variety of biological contexts, including the localization of Par6/aPKC to the apical cortex of neuroblasts [58]. More recent data have also implicated small Ras-like GTPases in regulating cortical polarity and spindle orientation. The Rap1/Rgl/Ral signaling network was shown to mediate those events through the regulation of the PDZ domain protein Canoe, which is a known binding partner of Pins [59, 60]. It is intriguing to hypothesize that Bnd could be part of a similar signaling network impinging on Dlg. Because the RA domain of Banderuola is not conserved in higher organisms, however, we favor the first hypothesis that rests on the conserved domains of the protein (ANK domains, FN3 domain, and Bnd motif). Hence, Bnd could act as an adaptor that mediates protein-protein interactions and regulates the function of binding partners such as Dlg. (P R) The enrichment of Dlg to the anterior cortex is disrupted. Although Dlg anterior localization is not affected in the heterozygous background (P, white), it is defective upon bnd LOF (Q, white; summarized in R). Spdo (red) labels the SOP cells, ph3 (white) marks the mitotic cells, Cnn staining (green) shows the alignment of the spindle, and DAPI marks DNA. Anterior is left. apkc: n = 17 (bnd D/+) and n = 38 (bnd D/D); Baz: n = 18 (bnd D/+) and n = 16 (bnd D/D); Pins: n = 27 (bnd D/+) and n = 15 (bnd D/D); Gai: n = 27 (bnd D/+) and n = 24 (bnd D/D); Mud: n = 30 (bnd D/+) and n = 30 (bnd D/D); Dlg: n = 35 (bnd D/+) and n = 24 (bnd D/D). Scale bars represent 5 mm.

Current Biology Vol 24 No 16 1818 A F Figure 4. Bnd Localizes at the Centrosomes, at the Spindle, and at Cortical Domains during Mitosis (A) Time-lapse confocal images of Bnd::GFP localization. Bnd::GFP (green) is initially diffuse in the cytoplasm, but at prophase, it gets enriched at the centrosomes (arrowheads) before NEBD. At metaphase, it associates with the centrosomes and spindle microtubules (arrowheads). It is also transiently enriched at cortical domains during mitosis (arrow). neur-gal4 drives the simultaneous expression of Bnd::GFP and His::RFP (red). Time is expressed in min:s and counted considering NEBD as reference point (t = 0:00). (B E 00 ) Time-lapse confocal images showing the colocalization of Bnd::CFP (blue) and Zeus::GFP (green), a microtubule marker. RFP::Pon.LD (red) marks Numb localization. Bnd localizes at the spindle from metaphase to anaphase (B D) and is enriched at the cleavage furrow at telophase (E). Bnd::CFP is also (legend continued on next page)

Banderuola Regulates Asymmetric Cell Division 1819 H I J Figure 5. banderuola LOF Affects Neuroblast Self-Renewal (A C 000 ) Staining of larval brain hemispheres with the neuroblast markers Miranda (Mira, green) and Deadpan (Dpn, red). In the bnd LOF background, the number of neuroblasts is reduced (B) compared to heterozygous condition (A). The number of neurons (Pros-positive cells, blue) is not affected. A genomic rescue of the bnd locus is able to restore normal neuroblast numbers in bnd mutant flies, as shown by the increase in Mira/Dpn-positive neuroblasts (C). (D E 0 )Inbnd LOF (E), there is a decrease in the number of mitotic neuroblasts (ph3, magenta) in the central brain area (D and E, yellow dashed line) compared to the heterozygous control (D). Note that the optic lobe neuroblasts are unaffected. (F G 0 ) There is no increase of proapoptotic Caspase3 activation (red) upon bnd LOF (G) compared to the heterozygous control (F). (H) Quantification of the number of neuroblasts in bnd heterozygous mutants and homozygous mutants and upon insertion in the bnd D/D background of a transgene carrying the bnd locus. Error bars show SD. (I) Quantification of neuroblast numbers in bnd heterozygous and homozygous mutants during larval development. Although the number of neuroblasts is comparable until the II instar stage, it starts decreasing progressively during the III instar stage. Error bars show SD. (J) Neuroblast mitotic index quantification. The percentage of mitotic neuroblasts is lower in bnd D/D larvae compared to bnd D/+ throughout all the larval stages examined. Error bars show SD. Scale bars represent 50 mm. Why Bnd is also found at centrosomes and at the spindle is harder to explain. In fact, Bnd is the only known protein apart from Mud that localizes to both the centrosome and the cell cortex during ACD. It could help in promoting the alignment of the spindle through the interaction with the Pins/Gai/Mud complex, but this cannot explain the entire phenotype because microtubules are not strictly required for polarity establishment during ACD [37, 42, 61, 62]. Although we could not detect a biochemical interaction between Bnd and Pins, Gai, or Mud, this interaction could be transient, or it could depend on polymerized microtubules. It will be compelling to verify the localization of the endogenous protein because this would consolidate the data derived from the protein overexpression experiments. Furthermore, this could allow us to unravel in detail the dynamics of Bnd cortical localization and its alignment with the SOP polarity axis, which could be concealed in overexpression conditions. localized at the centrosomes and at the cortex (B D). (F) Bnd::GFP expressed using neur-gal4 colocalizes with the centrosomal marker g-tubulin (blue) in fixed tissue staining. It is also possible to see areas of cortical enrichment on the anterior side. Sanpodo (red) marks SOP cells. Anterior is up in (A) (E) and left in (F). Scale bars represent 5 mm. See also Figure S5 and Movies S1 and S2.

Current Biology Vol 24 No 16 1820 I J K L M Figure 6. banderuola LOF Affects Polarity Establishment in Mitotic Neuroblasts (A B 00 ) apkc (green) and Miranda (red) do not localize asymmetrically in mitotic bnd D/D neuroblasts (B) compared to heterozygous ones (A). In particular, apkc is either weakly localized at the cortex or uniformly cortical (B and B 0 ). Cnn staining (green) marks the position of the mitotic spindle, and ph3 (white) marks the mitotic cells. (legend continued on next page)

Banderuola Regulates Asymmetric Cell Division 1821 The Antioncogenic Function of banderuola In neuroblasts, Bnd is required for self-renewal and asymmetric protein segregation and has an antioncogenic function that is redundant with Dlg. In bnd mutants, we observe defects leading to neuroblast loss. The remaining neuroblasts are misshapen, displaying abnormalities in the asymmetric protein segregation and reduced mitotic activity. Although the FRT site remaining in the bnd mutants prevents us from addressing this question through a clonal analysis, we favor the hypothesis that the phenotype is cell autonomous and is due to premature differentiation of neuroblasts. Indeed, this is consistent with the phenotype in the SOP lineage because genetic manipulations resulting in a piia to piib transformation (like Numb overexpression or Notch loss of function) often cause neuroblasts to divide symmetrically into two differentiating daughter cells. The localization of both the basal determinants and Dlg itself are affected in bnd mutants. Dlg is known to mediate the basal localization of cell fate determinants in Drosophila neuroblasts [14, 15, 53]. The abnormal localization of apkc in bnd mutant neuroblasts could also be explained as an effect of dlg LOF because apkc localization is affected in dlg mutants [63]. Thus, the various protein mislocalization phenotypes in bnd mutant neuroblasts could be explained by a model in which Bnd exerts its function solely by localizing Dlg. The tumor phenotypes, on the other hand, suggest that the two genes act in parallel. Overproliferation phenotypes are observed only upon LOF of both genes, and bnd LOF enhances the dlg RNAi phenotype. In fact, this type of genetic interaction has been described for pins and lgl before: whereas pins mutant neuroblasts underproliferate due to self-renewal failure, pins lgl double mutants have a massive overproliferation of neuroblasts due to an aberrant self-renewal program triggered by apkc [63]. A similar mechanism could underlie the overproliferation we observe upon double RNAi of bnd and dlg. An alternative explanation for the double knockdown phenotype is provided by the additional role that Dlg has in the telophase rescue pathway, which might be independent from bnd. This pathway is known to mediate the establishment of Pins/Gai cortical polarity, even in the absence of the Par complex, through a Dlg-dependent mechanism [42]. The pathway is active in wild-type neuroblasts but becomes essential only when components of the apical Par complex are missing [35]. It is possible that the telophase rescue pathway ensures the asymmetric segregation of cell fate determinants upon bnd RNAi. When dlg is inhibited as well, however, this pathway could be compromised, resulting in overproliferation and tumor formation. Possible Conservation of the Interaction with Dlg Dlg has four mammalian homologs. Like the Drosophila protein, they localize at the basolateral cortex in epithelia and have been shown to regulate cell polarity in various cell types [64, 65]. During rat astrocyte migration, for example, Dlg1 is required in association with APC for the polarization of the microtubule cytoskeleton at the leading edge of the migrating cell [66, 67]. Dlg-mediated polarity can be also considered a gatekeeper against tumor progression: Dlg1 is a target of oncoviral proteins and is often mislocalized or downregulated in late-stage tumors, implicating a causal connection between Dlg1 and cancer [68, 69]. As the interaction between Bnd and Dlg is conserved, Banderuola could be an evolutionarily conserved regulator of Dlg activity, and our studies may therefore be relevant for a variety of biological processes in higher organisms as well. Experimental Procedures Fly Strains Flies were raised on standard Drosophila media at 25 C. The following fly strains were used: pnr-gal4 (MD237) (Bloomington Drosophila Stock Center [BDSC]); pnr-gal4, phyllopod>>egfp::pon.ld [43]; neur-gal4, UAS-H2RFP [21]; P{XP}07120 and P{RB}01794 (Exelixis); UAS-Dcr2; insc-gal4, UAS- CD8::GFP [44]; insc-gal4 [29]; Zeus::GFP (BL6836); yw;; Zeus-GFP, pnr- Gal4 mw+, UAS-mRFP1::Pon.LD mw+/tm3, Sb. RNAi lines were obtained from the Vienna Drosophila RNAi Center (VDRC). The dlg sw and dlg HF321 mutant alleles were described in [54, 55] and were obtained from Y. Bellaiche (y dlg sw /Bascy) and from the BDSC (BL36278), respectively. Antibodies and Immunohistochemistry Primary antibodies used in this study were as follows: mouse anti-cut (2B10, 1:500; Developmental Studies Hybridoma Bank [DSHB]), rat anti-su(h) (1:100) (made following [70]), mouse anti-prospero (1:10; DSHB MR1A), rabbit anti-prospero (1:1,000) [71], mouse anti-phospho-histone H3 (Ser10) (1:1,000; Cell Signaling), rabbit anti-centrosomin (1:1,000; gift from T. Kaufman), guinea pig anti-sanpodo (1:1,000) [72], rabbit anti-numb (1:100) [38], rabbit anti-apkcz (1:500; sc-216; Santa Cruz), rabbit anti-baz (1:200) [21], rabbit anti-gai (1:100) [26], rabbit anti-mud (1:500) [73], mouse anti-discs Large (1:200 immunofluorescence/1:1,000 western blot; DSHB), rabbit anti-strabismus (1:200) [9], mouse anti-gtubulin (1:1,000; GTU-88; Abcam), rabbit anti-miranda (1:200) [29], mouse anti-prospero (1:10; DSHB), guinea pig anti-dpn (1:500) [74], rabbit anti-cleaved Caspase 3 (1:500; 5A1E; Cell Signaling), rabbit anti-gfp (1:2,000; Abcam), mouse anti-flag (1:2,000; Sigma M2), rabbit anti-myc (1:2,000; ab9106; Abcam). Secondary antibodies were conjugates of Alexa Fluor 488, Alexa Fluor 568, Alexa Fluor 647, and Alexa Fluor 405 (1:500; Invitrogen). For immunofluorescence, pupae were dissected in 8% paraformaldehyde (PFA), fixed for 20 30 min on ice, and stained as described [75]. Pupae were collected at 0 hr after puparium formation (APF) and aged for 15 hr at 25 C. For lineage analysis, pupae were collected at 0 hr APF and aged for 23 26 hr at 29 C. For neuroblast stainings, third instar larvae were dissected in PBS, fixed for 15 min in 5% PFA with 0.1% Triton X-100, and processed as described [29]. Live imaging of SOP cells was performed essentially as previously described [8]. Images were acquired on a Zeiss LSM510 Meta confocal microscope, and 3D movies and three-channel movies were acquired using a LSM710 Spectral confocal microscope. Immunoprecipitation For immunoprecipitations, embryos or cells were lysed in RIPA buffer (150 mm NaCl, 50 mm Tris-HCl [ph 8], 1 mm EDTA, 1% NP40, 0.5% Nadeoxycholate) supplemented with 10 mg/ml PMSF and Complete Protease Inhibitor Cocktail (Roche), cleared by centrifugation, and incubated with antibodies for 1 2 hr at 4 C. The immunocomplexes were then precipitated by using Protein G Sepharose 4B beads (Amersham) or GFP-Trap (C D 00 ) Inscuteable (green) and Miranda (red) do not localize asymmetrically in the bnd LOF background. Cnn staining (green) marks the position of the mitotic spindle, and ph3 (white) marks the mitotic cells. (E F 00 ) Numb (green) and Miranda (red) are not correctly segregated to the basal side in mitotic neuroblasts in the bnd LOF background (F) compared to the heterozygous (E). ph3 (white) marks the mitotic cells. (G H 00 ) Upon bnd LOF, Dlg (green) is not enriched at the apical cortex of mitotic neuroblasts, and the segregation of Miranda to the basal side is compromised (H, red). No defects are visible in the heterozygous controls (G). ph3 staining (red) marks mitotic cells. Scale bars of (A) (H) represent 10 mm. (I M) Quantification of the polarity phenotypes displayed in (A) (H). apkc: n = 117 (bnd D/+) and n = 50 (bnd D/D); Insc: n = 65 (bnd D/+) and n = 50 (bnd D/D); Numb: n = 57 (bnd D/+) and n = 20 (bnd D/D); Dlg: n = 54 (bnd D/+) and n = 42 (bnd D/D); Miranda: n = 164 (bnd D/+) and n = 51 (bnd D/D).

Current Biology Vol 24 No 16 1822 A B C M Figure 7. Bnd Interacts with Dlg, and the Two Proteins Are Functionally Connected (A) Coimmunoprecipitation (coip) of Bnd and Dlg in S2 cells. Bnd::Flag and Dlg::GFP were cotransfected in Drosophila S2 cells. Bnd::Flag coimmunoprecipitates with Dlg::GFP upon GFP immunoprecipitation. (B) CoIP of Dlg and Bnd in Drosophila embryos. Bnd::Flag is overexpressed using a heat shock promoter, and endogenous Dlg coimmunoprecipitates with Bnd::Flag in embryos. (C) The interaction between Bnd and Dlg is evolutionarily conserved as rat Dlg1::MYC coimmunoprecipitates with GFP-tagged mouse Bnd (ANKFN1) when overexpressed in 293T cells. (D G 00 ) bnd and dlg are displaying additive phenotypes. The number of neuroblasts (Mira-positive cells, red) is unaffected upon dlg RNAi (E) or bnd RNAi (F) compared to control (D), although the asymmetric partitioning of Miranda is defective (H J). Double RNAi of bnd and dlg (G) causes massive neuroblast (legend continued on next page)

Banderuola Regulates Asymmetric Cell Division 1823 (Chromotek) in the HEK293T cells experiments, washed three times with lysis buffer, and eluted by boiling in SDS sample buffer. Cell Culture S2 cells were cultured at 27 C in Schneider s Drosophila medium (Gibco), with 10% scomplemented FCS, penicillin (50 U/ml), and streptomycin (50 mg/ml). Cells were transfected with Cellfectin II reagent (Invitrogen), according to the manufacturer s protocol. UAS constructs were expressed by cotransfection with actin-gal4. HEK293T cells were cultured at 37 Cin DMEM, with 10% FBS, 1 mm L-Gln, penicillin (100 U/ml), and streptomycin (100 mg/ml). Cells were transfected using Turbofect (Fermentas), according to manufacturer s protocol. Constructs and Fly Transgenics The banderuola coding region (starting at position 256 of CG45058-RB) was amplified by PCR from embryonic cdna (0 9 hr) and verified by DNA sequencing. bnd expression constructs for cell culture and fly transgenesis expression were generated by subcloning the bnd CDS into destination vectors using Gateway technology (Invitrogen). For rescue experiments, the CH322-118J18 clone was used (BACPAC Resources Center). Transgenic flies were derived using standard methodology. ANKFN1 CDS was subcloned from ORFeome clone 100016073 (Source BioScience Life Sciences) using the Gateway technology. Rat Dlg constructs were obtained from S. Etienne-Manneville [76]. Supplemental Information Supplemental Information includes seven figures and two movies and can be found with this article online at http://dx.doi.org/10.1016/j.cub. 2014.06.059. Acknowledgments We wish to thank Elke Kleiner, Sara Farina Lopez, and Joseph Francis Gokcezade for technical assistance; all members of the J.A.K. laboratory for discussions; T. Kaufman, F. Schweisguth, Y. Bellaiche, S. Etienne-Manneville, and Yuh Nung Yan for sharing reagents; Maria Novatchkova for performing protein sequence alignment and conservation analysis; Marko Repic for help with the HEK293T cells experiments; and Christoph Jüschke, Spyros Goulas, and Frederik Wirtz-Peitz for comments on the manuscript. Work in the J.A.K. laboratory is supported by the Austrian Academy of Sciences, the EU Seventh Framework Programme network EuroSyStem, the Austrian Science Fund (grants I_552-B19 and Z_153_B09), and an advanced grant of the European Research Council. Received: September 12, 2012 Revised: March 8, 2014 Accepted: June 23, 2014 Published: July 31, 2014 References 1. Knoblich, J.A. (2008). Mechanisms of asymmetric stem cell division. Cell 132, 583 597. 2. Gönczy, P. (2008). 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SUPPLEMENTAL FIGURES A B -7 :09 0 :00 1 :50 2 :30 3 :31 5 :18 9 :26-2 :16 0 :00 1 :09 1 :36 2 :04 3 :00 7 :51 NEBD Anaphase Telophase/Cytokinesis Figure S1 (related to Figure 1): The asymmetric segregation of GFP::Pon is defective upon CG45058 (bnd) RNAi. (A, B) Confocal time-lapse images of mitotic SOP cells show that CG45058 knockdown causes defects in Numb reporter (EGFP::Pon.LD) localization and inheritance. The reporter is symmetrically localized or only weakly accumulates asymmetrically by nuclear envelope breakdown (NEBD) (A, B), although reporter localization is often rescued by anaphase or telophase. Abnormal shaped crescents, split in two parts (A, arrows) or accumulated at the cleavage furrow rather than at the anterior tip (A, arrowhead) can be observed. When such errors occur, there is often no defect visible at the two-cell stage (A, B). The time-lapse movies were performed using the pnr-gal4, phyllopod-egfp::pon.ld driver to express CG45058 RNAi (VDRC TID27759). Scale bar=5μm

A XP FRT FRT UAS white UAS P{XP}d07120 bnd locus RB white FRT hs-flp; bnd locus heat shock PBac{RB}Gclme01794 Hybrid PCR Two sided PCRs B Stock Eye color Hybrid PCR Two-sided PCR Viability w- W- negative negative viable {XP} 07120 W+ negative positive* viable {RB} 01794 W+ negative positive* viable bnd W- positive positive lethal bnd rescue W+ - - viable Figure S2 (related to Figure 2): Generation and analysis of a bnd deletion. (A) The bnd deletion has been generated by combining in trans stocks that carry P-element insertions bearing FRT sites with suitable orientation and located close to the gene. Upon hs-flp induction, the FRT sites were recombined and the bnd locus was deleted. (B) The recombination has been confirmed by eye color change and PCR on the resulting hybrid P-element and on the genomic surrounding (2 sided). Deleted stocks are positive for the hybrid and the two-sided PCRs, parental stocks only for the two-sided PCR. Deletion of the bnd locus is homozygous lethal, and a transgene carrying the bnd genomic locus (bnd rescue) restores viability.

Control (bnd Δ/+) bnd Δ/Δ A A B B C Stbm/Spdo ph3/dapi Stbm Stbm localization Stbm/Spdo ph3/dapi Stbm 100% 80% 60% 40% 20% Weak/No crescent Normal localization 0% bnd Δ/+ bnd Δ/Δ Figure S3 (related to Figure 3). Loss of bnd does not affect Strabismus localization in SOP cells (A-C) The PCP protein Strabismus (green) is correctly localized to the anterior cortex in SOP cells upon bnd LOF. In the bnd heterozygous control (A) Stbm accumulates to the anterior side of dividing SOP cells. In the bnd null background it is still possible to observe Stbm anterior crescent (B), and overall there is no striking difference between Stbm localization in bnd Δ/+ and bnd Δ/Δ cells. Spdo (red) labels the SOP cells; ph3 (white) marks the mitotic cells, DAPI marks DNA. Anterior is up. n = 25 (bnd Δ/+) and 17 (bnd Δ/Δ). Scale bar=5μm.

A B 100% 90% 80% CONTROL: RESCUE: 70% ; + / CyO (RFP) ; bnd Δ / TM6b X ; ; UAS-Bnd::GFP, bnd Δ / TM3,Sb ; insc-gal4 / CyO (RFP) ; bnd Δ / TM6b X ; ; UAS-Bnd::GFP, bnd Δ / TM3,Sb 60% 50% 40% 30% 20% TM3/TM6 bnd Δ/Δ bnd Δ/+ 10% 0% Control Rescue insc-gal4> UAS-Bnd::GFP bnd Δ/Δ Bnd::GFP/Mira/Dpn Mira C C C Dpn D Neuroblasts number 100 90 80 70 60 50 40 30 20 10 0 bnd Δ/+ bnd Δ/Δ bnd::gfp, bnd Δ/Δ Figure S4 (related to Figure 5). Bnd::GFP expression is able to rescue viability and the loss-of-neuroblasts phenotype in the bnd LOF background. (A) Scheme of the crossing strategy used to obtain progeny without (Control) or with (Rescue) the insc-gal4 driver in an identical genetic background. (B) Distribution of genotypes in the offspring from (A). The expression of UAS-Bnd::GFP driven by insc-gal4 is able to rescue viability, since the percentage of bnd Δ/Δ progeny is restored to mendelian ratios compared to controls, where it is virtually absent (viability less than 1% n = 136 for Control, 93 for the Rescue). (C) Staining of a larval brain hemisphere with the neuroblast markers Miranda (Mira, red) and Deadpan (Dpn, blue). Bnd::GFP expression is shown in green. UAS-Bnd::GFP expression mediated by insc-gal4 is able to restore normal neuroblast numbers and Miranda asymmetric localization in bnd LOF brains. Scale bar = 50 μm (D) Quantification of Mira/Dpn positive neuroblasts. Insc-Gal4 mediated expression of Bnd::GFP is able to rescue the loss-of-neuroblasts phenotype observed in bnd Δ/Δ brains, restoring the number of neuroblasts to a level comparable to the bnd Δ/+ background. Error bars = SD

A neur-gal4> UAS-His::RFP, UAS-Bnd::GFP B -8:00 0:00 4:26 7:06 8:42 9:27 Prophase NEBD Metaphase Anaphase Telophase Cytokinesis Figure S5 (related to Figure 4): Bnd localizes at the centrosomes and at apical cortical domains during mitosis. (A) Bnd::GFP is enriched at the apical cortex at the onset of mitosis and throughout prophase till NEDB, then the apical localization is lost. (B) Bnd::GFP is diffuse in the cytoplasm, but gets enriched at the centrosomes at prophase before Nuclear Envelope Breakdown (NEBD - arrowheads). At metaphase and anaphase, it associates with the centrosomes and spindle microtubules (arrows). It is also possible to observe a protein enrichment at the cleavage furrow. The time-lapse movies were performed using the neur-gal4 driver to express Bnd::GFP (green). The simultaneous expression of UAS-His::RFP (red) is used to follow the mitotic stages. Anterior is up. Scale bar=5μm.

bnd Δ/Δ bnd Δ/+ bnd Δ/Δ bnd Δ/+ Pins/pH3/Mira A A E Mira Pins localization 100% 80% 60% B B C Baz/pH3/Mira B C Baz C Weak/No crescent Normal localization 40% 20% 0% F Mira bnd Δ/+ bnd Δ/Δ Baz localization 100% 80% 60% D apkc/cnn/ph3/mira G A Pins D 40% D 20% 0% apkc/cnn G G Mira Weak/No crescent Normal localization Insc/Cnn/pH3/Mira H bnd Δ/+ bnd Δ/Δ Insc/Cnn H H Mira Figure S6 (related to Figure 6). bnd LOF affects polarity establishment in mitotic neuroblasts, but Miranda can still be asymmetrically segregated to the daughter cell in telophase (A-B) Pins (green) and Miranda (red) do not localize asymmetrically in the bnd LOF background (B) compared to heterozygous (A). ph3 (white) marks the mitotic cells. (C-D) Bazooka (green) and Miranda (red) do not localize asymmetrically in the bnd LOF background (D) compared to the heterozygous (A). ph3 (white) marks the mitotic cells. (E-F) Quantification of the polarity phenotypes displayed in A-D. Pins n = 35 (bnd Δ/+) and 36 (bnd Δ/Δ); Baz n = 25 (bnd Δ/+) and 26 (bnd Δ/Δ). (G-H) Miranda (red) can be asymmetrically inherited in the basal daughter in about 65% of bnd Δ/Δ telophase neuroblasts, despite the lack of apkc (G, green) and Insc (H, green) apical enrichment. Cnn staining (green) marks the position of the mitotic spindle, ph3 (white) marks the mitotic cells. Scale bar = 10μm (A-D).

insc-gal4> UAS-Bnd::GFP GFP/pH3/Dlg/Mira Dlg/pH3 A A A Bnd::GFP neur-gal4> UAS-Bnd::GFP GFP/pH3/Dlg/Spdo Dlg/pH3 B B B Bnd::GFP Figure S7 (Related to Figure 7). Bnd and Dlg transiently co-localize in Drosophila neuroblasts and SOP cells. (A) Upon overexpression in neuroblasts with the insc-gal4 driver, Bnd::GFP (green) localizes to the cytoplasm but is also enriched at the apical cortex (red arrowhead), where it co-localizes with Dlg (blue). The cell-fate determinant Miranda (red) gets segregated to the basal side. ph3 (blue) marks the mitotic cells. Scale bar = 10μm (B) When overexpressed in SOP cells with the neuralized-gal4 driver, Bnd::GFP (green) gets enriched at the anterior cortex (B, B ), partially co-localizing with Dlg (magenta). Spdo (red) labels the SOP cells; ph3 (magenta) marks the mitotic cells. Anterior is left. Scale bar = 5μm.