Report

Planar Cell Polarity Breaks the Symmetry of PAR Protein Distribution prior to Mitosis in Drosophila Sensory Organ Precursor Cells Graphical Abstract

Authors Charlotte Besson, Fred Bernard, ..., Khalil Mazouni, Franc¸ois Schweisguth

Correspondence [email protected]

In Brief Besson, Bernard, et al. combine live imaging with modeling to detect the onset of PAR planar polarization in Drosophila epithelial cells. PAR proteins become asymmetric in SOPs prior to mitosis independently of the mitotic kinase AuroraA. Planar cell polarity breaks the planar symmetry of PAR protein distribution.

Highlights d

PAR asymmetry in SOPs is set prior to mitosis

d

Planar cell polarity breaks PAR symmetry

Besson et al., 2015, Current Biology 25, 1–7 April 20, 2015 ª2015 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2015.02.073

Please cite this article in press as: Besson et al., Planar Cell Polarity Breaks the Symmetry of PAR Protein Distribution prior to Mitosis in Drosophila Sensory Organ Precursor Cells, Current Biology (2015), http://dx.doi.org/10.1016/j.cub.2015.02.073

Current Biology

Report Planar Cell Polarity Breaks the Symmetry of PAR Protein Distribution prior to Mitosis in Drosophila Sensory Organ Precursor Cells Charlotte Besson,1,2,3,6 Fred Bernard,1,2,6 Francis Corson,1,5 Herve´ Rouault,1,2,4 Elodie Reynaud,1,2 Alyona Keder,1,2 Khalil Mazouni,1,2 and Franc¸ois Schweisguth1,2,* 1Institut

Pasteur, rue du Dr Roux, 75015 Paris, France URA2578, rue du Dr Roux, 75015 Paris, France 3UPMC, Cellule Pasteur, rue du Dr Roux, 75015 Paris, France 4Present address: Janelia Farm, HHMI, Ashburn, VA 20147, USA 5Present address: LPS, ENS, rue d’Ulm, 75005 Paris, France 6Co-first author *Correspondence: [email protected] http://dx.doi.org/10.1016/j.cub.2015.02.073 2CNRS,

SUMMARY

During development, cell-fate diversity can result from the unequal segregation of fate determinants at mitosis [1]. Polarization of the mother cell is essential for asymmetric cell division (ACD). It often involves the formation of a cortical domain containing the PAR complex proteins Par3, Par6, and atypical protein kinase C (aPKC) [1–5]. In the fly notum, sensory organ precursor cells (SOPs) divide asymmetrically within the plane of the epithelium and along the body axis to generate two distinct cells [6–12]. Fate asymmetry depends on the asymmetric localization of the PAR complex. In the absence of planar cell polarity (PCP), SOPs divide with a random planar orientation but still asymmetrically, showing that PCP is dispensable for PAR asymmetry at mitosis [6, 13–15]. To study when and how the PAR complex localizes asymmetrically, we have used a quantitative imaging approach to measure the planar polarization of the proteins Bazooka (Baz, fly Par3), Par6, and aPKC in living pupae. By using imaging of functional GFP-tagged proteins with image processing and computational modeling, we find that Baz, Par6, and aPKC become planar polarized prior to mitosis in a manner independent of the AuroraA kinase and that PCP is required for the planar polarization of Baz, Par6, and aPKC during interphase. This indicates that a ‘‘mitosis rescue’’ mechanism establishes asymmetry at mitosis in PCP mutants. This study therefore identifies PCP as the initial symmetry-breaking signal for the planar polarization of PAR proteins in asymmetrically dividing SOPs. RESULTS AND DISCUSSION A Quantitative Live-Imaging Approach Asymmetric localization of the Baz-Par6-aPKC complex at the posterior pole of dividing SOPs is critical for the unequal segre-

gation of the fate determinants Numb and Neur [1, 12, 16]. Thus, deciphering when and how this complex becomes asymmetric are essential to understand fate asymmetry. Since formation of the Baz-Par6-aPKC complex is regulated by the AuroraA (AurA) mitotic kinase in SOPs [17], it is usually assumed that the Baz-Par6-aPKC complex forms and localizes asymmetrically at mitosis. To examine the dynamics of Baz, Par6, and aPKC asymmetry, we have developed a quantitative live-imaging approach using functional GFP-tagged proteins (Figures 1A– 1G). Specifically, we used a Par6-GFP genomic rescue construct [18] and a GFP-Baz protein-trap. Since GFP-Baz only reports on the distribution of two of the Baz isoforms [19], we also generated a BAC-encoded Baz-GFP protein that tags all isoforms. Last, we produced a BAC-encoded aPKC-GFP that also tags all aPKC isoforms. Genomic rescue assay showed that the BAC-encoded Baz-GFP and aPKC-GFP proteins are fully functional (see Supplemental Experimental Procedures). These four GFP-tagged proteins localized at the apical cell cortex in living pupae (Figures 1A–1D) and localized at the posterior pole of dividing SOPs (Figure S1) like the corresponding endogenous proteins. To study the polar distribution of Baz, Par6, and aPKC in individual cells, we moved away from partly subjective, hence error-prone, categorization, such as scoring ‘‘polar’’ versus ‘‘non-polar’’ cases, and developed a quantitative approach. Live imaging of developing pupae produced 4D (x,y,z,t) movies. After z-maximal projection of the apical planes, the contour of individual cells was extracted and an intensitynormalized polarity vector was calculated for each time point (Figure 1H; Figure S1; see Supplemental Experimental Procedures). The magnitude of this polarity vector revealed planar asymmetry at the cell cortex while the direction (a) gave the orientation of this planar asymmetry relative to the anteriorposterior (a-p) axis (Figure 1I). Thus, high-magnitude values and a posterior direction reflect planar polarization along the a-p axis. Using this method, the polarity vector was measured over time in both SOPs and epidermal (epi) cells (Figures 1H0 and 1H00 ). To compare polarization dynamics in different cells, a relative timescale was used with t = 0 corresponding to mitosis entry. This approach was used to measure the planar polarization of Baz, Par6, and aPKC.

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Please cite this article in press as: Besson et al., Planar Cell Polarity Breaks the Symmetry of PAR Protein Distribution prior to Mitosis in Drosophila Sensory Organ Precursor Cells, Current Biology (2015), http://dx.doi.org/10.1016/j.cub.2015.02.073

Figure 1. Measure of the Planar Polarization of the PAR Proteins (A–D) Live imaging of Baz-GFP (A), GFP-Baz (B), Par6-GFP (C), and aPKC-GFP (D) in the notum of 13.5 hr APF pupae (GFP, green). SOPs were identified using H2B-RFP (red). Scale bars, 20 mm. (E–G) Genomic structure (coding and non-coding exons, black and gray boxes, respectively; GFP, green; not all isoforms are shown) of Baz-GFP, GFP-Baz (protein-trap P-element, red), Par6-GFP, and aPKC-GFP. Scale bar, 5 kb. (H and I) Polarity vectors were measured using extracted cell contours from live imaging 4D movies. GFP-Baz polarity vectors of individual SOPs (black) are shown for t = 250 (H0 ) and t = 5 min (H00 ). The magnitude and direction of the mean polarity vector (red) is shown (I). In this and all other figures, anterior is left. See also Figure S1.

Planar Polarization at Interphase The planar polarization of Baz-GFP, GFP-Baz, Par6-GFP, and aPKC-GFP was measured from 13.5 to 17.5 hr after puparium formation (APF) in pupae mutant for baz, par6, and apkc, respectively (see Supplemental Experimental Procedures for detailed genotypes). The magnitude and direction were plotted over time (Figures 2A–2D, raw data; Figures 2A0 –2D0 , averaging over ten time points). At t = 225 (13.5 APF), the Baz-GFP, GFP-Baz, Par6-GFP, and aPKC-GFP vectors had low-magnitude values and were randomly oriented in both SOPs and epis (SOPs were identified using Histone2B-RFP expressed under the control of a SOP-specific enhancer). This indicated that these PAR complex proteins were not planar polarized. Interestingly, all four vectors had increased magnitude and oriented posteriorly over time in SOPs (Figures 2A–2D0 ) showing that Baz, Par6, and aPKC localized at the posterior apical cortex during late interphase consistent with the asymmetric and oriented mode of SOP division [6, 16]. By contrast, polarity vectors showed low magnitude and random direction in epi cells at all time points (Figures 2A–2D0 ). This suggested that Baz, Par6, and aPKC localized in a non-polar manner in epi cells, consistent with their symmetric mode of division [6]. Also, since Baz-GFP and GFP-Baz gave similar polarization profiles, they were used interchangeably afterward. While low-magnitude values and random direction suggested a non-polar distribution, different magnitudes values were measured in epi cells for Baz, Par6, and aPKC. Higher values could result from a weak and randomly oriented polarity or from stochastic fluctuations in protein distribution at the cell cortex, i.e., random patches (Figure 2E). Since the GFP signal along the cell cortex could be described as patches of higher intensity on top of a more or less uniform background (Figure 2F), these two possibilities could be discriminated using synthetic cells that were generated in silico. These synthetic cells comprised randomly located patches with similar statistics in patch number and intensities relative to the actual cells (Figure 2F). For epi cells,

the average magnitude of the polarity vectors computed from these synthetic cells was nearly identical to those measured for Baz, Par6, and aPKC (Figures 2A00 –2D00 ). Thus, random fluctuations in the GFP signal along the cell cortex alone could account for differences in magnitude values. Thus, our synthetic cell modeling approach clearly established that Baz, Par6, and aPKC are not planar polarized in epi cells. Similarly, the magnitude values measured in SOPs for Baz, Par6, and aPKC were also identical to those obtained from synthetic profiles at t = 225 (Figures 2A00 –2D00 ). This showed that Baz, Par6, and aPKC are not planar polarized at 13.5 hr APF. We further found that the measured values deviated over time from the synthetic profiles in SOPs (Figures 2A00 –2D00 ), indicating that randomly located patches could not account for the high-magnitude values measured from t = 125 onward. This difference between measured magnitudes and those calculated from the synthetic cells revealed the progressive planar polarization of Baz, Par6, and aPKC in SOPs. Thus, our quantitative image analysis combined with modeling clearly demonstrated that Baz, Par6, and aPKC are planar polarized prior to mitosis in SOPs. PAR Complex Formation and Polarization Baz, Par6, and aPKC are thought to form complexes at the posterior cortex of SOPs. Consistent with this view, all three proteins become asymmetrically distributed at around the same time, between t = 175 and t = 125. Furthermore, the polarization of Par6-GFP was abolished upon silencing of baz, and the polarization of aPKC-GFP was decreased in baz heterozygous pupae (Figures 3A and 3B; aPKC-GFP did not correctly localize at the cortex in baz mutant cells, see Figure 3C). Thus, asymmetric localization of Par6-aPKC prior to mitosis appeared to depend on proper Baz levels. Reciprocally, the asymmetric localization of Baz and Par6 was also dependent on proper Par6 levels (Figures 3E and 3F; in the complete absence of par6, cells had shape defects with a reduced apical domain, see Figure 3D). Thus, Baz, Par6, and aPKC appeared to be dependent on each other for their asymmetric distribution prior to mitosis. These data therefore indicated that the formation of the

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Please cite this article in press as: Besson et al., Planar Cell Polarity Breaks the Symmetry of PAR Protein Distribution prior to Mitosis in Drosophila Sensory Organ Precursor Cells, Current Biology (2015), http://dx.doi.org/10.1016/j.cub.2015.02.073

Figure 2. Baz, Par6, and aPKC Are Planar Polarized prior to Mitosis (A–D0 ) The magnitude (top) and direction (bottom) of the Baz-GFP (A and A0 : 44 SOPs, 42 epis), GFP-Baz (B and B0 : 50 SOPs, 47 epis), Par6-GFP (C and C0 : 83 SOPs, 78 epis), and aPKC-GFP (D and D0 : 35 SOPs, 34 epis) polarity vectors are shown in SOPs (red) and epis (green) from t = 250 min to mitosis (t = 0). Reference orientations (random, gray; posterior, purple) are indicated by dotted lines. Both raw data (A–D) and average (A0 –D0 ) profiles are shown. (A00 –D00 ) The magnitude of the calculated Baz-GFP (A00 ), GFP-Baz (B00 ), Par6-GFP (C00 ), and aPKC-GFP (D00 ) polarity vectors (light red and green, same data as in A0 –D0 ) are compared with those computed from synthetic cell (red and green dotted lines). Baz, Par6, and aPKC become planar polarized between t = 175 and t = 125 in SOPs. (E) Positive magnitude values of non-oriented polarity vectors may result from a random distribution of cortical patches or from a randomly oriented but polarized distribution. (F) To generate synthetic cell profiles, the length of the extracted contour was normalized and high-intensity patches were defined along this contour. The measured intensity and number of these patches were scored. Synthetic cells were created from this list of high-intensity patches.

Baz-Par6-aPKC complex is a limiting parameter for its polarization. Consistent with this, reducing the levels of Lethal(2) giant larvae (Lgl), a protein that prevents Baz binding to Par6-aPKC and inhibits aPKC activity [17], led to the earlier planar polarization of Par6-GFP (Figure 3G). Since planar polarization was observed 2 hr prior to SOP division, we wondered whether the mitotic kinase AurA could be

dispensable for this early polarization. Indeed, live imaging of aurA mutant pupae showed that the initial polarization of Par6GFP during interphase did not require this mitotic kinase (Figure 3H). We propose that the PAR complex forms and localizes asymmetrically during interphase in an AurA-independent manner and that AurA acts later at mitosis to amplify this asymmetry [17].

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Please cite this article in press as: Besson et al., Planar Cell Polarity Breaks the Symmetry of PAR Protein Distribution prior to Mitosis in Drosophila Sensory Organ Precursor Cells, Current Biology (2015), http://dx.doi.org/10.1016/j.cub.2015.02.073

Figure 3. Baz-, Par6-, and aPKC-Dependent but AurA-Independent Polarization (A, B, and E–H) Measured and synthetic cell profiles of the Par6-GFP (A, F–H), aPKC-GFP (B), and Baz-GFP (E) polarity vectors in bazRNAi (A: 20 SOPs, 18 epis), baz heterozygous (B: 22 SOPs, 23 epis), par-6 heterozygous (E: 31 SOPs, 30 epis; F: 31 SOPs, 31 epis), lgl heterozygous (G: 26 SOPs, 25 epis), and aurA mutant (H: 17 SOPs, 11 epis) SOPs (blue) and epis (orange). The corresponding wild-type SOP profiles (red) are also shown. Early planar polarization depends on proper levels of Baz, Par6, and aPKC but does not depend on aurA. (C) aPKC-GFP is largely cytoplasmic in baz mutant cells (that have two copies of aPKC-GFP) and baz heterozygous cells (that have one copy of aPKC-GFP; note that baz+/+ cells lack the aPKC-GFP transgene). Loss of cortical aPKC prevented us from extracting cell contours, hence studying planar polarization. (D) par-6 mutant cells (that have two copies of Baz-GFP; heterozygous cells have one copy of Baz-GFP; clone border in blue) showed reduced apical area. The wild-type cells (border of the twin clone in red) do not carry the Baz-GFP transgene.

PCP-Dependent Polarization and Mitosis Rescue We next investigated how asymmetry is established in SOPs during interphase. The anterior Gai-Pins complex is known to act redundantly with PCP to restrict the Baz-Par6-aPKC complex posteriorly in mitotic SOPs [13, 20, 21]. We therefore studied the potential role of Gai, Pins, and PCP in the early polarization of SOPs. We first found that the complete loss of Gai activity had a very minor effect on the polarization of Par6-GFP at late interphase (Figure 4A). Additionally, the silencing of pins did not affect the planar polarization of Par6-GFP and GFP-Baz (Fig-

ures 4B and 4C). Thus, the activities of Gai and Pins were not essential for the initial polarization of Baz and/or Par6. We next investigated the role of the PCP gene frizzled (fz). In fz mutant pupae, the measured magnitude of the Par6-GFP and Baz-GFP vectors remained low and did not significantly differ from those calculated in synthetic cells (Figures 4D and 4E), showing that planar polarization was lost. A similar result was obtained for GFP-Baz upon silencing of fz (data not shown). Also, the silencing of the PCP genes Van Gogh (Vang) and dishevelled (dsh) similarly abolished the planar polarization of Par6-GFP

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(legend on next page)

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and GFP-Baz (Figures 4F–4I). In all cases, the direction of the Baz and Par6 polarity vectors was randomized. These data clearly demonstrated that PCP is required for the initial planar polarization of Baz and Par6 in SOPs. We propose that the PAR complex reads the PCP information in SOPs to accumulate asymmetrically. How PCP regulates PAR complex distribution in SOPs remains to be determined [22, 23]. Also, since PCP operates across the notum to polarize all epithelial cells [14, 15], mechanisms restricting the interpretation of PCP to SOPs must exist (Figure 4J). Our finding that PCP provides a symmetry breaking input for the planar polarization of Baz, Par6, and aPKC was unexpected since PCP had previously been shown to be largely dispensable for SOP asymmetry at mitosis [6, 13]. Thus, a mitosis rescue mechanism must operate at mitosis to create asymmetry, albeit less efficiently and in a non-oriented manner, when it fails to be established by PCP prior to mitosis. A similar mitosis rescue phenomenon was observed in neuroblasts: while a microtubuledependent orientation cue normally persists from mothers to daughters to position the division axis, asymmetry can still form de novo at mitosis upon loss of this cue but with a random orientation relative to the previous division axis [24]. Since pins acts redundantly with fz to localize Baz asymmetrically in dividing SOPs [16], this mitosis rescue likely involves Pins. Pins is phosphorylated at mitosis by AurA, and this phosphorylation is important for its recruitment at the cortex by Discs-large (Dlg) to form an anterior Dlg-Pins complex [16, 25]. AurA also phosphorylates Par6 and Lgl and may thereby relieve the inhibition exerted by Lgl on the aPKC activity [17, 26, 27]. These data suggest that AurA may amplify the initial asymmetry established by PCP prior to mitosis and/or reinforce the possible input of PCP on asymmetry at mitosis. Accordingly, AurA would trigger mitosis rescue when the initial PCP-dependent asymmetry fails to be set up (Figures 4J and 4K). In summary, our study revealed that all three PAR proteins become asymmetrically localized in SOPs prior to mitosis in a mutually dependent manner and that PCP is required for their initial planar polarization. Future studies will address how planar polarization of the PAR complex is restricted to SOPs. We propose a model whereby first notum epithelial cells become planar polarized at larval stages; second, SOPs are selected within this epithelium at early pupal stages; this cellfate change allows Baz, Par6, and aPKC to read the PCP information and localize asymmetrically at interphase; third, at mitosis, mitotic kinases, possibly together with PCP, regulate the amplification of this initial asymmetry (Figures 4J and 4K). This model of cell-specific interpretation of PCP via the planar polarization of the PAR complex is likely conserved from flies to mammals [28].

SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures and one figure and can be found with this article online at http://dx.doi.org/ 10.1016/j.cub.2015.02.073. AUTHOR CONTRIBUTIONS F.S., C.B., and F.B. designed the project. H.R. conceived the polarity vector. F.C. developed the synthetic cell approach. K.M. created the BAC transgenes. C.B. and F.B. performed together most of the experiments. E.R. and A.K. performed together the experiments shown in Figures 2D, 4D, 4E, 4H, and 4I. All authors participated in the writing of the manuscript. ACKNOWLEDGMENTS We thank J. Knoblich, A. Spradling, D. Strutt, R. Ueda, the Bloomington Drosophila Stock Center (BDSC), Vienna Drosophila Research Center (VDRC), Drosophila Genomics Resource Center and Flybase for flies, DNA, and other resources. We thank R. Le Borgne and lab members for discussion and critical reading. This work was funded by grants from the Agence Nationale pour la Recherche (ANR12-BSV2-0010-01 and Labex Revive) and the Pasteur-Weizmann fondation. C.B. received doctoral fellowships from the MENRT and ARC. F.B. received an ARC post-doctoral fellowship. Received: December 5, 2014 Revised: January 31, 2015 Accepted: February 25, 2015 Published: April 2, 2015 REFERENCES 1. Knoblich, J.A. (2008). Mechanisms of asymmetric stem cell division. Cell 132, 583–597. 2. Motegi, F., and Seydoux, G. (2013). The PAR network: redundancy and robustness in a symmetry-breaking system. Philos. Trans. R. Soc. Lond. B Biol. Sci. 368, 20130010. 3. Cowan, C.R., and Hyman, A.A. (2007). Acto-myosin reorganization and PAR polarity in C. elegans. Development 134, 1035–1043. 4. Schober, M., Schaefer, M., and Knoblich, J.A. (1999). Bazooka recruits Inscuteable to orient asymmetric cell divisions in Drosophila neuroblasts. Nature 402, 548–551. 5. Wodarz, A., Ramrath, A., Kuchinke, U., and Knust, E. (1999). Bazooka provides an apical cue for Inscuteable localization in Drosophila neuroblasts. Nature 402, 544–547. 6. Gho, M., and Schweisguth, F. (1998). Frizzled signalling controls orientation of asymmetric sense organ precursor cell divisions in Drosophila. Nature 393, 178–181. 7. Gho, M., Bellaı¨che, Y., and Schweisguth, F. (1999). Revisiting the Drosophila microchaete lineage: a novel intrinsically asymmetric cell division generates a glial cell. Development 126, 3573–3584. 8. Hartenstein, V., and Posakony, J.W. (1989). Development of adult sensilla on the wing and notum of Drosophila melanogaster. Development 107, 389–405.

Figure 4. PCP Regulates the Early Planar Polarization of Baz and Par6 (A–I) Measured and synthetic cell profiles of the Par6-GFP (A, B, D–F, and H), GFP-Baz (C, G, and I), and Baz-GFP (E) polarity vectors in Gai mutant (A: 31 SOPS, 29 epis), pinsRNAi (B: 33 SOPS, 36 epis; C: 34 SOPs, 30 epis), fz mutant (D: 31 SOPs, 34 epis; E: 24 SOPs, 20 epis), vangRNAi (F: 25 SOPs, 24 epis; G: 41 SOPs, 33 epis), and dshRNAi (H: 36 SOPs, 35 epis; I: 20 SOPs, 17 epis) SOPs (blue) are compared to the corresponding wild-type profiles (red) and mutant epis (orange) as in Figure 3. The early planar polarization of Par6 and Baz was lost in the absence of PCP but remained unchanged upon loss and/or reduced Gai/Pins. (J–K00 ) Model. Prior to SOP specification (J), all notum cells are planar polarized with Vang anterior (yellow) and Fz posterior (green). Par proteins (red) localize at the apical cortex and are not planar polarized. Following SOP specification (J0 ), PAR proteins become planar polarized only in SOPs, leading to asymmetric division (J00 ). In PCP mutants, PCP is lost (K), PAR proteins are not planar polarized prior to mitosis (K0 ) and mitosis rescue restores asymmetry but not orientation along the body axis (K00 ).

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