Many of our organs, including the lungs and the intestine, are lined with a single layer of cells that separate the inside of the organ from the surrounding environment inside the body. These so-called epithelial cells form a tightly packed barrier and have a very characteristic organization.

The apical surface faces the outside world, while the basal surface faces the inner tissues. These different interfaces are reflected in the organization of the cells themselves. The shape, composition, and role of the apical cell surface are distinct from those of the basal surface, and they also contain different proteins. In some epithelial cells, the apical surface specializes and forms protruding structures called microvilli. Thus, epithelial cells are said to be polarized along this apical–basal axis. Over the last 30 years, many labs have identified and studied which proteins help epithelial cells become and stay polarized.

Previous biochemical experiments showed that these so-called polarity proteins interact with each other in many different ways. But it remains unclear whether some of these interactions are more important than others, and where exactly in the apical or basal membranes these interactions take place.

Mangeol et al. used super-resolution microscopy to observe the polarity of proteins at the apical membranes of both human and mouse cells from the small intestine to answer these questions. They focused on areas called tight junctions, where the intestinal cells connect with each other to form the barrier between the outside and the inside. First, all the polarity proteins clustered together in various formations, they were not distributed uniformly. For example, one protein called PAR3 was at the level of the tight junctions, whereas other proteins were closer to the apical surface and the outside world. Only two pairs of proteins – PAR6 and aPKC, and PALS1 and PATJ – formed stable clusters with each other. This finding was unexpected because previous biochemical experiments had predicted multiple interactions. Third, the PALS1/PATJ complexes stayed at the bottom of the microvilli protrusions, whereas PAR6/aPKC were inside the protrusions.

Taken together, these experiments reveal a detailed snapshot of how the polarity proteins themselves are organized at the apical surface of epithelial cells. Future work will be able to address how these protein complexes behave over time.

In epithelial tissues, cells coordinate their organization into a polarized sheet of cells. Each cell acquires an apico-basal organization and specialized lateral junctions, namely, tight junctions (TJs, also known as zonula occludens), adherens junctions, and desmosomes (Farquhar and Palade, 1963). This organization is key to the development, maintenance, and function of epithelial tissues.

Over the past two decades, several proteins have been discovered to be pivotal to epithelial polarization, such as PAR3, PAR6, aPKC (PAR complex), Crumbs, PATJ, PALS1 (Crumbs complex), Scribble, LGL, and DLG (Scribble complex) in mammals (for review, see Assémat et al., 2008; Pickett et al., 2019; Rodriguez-Boulan and Macara, 2014). These proteins are remarkably well conserved over the animal kingdom (Belahbib et al., 2018; Le Bivic, 2013). Deletion or depletion of one of these proteins usually results in dramatic developmental defects (Alarcon, 2010; Charrier et al., 2015; Hakanen et al., 2019; Lalli, 2012; Park et al., 2011; Sabherwal and Papalopulu, 2012; Tait et al., 2020; Whiteman et al., 2014).

In the quest to understand the role of polarity proteins, numerous genetic and biochemical studies have been carried out. We and others have found that these proteins interact to form multiprotein complexes. Pioneering studies defined three core complexes based on the discovery of protein interactions or localization: the PAR complex consisting of PAR3, PAR6, and aPKC proteins (Joberty et al., 2000; Lin et al., 2000), the Crumbs complex consisting of CRUMBS, PALS1, and PATJ (Bhat et al., 1999; Makarova et al., 2003; Roh et al., 2002b), and the Scribble complex consisting of Scribble, LGL, and DLG (Bilder et al., 2000). However, this view became more complex over the years as many interactions between proteins of different complexes can occur (Assémat et al., 2008; Hurd et al., 2003; Lemmers et al., 2004), and interactions of polarity proteins with cytoskeleton regulators and lateral junction proteins are common (Assémat et al., 2008; Chen and Macara, 2005; Itoh et al., 2001; Médina et al., 2002; Michel et al., 2005; Roh et al., 2002a; Takekuni et al., 2003; Tan et al., 2020). A current limitation in the understanding of polarization is that there is no clear hierarchy of the importance of these numerous interactions. Potential interactions revealed through biochemical assays do not necessarily reflect relevant interactions in cells and do not specify when nor where in the cell these interactions could be relevant.

Polarity proteins have been localized with classical light microscopy, and remarkably, they are often found concentrated at the apical junction, a key organizational landmark of epithelial cells. To understand how polarity proteins cooperate to orchestrate cell polarization, one needs to understand how precisely polarity proteins organize with respect to apical junctions or the cytoskeleton. However, except for a few limited cases (Hirose et al., 2002; Izumi et al., 1998; Tan et al., 2020), the precise localization of polarity proteins at these organizational landmarks is missing. Moreover, knowing how polarity proteins organize in relation to each other in the cell should enable us to decipher from their plentiful known potential interactions, which ones are more relevant in specific subregions of the cell.

To tackle these challenges, we decided to systematically localize with stimulated‐emission‐depletion (STED) microscopy the polarity proteins that are key to the establishment of the apical pole of epithelia: PAR3, aPKC, PAR6β, PATJ, PALS1, and CRB3A. These proteins localize at the apical junction region of epithelial cells. Because how proteins interact and localize is likely to depend on cell differentiation, we decided to focus here on mature epithelia, a state where we hypothesize that protein interactions and localization are stationary. Using human and murine intestine and Caco-2 cells, we first imaged endogenous polarity proteins with respect to the TJ to appreciate their overall organization in the region. Second, we localized these proteins two-by-two to uncover relevant apical polarity protein subcellular associations. Finally, we focused on the organization of polarity proteins with respect to the actin cytoskeleton. We find that polarity proteins localize in distinct subregions that do not reflect the canonical definition of polarity protein complexes. In addition, their localization with respect to the cytoskeleton emphasizes some emerging roles of polarity proteins as regulators of actin organization.

In this study, we have systematically localized polarity proteins with super-resolution microscopy in epithelial cells. We observed endogenous PAR3, aPKC, PAR6β, PATJ, PALS1, and CRB3A in the human intestine and Caco-2 cells, and PAR3 and PALS1 in the mouse intestine. We found the following (Figure 7): (1) all these polarity proteins organize as submicrometric clusters concentrated in the TJ region. PAR3 localizes at the TJ, aPKC, and PAR6β localize at the TJ level, but mostly apically of the TJ, while PATJ, PALS1, and CRB3A are apical of the TJ (Figures 1 and 2). (2) PAR6β-aPKC and PATJ-PALS1 form two pairs that are often respectively found in the same clusters (Figure 4A), strongly indicating that these respective proteins form a stable and major complex in this region of the cells (i.e., the PAR6-aPKC complex and the PALS1-PATJ complex). (3) Unexpectedly, PALS1-PATJ and PAR6β-aPKC clusters are segregated from each other (Figure 4A). Our data show that these clusters are concentrated in the first one or two rows of microvilli, with the PAR6β-aPKC complex localized at the base and within microvilli, whereas the PALS1-PATJ complex is localized at the base and in between these microvilli (Figures 5 and 6), further showing that the PAR6β-aPKC complex and the PALS1-PATJ complex are spatially separated in the cell. (4) CRB3 shows little association with any of the other polarity proteins (Figure 4B), questioning how PALS1-PATJ and PAR6β-aPKC are mechanistically recruited to the plasma membrane and localized to the apical surface.

Figure 7

Download asset Open asset

Previous studies were largely based on biochemical approaches. The first interactions found defined canonical polarity protein complexes, while subsequent studies highlighted the numerous potential interactions that can be found with such approaches, between polarity proteins of different complexes (Assémat et al., 2008; Bhat et al., 1999; Hurd et al., 2003; Joberty et al., 2000; Lemmers et al., 2004; Lin et al., 2000; Makarova et al., 2003; Roh et al., 2002b), as well as between polarity proteins and other interactors (Chen and Macara, 2005; Itoh et al., 2001; Médina et al., 2002; Michel et al., 2005; Roh et al., 2002a; Takekuni et al., 2003; Tan et al., 2020). Altogether, these studies provide a complex potential model of molecular interactions. However, in most cases, we do not know to what extent and where these interactions do occur in cells and whether they are transient or permanent. Notably, most of these previous studies used overexpression to identify the interactors of a given protein; this methodological limitation might have introduced biases in some cases. If we focus only on proteins observed in this article, these studies concluded that PAR6β can interact with PAR3, aPKC, PALS1, PATJ, and CRB3A; aPKC can additionally interact with PAR3, and PALS1 can interact with CRB3A and PATJ. Our results show that of these eight potential interactions, only two, namely, PAR6β-aPKC and PALS1-PATJ, are likely to be stable or spatially restricted, clarifying the current view on how polarity proteins interact in the apical junction area of the cell. We question the existence of the canonical Crumbs and PAR complexes as previously described and propose that only PAR6β-aPKC and PALS1-PATJ can be defined as major structural complexes. The other numerous possible interactions that have been claimed previously may exist transiently and our approach cannot rule out that they occur at other locations in the cell, but it questions their relevance to the understanding of the epithelial cell junctions.

The interaction between PAR3, PAR6, and aPKC is key to epithelial polarization (Horikoshi et al., 2009; Joberty et al., 2000) but the permanence of these interactions has been discussed in the past. In mammalian epithelial cells, PAR3, PAR6, and aPKC have been thought to interact at apical junctions as these proteins concentrate there, but only PAR6 and aPKC are found at the apical surface (Martin-Belmonte et al., 2007; Satohisa et al., 2005). Moreover, in a few non-mammalian systems, PAR3 was observed as segregated from PAR6 and aPKC at epithelial apical junctions: when observed with confocal microscopy, PAR3 is basal of PAR6 and aPKC in the apical junctions of Drosophila melanogaster embryos during cellularization (Harris and Peifer, 2005), as well as in chick neuroepithelial cells (Afonso and Henrique, 2006). Our data suggest that the segregation of PAR3 from PAR6-aPKC is likely to be a conserved principle of organization in polarized epithelia. Even if the interaction of PAR3 with PAR6-aPKC is central to polarization, it is likely to be transient. The mechanistic basis for the transient character of the interaction between PAR3 and PAR6-aPKC in mammalian epithelia may be similar to the Cdc-42-dependent mechanisms found in D. melanogaster (Morais-de-Sá et al., 2010) or C. elegans (Rodriguez et al., 2017; Wang et al., 2017). Interestingly, studies in C. elegans also found that polarity proteins can organize in clusters (Dickinson et al., 2017; Wang et al., 2017). Although this was observed in very different conditions, this points to a potentially conserved mechanism on how polarity proteins organize.

Our finding that PAR3 localizes at the TJ confirms previous observations using electron microscopy in rat small intestine (Izumi et al., 1998) and MDCK cells. One recent study found a small fraction of PAR3 at the level of the adherens junction (Tan et al., 2020). Even though STED allows for a much larger volume to be probed compared to electron microscopy, we did not observe PAR3 basal of the TJ. The localization of PAR3 may depend on the cell type as well as its maturation state, but interestingly PAR3 is never found in the region apical of the TJ, where we find the other polarity proteins.

Because CRB3 is a transmembrane protein and several studies reported its interaction with PALS1, it was thought to anchor PALS1 and PATJ to the apical membrane (Makarova et al., 2003; Roh et al., 2002b). Similarly, it is suggested in D. melanogaster that Crb recruits Par-6 and aPKC to the apical membrane (Morais-de-Sá et al., 2010). Our study suggests that the recruitment of PALS1, PATJ, PAR6β, and aPKC to the plasma membrane is unlikely to be due to CRB3A because CRB3A poorly colocalizes with these proteins. Nevertheless, our data suggest that PALS1-PATJ are localized at the plasma membrane, perhaps confined in this area by another set of interactors to be uncovered. This last observation is likely to be similar for PAR6-aPKC. We cannot rule out both for PALS1-PATJ and PAR6β-aPKC that the interaction with CRB3A could be transient, and that this transient interaction would be sufficient to localize these protein complexes in the apical surface area.

The importance of polarity proteins for the epithelial organization points to the fact that these proteins are likely to play a key role in the organization of the cytoskeleton. Several proteins having a role in actin regulation have been shown to interact with polarity proteins (Bazellières et al., 2018; Médina et al., 2002), but how polarity proteins could influence actin organization is largely unknown. The correlation of organization between the actin cytoskeleton and PAR6-aPKC and PALS1-PATJ clusters points to a potential structural or instructional role of these proteins in the cytoskeleton organization. These findings call for further investigations, including functional and structural approaches.

We have shown that our observations were not the consequence of methodological artifacts, as live imaging on PAR6β-Citrine CRISPR-Cas9 knock-in and immunolabelling gave very similar results on the same cells, and permeabilization with or without detergent allowed us to observe the same organization of PATJ (Figure 3). Nevertheless, because of the size of antibodies, the combination of primary and secondary antibodies displaces the fluorescent signal from the epitope it targets by about 25 nm. Can this displacement alter our conclusions regarding protein organization? The fluorescent densities observed with STED are very likely to be the result of the clustering of tens of fluorophores, therefore tens of antibodies recognizing tens of polarity proteins. Because these densities are created by tens of antibodies, we speculate that these antibodies are oriented isotropically in space. As a result, the use of antibodies might slightly increase the size of polarity protein clusters, but the fluorescence created by antibodies in images is well centered on polarity protein clusters, making antibodies a tool of choice for this study.

Are clusters of polarity proteins smaller than the diffraction limit? It is often not the case, as many fluorescent densities can be as large as twice the diffraction limit and often display complex shapes, indicating clusters of polarity proteins larger than 80 nm. It would be very valuable to understand how these clusters are organized at a smaller scale, and how other proteins are implicated, including cytoskeletal proteins. Moreover, single-molecule approaches used in Dickinson et al., 2017 could help to further identify the nature of polarity protein clusters and their stoichiometry.

In this study, we define endogenous polarity protein organization and how polarity proteins are likely to interact. The early concept of polarity protein complexes introduced by biochemical studies is impractical today because of the very large number of potential interactions between proteins discovered. Additionally, it omits important features, such as the dynamics of interaction and their reality in relation to cell subregions. Our study proposes a snapshot of the polarity organization in mature intestinal epithelial cells that calls for a novel, more dynamic definition of interactions between polarity proteins and associated proteins that will be needed to uncover the mechanistic basis of cell apico-basal polarization.

All data generated or analyzed during this study are included in the manuscript and supporting files.

1. 1. Afonso C
   2. Henrique D

   (2006) Par3 acts as a molecular organizer to define the apical domain of chick neuroepithelial cells

   Journal of Cell Science 119:4293–4304.

   https://doi.org/10.1242/jcs.03170

   • PubMed
   • Google Scholar
2. 1. Alarcon VB

   (2010) Cell polarity regulator PARD6B is essential for trophectoderm formation in the preimplantation mouse embryo

   Biology of Reproduction 83:347–358.

   https://doi.org/10.1095/biolreprod.110.084400

   • PubMed
   • Google Scholar
3. 1. Assémat E
   2. Bazellières E
   3. Pallesi-Pocachard E
   4. Le Bivic A
   5. Massey-Harroche D

   (2008) Polarity complex proteins

   Biochimica et Biophysica Acta 1778:614–630.

   https://doi.org/10.1016/j.bbamem.2007.08.029

   • PubMed
   • Google Scholar
4. 1. Bazellières E
   2. Aksenova V
   3. Barthélémy-Requin M
   4. Massey-Harroche D
   5. Le Bivic A

   (2018) Role of the Crumbs proteins in ciliogenesis, cell migration and actin organization

   Seminars in Cell & Developmental Biology 81:13–20.

   https://doi.org/10.1016/j.semcdb.2017.10.018

   • PubMed
   • Google Scholar
5. 1. Belahbib H
   2. Renard E
   3. Santini S
   4. Jourda C
   5. Claverie JM
   6. Borchiellini C
   7. Le Bivic A

   (2018) New genomic data and analyses challenge the traditional vision of animal epithelium evolution

   BMC Genomics 19:393.

   https://doi.org/10.1186/s12864-018-4715-9

   • PubMed
   • Google Scholar
6. 1. Bhat MA
   2. Izaddoost S
   3. Lu Y
   4. Cho KO
   5. Choi KW
   6. Bellen HJ

   (1999) Discs lost, a novel multi-PDZ domain protein

   Establishes and Maintains Epithelial Polarity. Cell 96:833–845.

   https://doi.org/10.1016/S0092-8674(00)80593-0

   • PubMed
   • Google Scholar
7. 1. Bilder D
   2. Li M
   3. Perrimon N

   (2000) Cooperative regulation of cell polarity and growth by Drosophila tumor suppressors

   Science 289:113–116.

   https://doi.org/10.1126/science.289.5476.113

   • PubMed
   • Google Scholar
8. 1. Chantret I
   2. Rodolosse A
   3. Barbat A
   4. Dussaulx E
   5. Brot-Laroche E
   6. Zweibaum A
   7. Rousset M

   (1994) Differential expression of sucrase-isomaltase in clones isolated from early and late passages of the cell line caco-2: evidence for glucose-dependent negative regulation

   Journal of Cell Science 107:213–225.

   https://doi.org/10.1242/jcs.107.1.213

   • PubMed
   • Google Scholar
9. 1. Charrier LE
   2. Loie E
   3. Laprise P

   (2015) Mouse crumbs3 sustains epithelial tissue morphogenesis in vivo

   Scientific Reports 5:1–16.

   https://doi.org/10.1038/srep17699

   • PubMed
   • Google Scholar
10. 1. Chen X
    2. Macara IG

    (2005) Par-3 controls tight junction assembly through the Rac exchange factor Tiam1

    Nature Cell Biology 7:262–269.

    https://doi.org/10.1038/ncb1226

    • PubMed
    • Google Scholar
11. 1. Dickinson DJ
    2. Schwager F
    3. Pintard L
    4. Gotta M
    5. Goldstein B

    (2017) A single-cell biochemistry approach reveals PAR complex dynamics during cell polarization

    Developmental Cell 42:416–434.

    https://doi.org/10.1016/j.devcel.2017.07.024

    • PubMed
    • Google Scholar
12. 1. Farquhar MG
    2. Palade GE

    (1963) Junctional complexes in various epithelia

    The Journal of Cell Biology 17:375–412.

    https://doi.org/10.1083/jcb.17.2.375

    • PubMed
    • Google Scholar
13. 1. Hakanen J
    2. Ruiz-Reig N
    3. Tissir F

    (2019) Linking cell polarity to cortical development and malformations

    Frontiers in Cellular Neuroscience 13:244.

    https://doi.org/10.3389/fncel.2019.00244

    • PubMed
    • Google Scholar
14. 1. Harris TJC
    2. Peifer M

    (2005) The positioning and segregation of apical cues during epithelial polarity establishment in Drosophila

    The Journal of Cell Biology 170:813–823.

    https://doi.org/10.1083/jcb.200505127

    • PubMed
    • Google Scholar
15. 1. Hayase J
    2. Kamakura S
    3. Iwakiri Y
    4. Yamaguchi Y
    5. Izaki T
    6. Ito T
    7. Sumimoto H

    (2013) The WD40 protein Morg1 facilitates par6-apkc binding to CRB3 for apical identity in epithelial cells

    The Journal of Cell Biology 200:635–650.

    https://doi.org/10.1083/jcb.201208150

    • PubMed
    • Google Scholar
16. 1. Hell SW
    2. Wichmann J

    (1994) Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy

    Opt Lett 19:780–782.

    https://doi.org/10.1364/ol.19.000780

    • PubMed
    • Google Scholar
17. 1. Hirose T
    2. Izumi Y
    3. Nagashima Y
    4. Tamai-Nagai Y
    5. Kurihara H
    6. Sakai T
    7. Suzuki Y
    8. Yamanaka T
    9. Suzuki A
    10. Mizuno K
    11. Ohno S

    (2002) Involvement of ASIP/PAR-3 in the promotion of epithelial tight junction formation

    Journal of Cell Science 115:2485–2495.

    https://doi.org/10.1242/jcs.115.12.2485

    • PubMed
    • Google Scholar
18. 1. Horikoshi Y
    2. Suzuki A
    3. Yamanaka T
    4. Sasaki K
    5. Mizuno K
    6. Sawada H
    7. Yonemura S
    8. Ohno S

    (2009) Interaction between Par-3 and the apkc-PAR-6 complex is indispensable for apical domain development of epithelial cells

    Journal of Cell Science 122:1595–1606.

    https://doi.org/10.1242/jcs.043174

    • PubMed
    • Google Scholar
19. 1. Hurd TW
    2. Gao L
    3. Roh MH
    4. Macara IG
    5. Margolis B

    (2003) Direct interaction of two polarity complexes implicated in epithelial tight junction assembly

    Nature Cell Biology 5:137–142.

    https://doi.org/10.1038/ncb923

    • PubMed
    • Google Scholar
20. 1. Itoh M
    2. Sasaki H
    3. Furuse M
    4. Ozaki H
    5. Kita T
    6. Tsukita S

    (2001) Junctional adhesion molecule (JAM) binds to Par-3: a possible mechanism for the recruitment of Par-3 to tight junctions

    The Journal of Cell Biology 154:491–497.

    https://doi.org/10.1083/jcb.200103047

    • PubMed
    • Google Scholar
21. 1. Izumi Y
    2. Hirose T
    3. Tamai Y
    4. Hirai S
    5. Nagashima Y
    6. Fujimoto T
    7. Tabuse Y
    8. Kemphues KJ
    9. Ohno S

    (1998) An atypical PKC directly associates and colocalizes at the epithelial tight junction with ASIP, a mammalian homologue of Caenorhabditis elegans polarity protein PAR-3

    The Journal of Cell Biology 143:95–106.

    https://doi.org/10.1083/jcb.143.1.95

    • PubMed
    • Google Scholar
22. 1. Joberty G
    2. Petersen C
    3. Gao L
    4. Macara IG

    (2000) The cell-polarity protein Par6 links Par3 and atypical protein kinase C to Cdc42

    Nature Cell Biology 2:531–539.

    https://doi.org/10.1038/35019573

    • PubMed
    • Google Scholar
23. 1. Kantardzhieva A
    2. Gosens I
    3. Alexeeva S
    4. Punte IM
    5. Versteeg I
    6. Krieger E
    7. Neefjes-Mol CA
    8. den Hollander AI
    9. Letteboer SJF
    10. Klooster J
    11. Cremers FPM
    12. Roepman R
    13. Wijnholds J

    (2005) MPP5 recruits MPP4 to the CRB1 complex in photoreceptors

    Investigative Ophthalmology & Visual Science 46:2192–2201.

    https://doi.org/10.1167/iovs.04-1417

    • PubMed
    • Google Scholar
24. 1. Lalli G

    (2012) Crucial polarity regulators in axon specification

    Essays in Biochemistry 53:55–68.

    https://doi.org/10.1042/bse0530055

    • PubMed
    • Google Scholar
25. 1. Le Bivic A

    (2013) Evolution and cell physiology. 4. why invent yet another protein complex to build junctions in epithelial cells?

    American Journal of Physiology. Cell Physiology 305:C1193–C1201.

    https://doi.org/10.1152/ajpcell.00272.2013

    • PubMed
    • Google Scholar
26. 1. Lemmers C
    2. Michel D
    3. Lane-Guermonprez L
    4. Delgrossi MH
    5. Médina E
    6. Arsanto JP
    7. Le Bivic A

    (2004) Crb3 binds directly to Par6 and regulates the morphogenesis of the tight junctions in mammalian epithelial cells

    Molecular Biology of the Cell 15:1324–1333.

    https://doi.org/10.1091/mbc.e03-04-0235

    • PubMed
    • Google Scholar
27. 1. Li Y
    2. Wei Z
    3. Yan Y
    4. Wan Q
    5. Du Q
    6. Zhang M

    (2014) Structure of crumbs tail in complex with the pals1 PDZ-SH3-GK tandem reveals a highly specific assembly mechanism for the apical crumbs complex

    PNAS 111:17444–17449.

    https://doi.org/10.1073/pnas.1416515111

    • PubMed
    • Google Scholar
28. 1. Lin D
    2. Edwards AS
    3. Fawcett JP
    4. Mbamalu G
    5. Scott JD
    6. Pawson T

    (2000) A mammalian PAR-3-PAR-6 complex implicated in Cdc42/Rac1 and aPKC signalling and cell polarity

    Nature Cell Biology 2:540–547.

    https://doi.org/10.1038/35019582

    • PubMed
    • Google Scholar
29. 1. Makarova O
    2. Roh MH
    3. Liu CJ
    4. Laurinec S
    5. Margolis B

    (2003) Mammalian crumbs3 is a small transmembrane protein linked to protein associated with lin-7 (Pals1)

    Gene 302:21–29.

    https://doi.org/10.1016/s0378111902010843

    • PubMed
    • Google Scholar
30. 1. Martin-Belmonte F
    2. Gassama A
    3. Datta A
    4. Yu W
    5. Rescher U
    6. Gerke V
    7. Mostov K

    (2007) Pten-Mediated apical segregation of phosphoinositides controls epithelial morphogenesis through Cdc42

    Cell 128:383–397.

    https://doi.org/10.1016/j.cell.2006.11.051

    • PubMed
    • Google Scholar
31. 1. Massey-Harroche D
    2. Delgrossi MH
    3. Lane-Guermonprez L
    4. Arsanto JP
    5. Borg JP
    6. Billaud M
    7. Le Bivic A

    (2007) Evidence for a molecular link between the tuberous sclerosis complex and the Crumbs complex

    Human Molecular Genetics 16:529–536.

    https://doi.org/10.1093/hmg/ddl485

    • PubMed
    • Google Scholar
32. 1. Médina E
    2. Williams J
    3. Klipfell E
    4. Zarnescu D
    5. Thomas CM
    6. Le Bivic A

    (2002) Crumbs interacts with moesin and beta (heavy) -spectrin in the apical membrane skeleton of Drosophila

    The Journal of Cell Biology 158:941–951.

    https://doi.org/10.1083/jcb.200203080

    • PubMed
    • Google Scholar
33. 1. Michel D
    2. Arsanto J-P
    3. Massey-Harroche D
    4. Béclin C
    5. Wijnholds J
    6. Le Bivic A

    (2005) Patj connects and stabilizes apical and lateral components of tight junctions in human intestinal cells

    Journal of Cell Science 118:4049–4057.

    https://doi.org/10.1242/jcs.02528

    • PubMed
    • Google Scholar
34. 1. Morais-de-Sá E
    2. Mirouse V
    3. St Johnston D

    (2010) Apkc phosphorylation of bazooka defines the apical/lateral border in Drosophila epithelial cells

    Cell 141:509–523.

    https://doi.org/10.1016/j.cell.2010.02.040

    • PubMed
    • Google Scholar
35. 1. Park B
    2. Alves CH
    3. Lundvig DM
    4. Tanimoto N
    5. Beck SC
    6. Huber G
    7. Richard F
    8. Klooster J
    9. Andlauer TFM
    10. Swindell EC
    11. Jamrich M
    12. Le Bivic A
    13. Seeliger MW
    14. Wijnholds J

    (2011) Pals1 is essential for retinal pigment epithelium structure and neural retina stratification

    The Journal of Neuroscience 31:17230–17241.

    https://doi.org/10.1523/JNEUROSCI.4430-11.2011

    • PubMed
    • Google Scholar
36. 1. Pickett MA
    2. Naturale VF
    3. Feldman JL

    (2019) A polarizing issue: diversity in the mechanisms underlying apico-basolateral polarization in vivo

    Annual Review of Cell and Developmental Biology 35:285–308.

    https://doi.org/10.1146/annurev-cellbio-100818-125134

    • PubMed
    • Google Scholar
37. 1. Pinto M
    2. Robine-Leon S
    3. Appay MD
    4. Kedinger M
    5. Triadou N
    6. Dussaulx E
    7. Lacroix B
    8. Simon-Assmann P
    9. Haffen K
    10. Fogh J
    11. Zweibaum A

    (1983)

    Enterocyte-like differentiation and polarization of the human colon carcinoma cell line caco-2 in culture

    Biology of the Cell 47:323–330.

    • Google Scholar
38. 1. Rodriguez J
    2. Peglion F
    3. Martin J
    4. Hubatsch L
    5. Reich J
    6. Hirani N
    7. Gubieda AG
    8. Roffey J
    9. Fernandes AR
    10. St Johnston D
    11. Ahringer J
    12. Goehring NW

    (2017) Apkc cycles between functionally distinct PAR protein assemblies to drive cell polarity

    Developmental Cell 42:400–415.

    https://doi.org/10.1016/j.devcel.2017.07.007

    • PubMed
    • Google Scholar
39. 1. Rodriguez-Boulan E
    2. Macara IG

    (2014) Organization and execution of the epithelial polarity programme

    Nature Reviews. Molecular Cell Biology 15:225–242.

    https://doi.org/10.1038/nrm3775

    • PubMed
    • Google Scholar
40. 1. Roh MH
    2. Liu CJ
    3. Laurinec S
    4. Margolis B

    (2002a) The carboxyl terminus of zona occludens-3 binds and recruits a mammalian homologue of discs lost to tight junctions

    The Journal of Biological Chemistry 277:27501–27509.

    https://doi.org/10.1074/jbc.M201177200

    • PubMed
    • Google Scholar
41. 1. Roh MH
    2. Makarova O
    3. Liu CJ
    4. Shin K
    5. Lee S
    6. Laurinec S
    7. Goyal M
    8. Wiggins R
    9. Margolis B

    (2002b) The maguk protein, pals1, functions as an adapter, linking mammalian homologues of crumbs and discs lost

    The Journal of Cell Biology 157:161–172.

    https://doi.org/10.1083/jcb.200109010

    • PubMed
    • Google Scholar
42. 1. Sabherwal N
    2. Papalopulu N

    (2012) Apicobasal polarity and cell proliferation during development

    Essays in Biochemistry 53:95–109.

    https://doi.org/10.1042/bse0530095

    • PubMed
    • Google Scholar
43. 1. Sandoz J
    2. Nagy Z
    3. Catez P
    4. Caliskan G
    5. Geny S
    6. Renaud JB
    7. Concordet JP
    8. Poterszman A
    9. Tora L
    10. Egly JM
    11. Le May N
    12. Coin F

    (2019) Functional interplay between TFIIH and KAT2A regulates higher-order chromatin structure and class II gene expression

    Nature Communications 10:1288.

    https://doi.org/10.1038/s41467-019-09270-2

    • Google Scholar
44. 1. Satohisa S
    2. Chiba H
    3. Osanai M
    4. Ohno S
    5. Kojima T
    6. Saito T
    7. Sawada N

    (2005) Behavior of tight-junction, adherens-junction and cell polarity proteins during HNF-4alpha-induced epithelial polarization

    Experimental Cell Research 310:66–78.

    https://doi.org/10.1016/j.yexcr.2005.06.025

    • PubMed
    • Google Scholar
45. 1. Schnell U
    2. Dijk F
    3. Sjollema KA
    4. Giepmans BNG

    (2012) Immunolabeling artifacts and the need for live-cell imaging

    Nature Methods 9:152–158.

    https://doi.org/10.1038/nmeth.1855

    • PubMed
    • Google Scholar
46. 1. Stadler C
    2. Rexhepaj E
    3. Singan VR
    4. Murphy RF
    5. Pepperkok R
    6. Uhlén M
    7. Simpson JC
    8. Lundberg E

    (2013) Immunofluorescence and fluorescent-protein tagging show high correlation for protein localization in mammalian cells

    Nature Methods 10:315–323.

    https://doi.org/10.1038/nmeth.2377

    • PubMed
    • Google Scholar
47. 1. Steponkus PL
    2. Lynch DV

    (1989) Freeze/thaw-induced destabilization of the plasma membrane and the effects of cold acclimation

    Journal of Bioenergetics and Biomembranes 21:21–41.

    https://doi.org/10.1007/BF00762210

    • PubMed
    • Google Scholar
48. 1. Tait CM
    2. Chinnaiya K
    3. Manning E
    4. Murtaza M
    5. Ashton JP
    6. Furley N
    7. Hill CJ
    8. Alves CH
    9. Wijnholds J
    10. Erdmann KS
    11. Furley A
    12. Rashbass P
    13. Das RM
    14. Storey KG
    15. Placzek M

    (2020) Crumbs2 mediates ventricular layer remodelling to form the spinal cord central canal

    PLOS Biology 18:e3000470.

    https://doi.org/10.1371/journal.pbio.3000470

    • PubMed
    • Google Scholar
49. 1. Takekuni K
    2. Ikeda W
    3. Fujito T
    4. Morimoto K
    5. Takeuchi M
    6. Monden M
    7. Takai Y

    (2003) Direct binding of cell polarity protein PAR-3 to cell-cell adhesion molecule nectin at neuroepithelial cells of developing mouse

    The Journal of Biological Chemistry 278:5497–5500.

    https://doi.org/10.1074/jbc.C200707200

    • PubMed
    • Google Scholar
50. 1. Tan B
    2. Yatim SMJM
    3. Peng S
    4. Gunaratne J
    5. Hunziker W
    6. Ludwig A

    (2020) The mammalian Crumbs complex defines a distinct polarity domain apical of epithelial tight junctions

    Current Biology 30:2791–2804.

    https://doi.org/10.1016/j.cub.2020.05.032

    • PubMed
    • Google Scholar
51. 1. Vacca B
    2. Bazellières E
    3. Nouar R
    4. Harada A
    5. Massey-Harroche D
    6. Le Bivic A

    (2014) Drebrin E depletion in human intestinal epithelial cells mimics Rab8a loss of function

    Human Molecular Genetics 23:2834–2846.

    https://doi.org/10.1093/hmg/ddt670

    • PubMed
    • Google Scholar
52. 1. Wang SC
    2. Low TYF
    3. Nishimura Y
    4. Gole L
    5. Yu W
    6. Motegi F

    (2017) Cortical forces and CDC-42 control clustering of PAR proteins for Caenorhabditis elegans embryonic polarization

    Nature Cell Biology 19:988–995.

    https://doi.org/10.1038/ncb3577

    • PubMed
    • Google Scholar
53. 1. Whiteman EL
    2. Fan S
    3. Harder JL
    4. Walton KD
    5. Liu CJ
    6. Soofi A
    7. Fogg VC
    8. Hershenson MB
    9. Dressler GR
    10. Deutsch GH
    11. Gumucio DL
    12. Margolis B

    (2014) Crumbs3 is essential for proper epithelial development and viability

    Molecular and Cellular Biology 34:43–56.

    https://doi.org/10.1128/MCB.00999-13

    • PubMed
    • Google Scholar
54. 1. Wu Y
    2. Eghbali M
    3. Ou J
    4. Lu R
    5. Toro L
    6. Stefani E

    (2010) Quantitative determination of spatial protein-protein correlations in fluorescence confocal microscopy

    Biophysical Journal 98:493–504.

    https://doi.org/10.1016/j.bpj.2009.10.037

    • PubMed
    • Google Scholar

Author details

1. Pierre Mangeol

   Aix-Marseille University, CNRS, UMR7288, Developmental Biology Institute of Marseille (IBDM), Marseille, France

   Contribution

   Conceptualization, Resources, Data curation, Software, Formal analysis, Funding acquisition, Investigation, Visualization, Methodology, Writing - original draft, Writing – review and editing

   For correspondence

   pierre.mangeol@univ-amu.fr

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8305-7322

2. Dominique Massey-Harroche

   Aix-Marseille University, CNRS, UMR7288, Developmental Biology Institute of Marseille (IBDM), Marseille, France

   Contribution

   Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

3. Fabrice Richard

   Aix-Marseille University, CNRS, UMR7288, Developmental Biology Institute of Marseille (IBDM), Marseille, France

   Contribution

   Methodology

   Competing interests

   No competing interests declared

4. Jean-Paul Concordet

   Laboratoire Structure et Instabilité des Génomes, Muséum National d’Histoire Naturelle (MNHN), Institut National de la Santé et de la Recherche Médicale (INSERM), U1154, Centre National de la Recherche Scientifique (CNRS), Paris, France

   Contribution

   Methodology

   Competing interests

   No competing interests declared

5. Pierre-François Lenne

   Aix-Marseille University, CNRS, UMR7288, Developmental Biology Institute of Marseille (IBDM), Marseille, France

   Contribution

   Conceptualization, Supervision, Funding acquisition, Validation, Methodology, Writing – review and editing

   Contributed equally with

   André Le Bivic

   For correspondence

   pierre-francois.lenne@univ-amu.fr

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1066-7506

6. André Le Bivic

   Aix-Marseille University, CNRS, UMR7288, Developmental Biology Institute of Marseille (IBDM), Marseille, France

   Contribution

   Conceptualization, Supervision, Funding acquisition, Validation, Methodology, Writing – review and editing

   Contributed equally with

   Pierre-François Lenne

   For correspondence

   andre.le-bivic@univ-amu.fr

   Competing interests

   No competing interests declared

• 1,709

  views

• 337

  downloads

• 12

  citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.