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

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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.

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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

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