Super-resolution imaging uncovers the nanoscopic segregation of polarity proteins in epithelia

  1. Pierre Mangeol
  2. Dominique Massey-Harroche
  3. Fabrice Richard
  4. Jean-Paul Concordet
  5. Pierre-François Lenne
  6. André Le Bivic

Reviewed by

Read the full article See related articles

Discuss this preprint

Start a discussion What are Sciety discussions?

Listed in

Log in to save this article

Abstract

Epithelial tissues acquire their integrity and function through the apico-basal polarization of their constituent cells. Proteins of the PAR and Crumbs complexes are pivotal to epithelial polarization, but the mechanistic understanding of polarization is challenging to reach, largely because numerous potential interactions between these proteins and others have been found, without a clear hierarchy in importance. We identify the regionalized and segregated organization of members of the PAR and Crumbs complexes at epithelial apical junctions by imaging endogenous proteins using stimulated‐emission‐depletion microscopy on Caco-2 cells, and human and murine intestinal samples. Proteins organize in submicrometric clusters, with PAR3 overlapping with the tight junction (TJ) while PALS1-PATJ and aPKC-PAR6β form segregated clusters that are apical of the TJ and present in an alternated pattern related to actin organization. CRB3A is also apical of the TJ and partially overlaps with other polarity proteins. Of the numerous potential interactions identified between polarity proteins, only PALS1-PATJ and aPKC-PAR6β are spatially relevant in the junctional area of mature epithelial cells, simplifying our view of how polarity proteins could cooperate to drive and maintain cell polarity.

Article activity feed

  1. Version published to 10.7554/elife.62087 on eLife
  2. eLife

    ###Reviewer #3:

    The study by Mangeol et al. aims to dissect the localisations, interactions and hierarchical order of apical protein complexes crucial to the generation and maintenance of epithelial polarity in epithelial tissues.

    They analyse by super-resolution microscopy (STORM) three different mature epithelia, human and mouse intestine as well as mature Caco-2 cells in culture. Using immunofluorescence labeling of endogenous proteins, they compare individual components to markers of tight junctions, to each other and to the actin cytoskeleton. They identify defined clusters in defined sub regions of the apical domain of the analysed cells, raising interesting questions for future analyses.

    The subject matter of the study, the generation and maintenance of epithelial polarity and the role of apical polarity complexes, is clearly a very important one, especially as most organ systems are epithelial in nature. And despite decades of study, many questions are still unresolved.

    The imaging performed in this study is skilful and beautifully presented. The imaging achieving, according to the authors, an isotropic resolution of about 80nm is impressive. Because of this great gain in resolution compared to other studies of similar components I have a couple of technical questions or comments:

    1. I would very much appreciate some comments or thoughts on the fact that polarity proteins were revealed using antibodies. Antibodies are in the range of 10-15nm in length, so with an isotropic resolution of 80 nm, this might have to be taken into account when using primary and secondary antibodies to reveal proteins. In particular, monoclonal versus polyclonal antibodies might have differing effects on localisation precision.

    2. The authors use rather high concentrations of detergent (1% SDS or 1% Triton X-100) for permeabilisation according to their protocols. Are they not worried that this might affect tissue integrity and protein distribution?

    The authors rightly point out where their study fits within what has been attempted by other labs previously in order to understand and dissect apical polarity complex function. They clearly define interesting aspects, such as PALS1-PATJ and aPKC-PAR6 forming independent clusters, and the lack of colocalisation and thus maybe association with Crumbs3. In contrast to the last sentence statement of their abstract 'This organization at the nanoscale level significantly simplifies our view on how polarity proteins could cooperate to drive and maintain cell polarity.' I cannot yet see what these results simplify about our understanding of apical polarity complexes and even more so what the authors' new model is of how the complexes work. This needs to be spelt out more clearly, please. And I would also point out that, in part, other studies have pointed in the same direction. The recent paper by the Ludwig lab (Tan et al. 2020 Current Biology 30, 2791-2804) points in part in a similar direction, identifying a vertebrate 'marginal zone' similar to the one already known from invertebrate epithelia, as well as identifying basal to this an apical and basal tight junction area. Furthermore, as the authors themselves discuss in the discussion, the 'splitting away' of Par3 has been observed in Drosophila epithelia (embryonic, follicle cells and eye disc), and should maybe be introduced already at an earlier point of the paper. Furthermore, papers by Wang et al. and Dickinson et al., that also analyse PAR complex clustering should be cited and mentioned in the introduction/discussion (Wang, S.-C., Low, T. Y. F., Nishimura, Y., Gole, L., Yu, W., & Motegi, F. (2017). Cortical forces and CDC-42 control clustering of PAR proteins for Caenorhabditis elegans embryonic polarization. Nature Cell Biology, 19(8), 988-995. http://doi.org/10.1016/S0960-9822(99)80042-6; Dickinson, D. J., Schwager, F., Pintard, L., Gotta, M., & Goldstein, B. (2017). A Single-Cell Biochemistry Approach Reveals PAR Complex Dynamics during Cell Polarization, 1-42. http://doi.org/10.1016/j.devcel.2017.07.024).

    I am also a bit confused by the analysis presented in Figure 5 with regards to colocalisation of components with apical F-actin structures and the deduction from these and the EM data that some components, aPKC/Par6, localise to 'the first row of' microvilli near junctions whilst PALS1-PATJ localise near the base of said microvilli. How would localisation to the apical plasma membrane outside of or within microvilli be restricted to only the ones near junctions? There is not only F-actin in microvilli but also all over and near the apical cortex, so what distinguished the ability of aPKC/PAR6 to bind to actin in microvilli? The PATJ knock-down results are interesting, and I agree suggestive of some interaction between the complexes and actin organisation. But without further analyses as to what other components might be affected in their localisation in this situation, it is hard to judge whether the effect on actin is a direct or rather indirect one, so I am unsure as to what these images add without more in depth follow-up.

    Some more specific comments:

    Figure 1: It would be good to show and demonstrate that Occludin and ZO-1 labeling are completely interchangeable in terms of localisation precision.

    Figure 3: I do understand the authors' rationale for analysing the localisation in the orientation (planar versus apical-basal) that reveals the largest distance, but it would be good to nonetheless show the other orientation for completeness (maybe as supplementary).

  3. eLife

    ###Reviewer #2:

    The manuscript addresses a fundamental problem: the organisation of epithelial polarity determinants at the apical domain of human epithelial cells. The authors use STED microscopy to examine antibody-stained fixed Caco2 cells. My major concern is that the process of fixation and immunostaining may introduce artefacts that are causing the segregated dots to appear. This issue could be addressed by using CRISPR-knockin GFP versions of some of the proteins studied, which is technically straightforward to perform these days, and would allow the conclusions to be drawn with full confidence.

  4. eLife

    ###Reviewer #1:

    Mangeol et al investigate the nanoscale organization of apical-basal polarity complexes using super-resolution microscopy approaches (STED) in polarized intestinal epithelial cells, both in culture and from in vivo tissue samples. They provide a careful characterization of Par3-Par6-aPKC and Patj-Pals1-Crb3a localization relative to tight junctions in both planar and apical-basal axes. They find that each protein localizes in the near vicinity of the tight junction, in a clustered organization. Through pairwise colocalization analyses, they observe significant separation of polarity proteins that are generally considered to be part of the same molecular complex based on biochemical assays. Specifically, PAR3 is not associated with aPKC or PAR6, and CRB3a colocalizes poorly with all other polarity proteins.

    Overall, this paper provides a thorough description of polarity protein localization at the submicron scale. The data are presented in a clear and convincing manner and the conclusions are largely consistent with the data. The unexpected separation of polarity proteins suggests that some of the previously described biochemical interactions may be transient, warranting further investigation comparing different stages of polarization. These findings will be of interest to those in the field of cell polarity.

    Comments/concerns:

    1. All of the results depend on antibody quality, specificity, and antigenicity but no antibody validation provided (with the exception of PATJ). If one primary antibody is less specific than the others, the colocalization data will be heavily skewed, appearing not to be colocalized. Perhaps this can explain why Crb3a fails to colocalize with the other proteins? Validating the results with a second primary antibody or an endogenously tagged GFP-fusion protein would alleviate this concern.

    2. The authors show that CRB3a doesn't colocalize PALS or PATJ, suggesting another transmembrane protein recruits them to the membrane. Could this function be provided by another CRB family member or is CRB3a the only one expressed in intestinal epithelia?

    3. The super-resolution characterization of actin organization is not as extensive or convincing as the description of polarity protein localization. A closer examination of actin organization relative to PATJ and aPKC at junctional, apical, and villi positions would strengthen the findings in Figure 5.

    4. In some cases the number of biological replicates is small. Only one mouse sample was used, and the quantifications of junctions are performed across just 1 or 2 cell culture replicates (although more replicates were performed, just not used for quantification). Therefore, the data reflect the variability across junctions (violin plots in Figs 1-2) but they don't reflect the variability across biological replicates. This also means the p-value in Figure 5 was calculated using n=number of junctions rather than n=experimental replicates, which would be a more appropriate comparison of means. Quantifying the data across 3 biological replicates to show the variability across experiments would greatly strengthen the results and conclusions.

  5. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.

    This manuscript is in revision at eLife.

    ###Summary:

    The manuscript addresses a fundamental problem: the organisation of epithelial polarity determinants at the apical domain of human epithelial cells. Mangeol et al investigate this question using super-resolution microscopy approaches (STED) in polarised intestinal epithelial cells. Using immunofluorescence labeling of endogenous proteins, they provide a careful characterization of Par3-Par6-aPKC and Patj-Pals1-Crb3a localization relative to tight junctions. They find that each protein localizes in the near vicinity of the tight junction, in a clustered organization. Through pairwise colocalization analyses, they observe significant separation of polarity proteins that are generally considered to be part of the same molecular complex based on biochemical assays. Specifically, PAR3 is not associated with aPKC or PAR6, and CRB3a colocalizes poorly with all other polarity proteins, raising interesting questions for future analyses.

    The imaging performed in this study is skillful and beautifully presented and, achieving an isotropic resolution of about 80nm, is impressive. However, because of this great gain in resolution compared to other studies of similar components, the major concern of all three reviewers is that the process of fixation and immunostaining may introduce artefacts that are causing the segregated dots to appear. Variable antibody quality and insufficient validation of antibody specificity raise additional concerns about the observed patterns of localization.

  7. Version published to 10.1101/2020.08.12.248674 on bioRxiv