Centrosome Migration and Apical Membrane Formation in Polarized Epithelial Cells: Insights from the MDCK Cyst Model
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eLife Assessment
Wang et al's study addresses an important critical gap in our understanding of de novo epithelial polarization using MDCK cell doublets surrounded by ECM, providing convincing evidence through imaging and depletion studies on the role of conserved polarity proteins and the centrosome during this process. While the authors propose a clear hierarchical model, there is a need for further exploration of how microtubule organization contributes to this process. Specifically, live cell imaging of microtubules under mutants and their included ECM conditions, along with a more precise temporal mapping of microtubule dynamics in relation to proteins like Gp135, would strengthen the study's conclusions.
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Abstract
Polarization is crucial for the proper functioning of epithelial cells. Early polarization features include the trafficking and enrichment of polarity molecules to form the apical membrane (AM) or cell-cell junctions, as well as the apical positioning of the centrosome. However, the dependencies among polarity molecules, AM formation, and centrosome positioning remain poorly understood. In conventional Matrigel-cultured epithelial cells, de novo polarization can occur when a single cell divides. At the exit of mitosis, centrosomes move to the location where the apical membrane will form, raising the question of the role of the centrosome in epithelial polarization. We perturb centrosomes and polarity regulators in Matrigel-cultured cells and also manipulate polarity direction in non-conventional culture to examine the relationship between polarity features. Surprisingly, the centrosome is not essential for AM formation but promotes formation efficiency. The polarity regulator Par3, rather than the trafficking of AM components, affects centrosome positioning. In non-conventional cultures, the centrosome migration is opposite to that of the AM direction, and Par3 exhibits a different pattern from Matrigel culture. Taken together, our work shows that polarity indicated by centrosome position is not universal and elucidates the upstream-downstream relationship between centrosome positioning and other polarization features, providing insights into epithelial polarization.
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eLife Assessment
Wang et al's study addresses an important critical gap in our understanding of de novo epithelial polarization using MDCK cell doublets surrounded by ECM, providing convincing evidence through imaging and depletion studies on the role of conserved polarity proteins and the centrosome during this process. While the authors propose a clear hierarchical model, there is a need for further exploration of how microtubule organization contributes to this process. Specifically, live cell imaging of microtubules under mutants and their included ECM conditions, along with a more precise temporal mapping of microtubule dynamics in relation to proteins like Gp135, would strengthen the study's conclusions.
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Reviewer #1 (Public review):
Summary:
Wang, Po-Kai, et al., utilized the de novo polarization of MDCK cells cultured in Matrigel to assess the interdependence between polarity protein localization, centrosome positioning, and apical membrane formation. They show that the inhibition of Plk4 with Centrinone does not prevent apical membrane formation, but does result in its delay, a phenotype the authors attribute to the loss of centrosomes due to the inhibition of centriole duplication. However, the targeted mutagenesis of specific centrosome proteins implicated in the positioning of centrosomes in other cell types (CEP164, ODF2, PCNT, and CEP120) did not affect centrosome positioning in 3D cultured MDCK cells. A screen of proteins previously implicated in MDCK polarization revealed that the polarity protein Par-3 was upstream of …
Reviewer #1 (Public review):
Summary:
Wang, Po-Kai, et al., utilized the de novo polarization of MDCK cells cultured in Matrigel to assess the interdependence between polarity protein localization, centrosome positioning, and apical membrane formation. They show that the inhibition of Plk4 with Centrinone does not prevent apical membrane formation, but does result in its delay, a phenotype the authors attribute to the loss of centrosomes due to the inhibition of centriole duplication. However, the targeted mutagenesis of specific centrosome proteins implicated in the positioning of centrosomes in other cell types (CEP164, ODF2, PCNT, and CEP120) did not affect centrosome positioning in 3D cultured MDCK cells. A screen of proteins previously implicated in MDCK polarization revealed that the polarity protein Par-3 was upstream of centrosome positioning, similar to other cell types.
Strengths:
The investigation into the temporal requirement and interdependence of previously proposed regulators of cell polarization and lumen formation is valuable to the community. Wang et al., have provided a detailed analysis of many of these components at defined stages of polarity establishment. Furthermore, the generation of PCNT, p53, ODF2, Cep120, and Cep164 knockout MDCK cell lines is likely valuable to the community.
Weaknesses:
Additional quantifications would highly improve this manuscript, for example it is unclear whether the centrosome perturbation affects gamma tubulin levels and therefore microtubule nucleation, it is also not clear how they affect the localization of the trafficking machinery/polarity proteins. For example, in Figure 4, the authors measure the intensity of Gp134 at the apical membrane initiation site following cytokinesis, but there is no measure of Gp134 at the centrosome prior to this.
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Reviewer #2 (Public review):
Summary:
The authors decoupled several players that are thought to contribute to the establishment of epithelial polarity and determined their causal relationship. This provides a new picture of the respective roles of junctional proteins (Par3), the centrosome, and endomembrane compartments (Cdc42, Rab11, Gp135) from upstream to downstream.
Their conclusions are based on live imaging of all players during the early steps of polarity establishment and on the knock-down of their expression in the simplest ever model of epithelial polarity: a cell doublet surrounded by ECM.The position of the centrosome is often taken as a readout for the orientation of the cell polarity axis. There is a long-standing debate about the actual role of the centrosome in the establishment of this polarity axis. Here, using a …
Reviewer #2 (Public review):
Summary:
The authors decoupled several players that are thought to contribute to the establishment of epithelial polarity and determined their causal relationship. This provides a new picture of the respective roles of junctional proteins (Par3), the centrosome, and endomembrane compartments (Cdc42, Rab11, Gp135) from upstream to downstream.
Their conclusions are based on live imaging of all players during the early steps of polarity establishment and on the knock-down of their expression in the simplest ever model of epithelial polarity: a cell doublet surrounded by ECM.The position of the centrosome is often taken as a readout for the orientation of the cell polarity axis. There is a long-standing debate about the actual role of the centrosome in the establishment of this polarity axis. Here, using a minimal model of epithelial polarization, a doublet of daugthers MDCK cultured in Matrigel, the authors made several key observations that bring new light to our understanding of a mechanism that has been studied for many years without being fully explained:
(1) They showed that centriole can reach their polarized position without most of their microtubule-anchoring structures. These observations challenge the standard model according to which centrosomes are moved by the production and transmission of forces along microtubules.
(2) (However) they showed that epithelial polarity can be established in the absence of centriole.
(3) (Somehow more expectedly) they also showed that epithelial polarity can't be established in the absence of Par3.
(4) They found that most other polarity players that are transported through the cytoplasm in lipid vesicles, and finally fused to the basal or apical pole of epithelial cells, are moved along an axis which is defined by the position of centrosome and orientation of microtubules.
(5) Surprisingly, two non-daughters cells that were brought in contact (for 6h) could partially polarize by recruiting a few Par3 molecules but not the other polarity markers.
(6) Even more surprisingly, in the absence of ECM, Par 3 and centrosomes could move to their proper position close to the intercellular junction after cytokinesis but other polarity markers (at least GP135) localized to the opposite, non-adhesive, side. So the polarity of the centrosome-microtubule network could be dissociated from the localisation of GP135 (which was believed to be transported along this network).
Strengths:
(1) The simplicity and reproducibility of the system allow a very quantitative description of cell polarity and protein localisation.
(2) The experiments are quite straightforward, well-executed, and properly analyzed.
(3) The writing is clear and conclusions are convincing.
Weaknesses:
(1) The simplicity of the system may not capture some of the mechanisms involved in the establishment of cell polarity in more physiological conditions (fluid flow, electrical potential, ion gradients,...).
(2) The absence of centriole in centrinone-treated cells might not prevent the coalescence of centrosomal protein in a kind of MTOC which might still orient microtubules and intracellular traffic. How are microtubules organized in the absence of centriole? If they still form a radial array, the absence of a centriole at the center of it somehow does not conflict with classical views in the field.
(3) The mechanism is still far from clear and this study shines some light on our lack of understanding. Basic and key questions remain:
a) How is the centrosome moved toward the Par3-rich pole? This is particularly difficult to answer if the mechanism does not imply the anchoring of MTs to the centriole or PCM.
b) What happens during cytokinesis that organises Par3 and intercellular junction in a way that can't be achieved by simply bringing two cells together? In larger epithelia cells have neighbours that are not daughters, still, they can form tight junctions with Par3 which participates in the establishment of cell polarity as much as those that are closer to the cytokinetic bridge (as judged by the overall cell symmetry). Is the protocol of cell aggregation fully capturing the interaction mechanism of non-daughter cells? -
Reviewer #3 (Public review):
Here, Wang et al. aim to clarify the role of the centrosome and conserved polarity regulators in apical membrane formation during the polarization of MDCK cells cultured in 3D. Through well-presented and rigorous studies, the authors focused on the emergence of polarity as a single MDCK cell divided in 3D culture to form a two-cell cyst with a nascent lumen. Focusing on these very initial stages, rather than in later large cyst formation as in most studies, is a real strength of this study. The authors found that conserved polarity regulators Gp135/podocalyxin, Crb3, Cdc42, and the recycling endosome component Rab11a all localize to the centrosome before localizing to the apical membrane initiation site (AMIS) following cytokinesis. This protein relocalization was concomitant with a repositioning of …
Reviewer #3 (Public review):
Here, Wang et al. aim to clarify the role of the centrosome and conserved polarity regulators in apical membrane formation during the polarization of MDCK cells cultured in 3D. Through well-presented and rigorous studies, the authors focused on the emergence of polarity as a single MDCK cell divided in 3D culture to form a two-cell cyst with a nascent lumen. Focusing on these very initial stages, rather than in later large cyst formation as in most studies, is a real strength of this study. The authors found that conserved polarity regulators Gp135/podocalyxin, Crb3, Cdc42, and the recycling endosome component Rab11a all localize to the centrosome before localizing to the apical membrane initiation site (AMIS) following cytokinesis. This protein relocalization was concomitant with a repositioning of centrosomes towards the AMIS. In contrast, Par3, aPKC, and the junctional components E-cadherin and ZO1 localize directly to the AMIS without first localizing to the centrosome. Based on the timing of the localization of these proteins, these observational studies suggested that Par3 is upstream of centrosome repositioning towards the AMIS and that the centrosome might be required for delivery of apical/luminal proteins to the AMIS.
To test this hypothesis, the authors generated numerous new cell lines and/or employed pharmacological inhibitors to determine the hierarchy of localization among these components. They found that removal of the centrosome via centrinone treatment severely delayed and weakened the delivery of Gp135 to the AMIS and single lumen formation, although normal lumenogenesis was apparently rescued with time. This effect was not due to the presence of CEP164, ODF2, CEP120, or Pericentrin. Par3 depletion perturbed the repositioning of the centrosome towards the AMIS and the relocalization of the Gp135 and Rab11 to the AMIS, causing these proteins to get stuck at the centrosome. Finally, the authors culture the MDCK cells in several ways (forced aggregation and ECM depleted) to try and further uncouple localization of the pertinent components, finding that Par3 can localize to the cell-cell interface in the absence of cell division. Par3 localized to the edge of the cell-cell contacts in the absence of ECM and this localization was not sufficient to orient the centrosomes to this site, indicating the importance of other factors in centrosome recruitment.
Together, these data suggest a model where Par3 positions the centrosome at the AMIS and is required for the efficient transfer of more downstream polarity determinants (Gp135 and Rab11) to the apical membrane from the centrosome. The authors present solid and compelling data and are well-positioned to directly test this model with their existing system and tools. In particular, one obvious mechanism here is that centrosome-based microtubules help to efficiently direct the transport of molecules required to reinforce polarity and/or promote lumenogenesis. This model is not really explored by the authors except by Pericentrin and subdistal appendage depletion and the authors do not test whether these perturbations affect centrosomal microtubules. Exploring the role of microtubules in this process could considerably add to the mechanisms presented here. In its current state, this paper is a careful observation of the events of MCDK polarization and will fill a knowledge gap in this field. However, the mechanism could be significantly bolstered with existing tools, thereby elevating our understanding of how polarity emerges in this system.
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