Discussion

Understanding how epithelial cells establish and maintain the axis of polarity is a fundamental issue for our comprehension of how epithelial tissue organize themselves into tubes and cysts. In the present study, we have demonstrated that in MDCK cysts the midbody remnant of the first cellular division is the earliest polarization signal (Model in Fig. IV.7a). The position of the first post-mitotic midbody defines the location of lumen formation and establishes the axis of polarity, converting the membrane where it is positioned into the AMIS. Moreover, midbody remnants are also maintained in the following division, marking the edge of the lumen when it physically opens. It has been shown that the resolution of the membrane connection between two daughter cells, during abscission, requires targeting and fusion of exocyst vesicles with the intracellular-bridge plasma membrane²⁷. Several subunits of the exocyst complex, including Sec8, localized to the midbody ring during abscission and formed a ring-like structure²⁷. Thus, the selective localization of key tethering factors (such as Sec8) of the exocyst complex at the midbody during abscission could be the link between midbody remnant and AMIS specification via polarized exocytosis.

We provide evidence for this model by showing that a perturbation of midbody remnant fate is associated with major defects in MDCK cyst morphogenesis. The overexpression of the phosphatase PRL-3 alters the fate of the post-mitotic midbody from release into the lumenal space to cytoplasmic or lateral plasma membrane retention (Model in Fig. IV.7b). The site where the midbody remnant is retained became first an ectopic AMIS, enriched in Par3 and surrounded by Sec8, and later on an ectopic lumen that stained positive for all the classical luminal markers (Model in Fig. IV.7b). Together, our results show that midbody remnants themselves are a signal sufficient to drive the entire pathway of de novo lumen formation, leading to the opening of completely specified lumens, independently of the position where they are located.

Recently, it has been suggested that midbody remnants could participate in non-cytokinetic functions, such as cell fate determination and cell polarity specification^13,23,25. In particular, it has been shown that cytokinesis remnants are the earliest landmarks of neuronal polarity in Drosophila melanogaster in vivo²⁴. Moreover, in Drosophila follicular epithelium, apical midbody localization provides a positional cue critical for the formation of the apico-basal axis of the tissue, as

confirmed by disruption of the epithelial architecture in case of midbody ectopic localization²⁵. Our results strongly support a function of midbody remnants in polarization, showing that during cystogenesis, midbody remnants are the earliest signal of polarization. Interestingly, a link between cytokinesis and polarization has been also described in unicellular organisms, such as budding yeast, where the post-mitotic bud scar contributes to cell polarity and to establishing the division axis for the next cell division²⁸.

Figure IV.7. Midbody remnants are the earliest signal of polarization, determining intrinsically the lumen position. (a) Schematic representation of different stages of lumenogenesis in MDCK cysts. During the first cytokinesis, members of the exocysts complex are delivered to the midbody to complete the abscission. Midbody remnants define the AMIS (apical membrane initiation site) that is established by polarized exocytosis. Expansion then allows opening of the luminal space, midbody remnants mark the edge of the lumen. (b) PRL-3 overexpression affects lumenogenesis by altering post-mitotic midbody fate. After the first cytokinesis the post-mitotic midbody is internalized and retained in the cytoplasm. This site becomes a wrongly localized and PRL-3 enriched AMIS where apical determinants are delivered by exocytosis.

Expansion then allows opening of ectopic lumens in the position where the midbody remnant is retained. Red lines, microtubules; yellow circles, midbody and midbody remnants; small red circles, exocysts subunits; blue ovals, nuclei; empty red circles, exocysts vescicles.

Molecular details of how the fate of midbody remnants is determined will be illuminated by the identification of the PRL-3 substrate in this process, which could be a key player in the establishment the lumen. Thus far, several protein substrates and a non-protein substrate have been suggested, but none has been confirmed yet as the substrate that accounts for the pleiotropic effects on cellular signaling caused by PRL-3 overexpression¹². Indeed, the identification of PRL-3 substrates has remained extremely challenging since its discovery over a decade ago^11,12. In the context of lumen formation, the suggested PRL-3 substrates PI(4,5)P2 and Ezrin are particularly attractive candidates as they are known determinants of cellular polarity¹⁶. We did not observe significant changes in the phosphorylation status of either one of these proposed substrates with the methods applied here. Rapid changes of tightly controlled phosphorylation levels may however be difficult to detect, and it will take the development of elaborate, sensitive methods such as phosphoproteomics at distinct time points in 3D cell culture to make progress in this area. Acute changes, for example in the concentration of PI(4,5)P2 at relevant locations as seen in other processes such as endocytosis and exocytosis²⁹, could account for the alteration of midbody fate with its dramatic phenotypic consequences. Alternatively, hitherto unknown effects caused by protein-protein interactions that depend on the intact WT phosphatase (as opposed to the C104S mutant, which seems to be structurally less stable³⁰) could be the critical factor in altering midbody fate.

The question of whether PRL-3 is a bona-fide oncogene has not yet been addressed in detail. In a recent report³¹, the authors observed elevated expression of PRL-3 in the colon of WT mice immediately following the treatment with a carcinogen. In addition, WT mice expressed 3 at an elevated level in primary tumors and PRL-3 knock out mice developed 50% fewer tumors following treatment with carcinogens³¹. These findings suggest strongly that PRL-3 could play a role in colon tumorigenesis,aside from its known role in the progression of late stage cancer and metastasis^11,12. Our data strongly support these findings in that the simple over-expression of PRL-3 in a non-cancerous background causes detrimental disorganization of epithelial structures. Moreover, it is known that one of the earliest events following the acquisition of oncogenic mutations by an epithelial cell is the formation of multilayered and disorganized structures⁸. This resembles the phenotype exhibited by PRL-3 overexpressing multilayered cells that grow into the cyst lumen in a disorganized way. In addition to the disruption of cell-cell contacts, cell-matrix

adhesion and spindle orientation⁸, switching the fate of midbody remnants could therefore be another reason for loss of epithelial organization in cancer.

We identified midbody remnants as the earliest signal of polarization in cystogenesis, and that PRL-3 overexpression alters the fate of the midbody remnants.

These findings not only offer an explanation for the long unanswered question of how the position of the AMIS is specified, but also suggest a novel mechanism for the loss of epithelial organization in cancer, including the identification of PRL-3 as an enzyme involved in this mechanism. Studying these mechanisms in vivo in epithelial organ formation and cancers originating from epithelial tissues will be major challenges in developmental and cancer cell biology in the future.

IV.5. Methods