The establishment of apical–basal polarity within a single cell and throughout a growing tissue is a key feature of epithelial morphogenesis. To examine the underlying mechanisms, the human intestinal epithelial cell line Caco-2 was grown in a three-dimensional matrix to generate a cystlike structure, where the apical surface of each epithelial cell faces a fluid-filled central lumen. A discrete apical domain is established as early as the first cell division and between the two daughter cells. During subsequent cell divisions, the apical domain of each daughter cell is maintained at the center of the growing structure through a combination of mitotic spindle orientation and asymmetric abscission. Depletion of Cdc42 does not prevent the establishment of apical–basal polarity in individual cells but rather disrupts spindle orientation, leading to inappropriate positioning of apical surfaces within the cyst. We conclude that Cdc42 regulates epithelial tissue morphogenesis by controlling spindle orientation during cell division.

The cell cycle is exquisitely controlled by multiple sequential regulatory inputs to ensure fidelity. Here we demonstrate that the final step in division, the physical separation of daughter cells, is controlled by a member of the PKC gene superfamily. Specifically, we have identified three phosphorylation sites within PKCepsilon that control its association with 14-3-3. These phosphorylations are executed by p38 MAP kinase (Ser 350), GSK3 (Ser 346) and PKC itself (Ser 368). Integration of these signals is essential during mitosis because mutations that prevent phosphorylation of PKCepsilon and/or PKCepsilon binding to 14-3-3 also cause defects in the completion of cytokinesis. Using chemical genetic and dominant-negative approaches it is shown that selective inhibition of PKCepsilon halts cells at the final stages of separation. This arrest is associated with persistent RhoA activation at the midbody and a delay in actomyosin ring dissociation. This study therefore identifies a new regulatory mechanism that controls exit from cytokinesis, which has implications for carcinogenesis.

Cdc42 plays an evolutionarily conserved role in promoting cell polarity and is indispensable during epithelial morphogenesis. To further investigate the role of Cdc42, we have used a three-dimensional matrigel model, in which single Caco-2 cells develop to form polarized cysts. Using this system, we previously reported that Cdc42 controls mitotic spindle orientation during cell division to correctly position the apical surface in a growing epithelial structure. In the present study, we have investigated the specific downstream effectors through which Cdc42 controls this process. Here, we report that Par6B and its binding partner, atypical protein kinase C (aPKC), are required to regulate Caco-2 morphogenesis. Depletion or inhibition of Par6B or aPKC phenocopies the loss of Cdc42, inducing misorientation of the mitotic spindle, mispositioning of the nascent apical surface, and ultimately, the formation of aberrant cysts with multiple lumens. Mechanistically, Par6B and aPKC function interdependently in this context. Par6B localizes to the apical surface of Caco-2 cysts and is required to recruit aPKC to this compartment. Conversely, aPKC protects Par6B from proteasomal degradation, in a kinase-independent manner. In addition, we report that depletion or inhibition of aPKC induces robust apoptotic cell death in Caco-2 cells, significantly reducing both cyst size and number. Cell survival and apical positioning depend upon different thresholds of aPKC expression, suggesting that they are controlled by distinct downstream pathways. We conclude that Par6B and aPKC control mitotic spindle orientation in polarized epithelia and, furthermore, that aPKC coordinately regulates multiple processes to promote morphogenesis.Cdc42 is a small GTPase that plays a fundamental role in promoting cell polarity across evolution and in a wide range of biological contexts (1). For instance, Cdc42 is required for bud emergence in Saccharomyces cerevisiae (2), asymmetric cell division in Caenorhabditis elegans (3) and directed cell migration in astrocytes (4). In epithelial cells, Cdc42 is indispensable during morphogenesis, controlling tight junction formation (5-7), the delivery of basolateral proteins (8), and apical surface formation through the regulated trafficking of vacuolar apical components (9). To further investigate the role of Cdc42, we have exploited a three-dimensional model of epithelial morphogenesis, in which single Caco-2 cells are cultured in matrigel to form polarized cysts (Fig. 1A). Using this system, we previously reported that Cdc42 controls mitotic spindle orientation to correctly position the nascent apical surface in a growing cyst (10). These findings highlight an intriguing relationship between epithelial mitosis and morphogenesis, which couples cell proliferation to tissue architecture.A major challenge now is to determine the specific upstream regulators and downstream effectors through which Cdc42 controls epithelial morphogenesis. Recent work has focused on the identification of specific guanine nucleotid...

Entosis is a form of epithelial cell cannibalism that is prevalent in human cancer, typically triggered by loss of matrix adhesion. Here, we report an alternative mechanism for entosis in human epithelial cells, driven by mitosis. Mitotic entosis is regulated by Cdc42, which controls mitotic morphology. Cdc42 depletion enhances mitotic deadhesion and rounding, and these biophysical changes, which depend on RhoA activation and are phenocopied by Rap1 inhibition, permit subsequent entosis. Mitotic entosis occurs constitutively in some human cancer cell lines and mitotic index correlates with cell cannibalism in primary human breast tumours. Adherent, wild-type cells can act efficiently as entotic hosts, suggesting that normal epithelia may engulf and kill aberrantly dividing neighbours. Finally, we report that Paclitaxel/taxol promotes mitotic rounding and subsequent entosis, revealing an unconventional activity of this drug. Together, our data uncover an intriguing link between cell division and cannibalism, of significance to both cancer and chemotherapy.DOI: http://dx.doi.org/10.7554/eLife.27134.001