ROCK1 but not ROCK2 contributes to RhoA signaling and NMIIA-mediated contractility at the epithelial zonula adherens

Rho kinases (ROCK1 and ROCK2) function downstream of the small GTPase RhoA to drive actomyosin cytoskeletal remodeling. It has often been believed that ROCK1 and ROCK2 may be functionally redundant, as they share a highly conserved kinase domain. However, in this study, we report differential functional effects for these ROCKs at the epithelial zonula adherens (ZA). Using specific siRNA, we found that ROCK1 depletion disrupted cadherin organization at the ZA, accompanied by loss of F-actin and NMIIA, whereas ROCK2 knockdown had no significant effect. Further, ROCK1, but not ROCK2, was necessary to stabilize GTP-RhoA at the ZA, thereby sustaining junctional tension and inhibiting intraepithelial cell movement. We also found that nonmuscle myosin IIA is a major determinant of ROCK1 cortical stability. Thus, despite sharing the catalytic domain with ROCK2, ROCK1 appears to be the dominant kinase essential for junctional integrity and contractile tension at epithelial ZA

Mechanosensing and mechanotransduction at cell-cell junctions

Cell adhesion systems are defined by their ability to resist detachment force. Our understanding of the biology of cell-cell adhesions has recently been transformed by the realization that many of the forces that act on those adhesions are generated by the cells that they couple together; and that force at adhesive junctions can be sensed to regulate cell behavior. Here, we consider the mechanisms responsible for applying force to cell-cell junctions and the mechanosensory pathways that detect those forces. We focus on cadherins, as these are the best-studied examples to date, but it is likely that similar principles will apply to other molecular systems that can engage with force-generators within cells and physically couple those cells together

In life there is death: How epithelial tissue barriers are preserved despite the challenge of apoptosis

Apoptosis is a ubiquitous mode of programmed cell death that is found in healthy organs and can be stimulated by many toxic stresses. When it occurs in epithelia, apoptosis presents major challenges to tissue integrity. Apoptotic corpses can promote inflammatory and autoimmune responses if they are retained, and the cellular fragmentation that accompanies apoptosis can potentially compromise the epithelial barrier. Here we discuss 2 homeostatic mechanisms that allow epithelia to circumvent these potential risks: clearance of apoptotic corpses by professional and non-professional phagocytes and physical expulsion of apoptotic cells by apical extrusion. Extrusion and phagocytosis may represent complementary responses that preserve epithelial integrity despite the inevitable challenge of apoptosis

Snail induces epithelial cell extrusion by regulating RhoA contractile signalling and cell–matrix adhesion

Cell extrusion is a morphogenetic process that is implicated in epithelial homeostasis and elicited by stimuli ranging from apoptosis to oncogenic transformation. To explore whether the morphogenetic transcription factor Snail (SNAI1) induces extrusion, we inducibly expressed a stabilized Snail transgene in confluent MCF-7 monolayers. When expressed in small clusters (less than three cells) within otherwise wild-type confluent monolayers, Snail expression induced apical cell extrusion. In contrast, larger clusters or homogenous cultures of Snail cells did not show enhanced apical extrusion, but eventually displayed sporadic basal delamination. Transcriptomic profiling revealed that Snail did not substantively alter the balance of epithelial and mesenchymal genes. However, we identified a transcriptional network that led to upregulated RhoA signalling and cortical contractility in cells expressing Snail. Enhanced contractility was necessary, but not sufficient, to drive extrusion, suggesting that Snail collaborates with other factors. Indeed, we found that the transcriptional downregulation of cell–matrix adhesion cooperates with contractility to mediate basal delamination. This provides a pathway for Snail to influence epithelial morphogenesis independently of classic epithelial-to-mesenchymal transition

A mechanosensitive RhoA pathway that protects epithelia against acute tensile stress

Adherens junctions are tensile structures that couple epithelial cells together. Junctional tension can arise from cell-intrinsic application of contractility or from the cell-extrinsic forces of tissue movement. Here, we report a mechanosensitive signaling pathway that activates RhoA at adherens junctions to preserve epithelial integrity in response to acute tensile stress. We identify Myosin VI as the force sensor, whose association with E-cadherin is enhanced when junctional tension is increased by mechanical monolayer stress. Myosin VI promotes recruitment of the heterotrimeric G alpha 12 protein to E-cadherin, where it signals for p114 RhoGEF to activate RhoA. Despite its potential to stimulate junctional actomyosin and further increase contractility, tension-activated RhoA signaling is necessary to preserve epithelial integrity. This is explained by an increase in tensile strength, especially at the multicellular vertices of junctions, that is due to mDia1-mediated actin assembly

BECLIN1 is essential for intestinal homeostasis involving autophagy-independent mechanisms through its function in endocytic trafficking

Abstract Autophagy-related genes have been closely associated with intestinal homeostasis. BECLIN1 is a component of Class III phosphatidylinositol 3-kinase complexes that orchestrate autophagy initiation and endocytic trafficking. Here we show intestinal epithelium-specific BECLIN1 deletion in adult mice leads to rapid fatal enteritis with compromised gut barrier integrity, highlighting its intrinsic critical role in gut maintenance. BECLIN1-deficient intestinal epithelial cells exhibit extensive apoptosis, impaired autophagy, and stressed endoplasmic reticulum and mitochondria. Remaining absorptive enterocytes and secretory cells display morphological abnormalities. Deletion of the autophagy regulator, ATG7, fails to elicit similar effects, suggesting additional novel autophagy-independent functions of BECLIN1 distinct from ATG7. Indeed, organoids derived from BECLIN1 KO mice show E-CADHERIN mislocalisation associated with abnormalities in the endocytic trafficking pathway. This provides a mechanism linking endocytic trafficking mediated by BECLIN1 and loss of intestinal barrier integrity. Our findings establish an indispensable role of BECLIN1 in maintaining mammalian intestinal homeostasis and uncover its involvement in endocytic trafficking in this process. Hence, this study has important implications for our understanding of intestinal pathophysiology

BECLIN1 is essential for intestinal homeostasis involving autophagy-independent mechanisms through its function in endocytic trafficking.

Autophagy-related genes have been closely associated with intestinal homeostasis. BECLIN1 is a component of Class III phosphatidylinositol 3-kinase complexes that orchestrate autophagy initiation and endocytic trafficking. Here we show intestinal epithelium-specific BECLIN1 deletion in adult mice leads to rapid fatal enteritis with compromised gut barrier integrity, highlighting its intrinsic critical role in gut maintenance. BECLIN1-deficient intestinal epithelial cells exhibit extensive apoptosis, impaired autophagy, and stressed endoplasmic reticulum and mitochondria. Remaining absorptive enterocytes and secretory cells display morphological abnormalities. Deletion of the autophagy regulator, ATG7, fails to elicit similar effects, suggesting additional novel autophagy-independent functions of BECLIN1 distinct from ATG7. Indeed, organoids derived from BECLIN1 KO mice show E-CADHERIN mislocalisation associated with abnormalities in the endocytic trafficking pathway. This provides a mechanism linking endocytic trafficking mediated by BECLIN1 and loss of intestinal barrier integrity. Our findings establish an indispensable role of BECLIN1 in maintaining mammalian intestinal homeostasis and uncover its involvement in endocytic trafficking in this process. Hence, this study has important implications for our understanding of intestinal pathophysiology