Rac1-PAK1 regulation of Rab11 cycling promotes junction destabilization.

Rac1 GTPase is hyperactivated in tumors and contributes to malignancy. Rac1 disruption of junctions requires its effector PAK1, but the precise mechanisms are unknown. Here, we show that E-cadherin is internalized via micropinocytosis in a PAK1-dependent manner without catenin dissociation and degradation. In addition to internalization, PAK1 regulates E-cadherin transport by fine-tuning Rab small GTPase function. PAK1 phosphorylates a core Rab regulator, RabGDIß, but not RabGDIα. Phosphorylated RabGDIß preferentially associates with Rab5 and Rab11, which is predicted to promote Rab retrieval from membranes. Consistent with this hypothesis, Rab11 is activated by Rac1, and inhibition of Rab11 function partially rescues E-cadherin destabilization. Thus, Rac1 activation reduces surface cadherin levels as a net result of higher bulk flow of membrane uptake that counteracts Rab11-dependent E-cadherin delivery to junctions (recycling and/or exocytosis). This unique small GTPase crosstalk has an impact on Rac1 and PAK1 regulation of membrane remodeling during epithelial dedifferentiation, adhesion, and motility.

Junction Mapper is a novel computer vision tool to decipher cell-cell contact phenotypes.

Stable cell-cell contacts underpin tissue architecture and organization. Quantification of junctions of mammalian epithelia requires laborious manual measurements that are a major roadblock for mechanistic studies. We designed Junction Mapper as an open access, semi-automated software that defines the status of adhesiveness via the simultaneous measurement of pre-defined parameters at cell-cell contacts. It identifies contacting interfaces and corners with minimal user input and quantifies length, area and intensity of junction markers. Its ability to measure fragmented junctions is unique. Importantly, junctions that considerably deviate from the contiguous staining and straight contact phenotype seen in epithelia are also successfully quantified (i.e. cardiomyocytes or endothelia). Distinct phenotypes of junction disruption can be clearly differentiated among various oncogenes, depletion of actin regulators or stimulation with other agents. Junction Mapper is thus a powerful, unbiased and highly applicable software for profiling cell-cell adhesion phenotypes and facilitate studies on junction dynamics in health and disease.

Cooperation of distinct Rac-dependent pathways to stabilise E-cadherin adhesion.

The precise mechanisms via which Rac1 is activated by cadherin junctions are not fully known. In keratinocytes Rac1 activation by cadherin junctions requires EGFR signalling, but how EGFR does so is unclear. To address which activator could mediate E-cadherin signalling to Rac1, we investigated EGFR and two Rac1 GEFs, SOS1 and DOCK180. EGFR RNAi prevented junction-induced Rac1 activation and led to fragmented localization of E-cadherin at cadherin contacts. In contrast, depletion of another EGFR family member, ErbB3, did not interfere with either process. DOCK180 RNAi, but not SOS1, prevented E-cadherin-induced Rac1 activation. However, in a strong divergence from EGFR RNAi phenotype, DOCK180 depletion did not perturb actin recruitment or cadherin localisation at junctions. Rather, reduced DOCK180 levels impaired the resistance to mechanical stress of pre-formed cell aggregates. Thus, within the same cell type, EGFR and DOCK180 regulate Rac1 activation by newly-formed contacts, but control separate cellular events that cooperate to stabilise junctions.

ROCK1 and ROCK2 regulate epithelial polarisation and geometric cell shape.

BACKGROUND INFORMATION: Cell-cell adhesion and contraction play an essential role in the maintenance of geometric shape and polarisation of epithelial cells. However, the molecular regulation of contraction during cell elongation leading to epithelial polarisation and acquisition of geometric cell shape is not clear. RESULTS: Upon induction of cell-cell adhesion, we find that human keratinocytes acquire specific geometric shapes favouring hexagons, by re-modelling junction length/orientation and thus neighbour allocation. Acquisition of geometric shape correlates temporally with epithelial polarisation, as shown by an increase in lateral height. ROCK1 and ROCK2 are important regulators of myosin II contraction, but their specific role in epithelial cell shape has not been addressed. Depletion of ROCK proteins interferes with the correct proportion of hexagonal cell shapes and full elongation of lateral domain. Interestingly, ROCK proteins are not essential for maintenance of circumferential thin bundles, the main contractile epithelial F-actin pool. Instead, ROCK1 or ROCK2 regulates thin bundle contraction and positioning along the lateral domain, an important event for the stabilisation of the elongating lateral domain. Mechanistically, E-cadherin clustering specifically leads to ROCK1/ROCK2-dependent inactivation of myosin phosphatase and phosphorylation of myosin regulatory light chain. These events correlate temporally with the increase in lateral height and thin bundle compaction towards junctions. CONCLUSION: We conclude that ROCK proteins are necessary for acquisition of elongated and geometric cell shape, two key events for epithelial differentiation.

Quantitative and robust assay to measure cell-cell contact assembly and maintenance.

Epithelial junction formation and maintenance are multistep processes that rely on the clustering of macromolecular complexes. These events are highly regulated by signalling pathways that involve Rho small GTPases. Usually, when analysing the contribution of different components of Rho-dependent pathways to cell-cell adhesion, the localisation of adhesion receptors at junctions is evaluated by immunofluorescence. However, we find that this method has limitations on the quantification (dynamic range), ability to detect partial phenotypes and to differentiate between the participation of a given regulatory protein in assembly and/or maintenance of cell-cell contacts.In this chapter, we describe a suitable method, the aggregation assay, in which we adapted a quantitative strategy to allow objective and reproducible detection of partial phenotypes. Importantly, this methodology estimates the ability of cells to form junctions and their resistance to mechanical shearing forces (stabilisation).

Newly formed E-cadherin contacts do not activate Cdc42 or induce filopodia protrusion in human keratinocytes.

BACKGROUND INFORMATION: The appropriate regulation of cell-cell adhesion is an important event in the homoeostasis of different cell types. In epithelial cells, tight adhesion mediated by E-cadherin receptors is essential for the differentiation and functionality of epithelial sheets. Upon assembly of cadherin-mediated cell-cell contacts, it is well established that the small GTPases Rho and Rac are activated and are necessary for junction stability. However, the role of the small GTPase Cdc42 in cadherin adhesion is less clear. Cdc42 can be activated by E-cadherin in a breast tumour cell line, but the requirement for Cdc42 function for new junction assembly or maintenance has been contradictory. Cdc42 participation in cell-cell contacts has been inferred from the presence of filopodia, the typical F-actin structure induced by Cdc42 activation, as cells approach each other to establish cell-cell contacts. Yet, under these conditions, the contribution of migration to filopodia protrusion cannot be excluded and the results are difficult to interpret. RESULTS: In the present study, we set out to address (a) whether Cdc42 is activated by new E-cadherin cell-cell contacts when junction assembly occurs without prior migration and (b) whether Cdc42 function is necessary for cadherin stability. We found that junction formation in confluent keratinocytes or upon E-cadherin clustering decreased Cdc42-GTP levels. In the absence of serum- and migration-induced Cdc42 activation, we demonstrated that cell-cell contacts do not induce filopodia or require Cdc42 function to assemble. CONCLUSION: We conclude that Cdc42 does not participate in the early events that initiate stable cadherin adhesion in keratinocytes. Yet, it is feasible that Cdc42 may be activated at later time points or by other receptors. Cdc42 can then participate in additional functions during polarization, such as Golgi re-positioning or basolateral trafficking.

RhoE Is required for keratinocyte differentiation and stratification.

The molecular mechanism via which keratinocyte differentiation assembles multiple layers of cells (stratification) is poorly understood. We describe here a novel function of the Rho family member RhoE as a regulator of epidermal morphogenesis. RhoE protein levels are specifically and transiently up-regulated upon keratinocyte differentiation. RhoE up-regulation requires the activity of Rho kinase (ROCK) I, suggesting that both RhoE and ROCKI are important during keratinocyte differentiation. RhoE overexpression results in a striking enlargement of cell size and the number of stratified cells. In contrast, RhoE depletion induces hyperproliferation and delays initiation of keratinocyte differentiation. Interestingly, up-regulation of RhoE protein is seen primarily in basal, undifferentiated cells, in which commitment to differentiation and stratification takes place. RhoE activation in basal cells negatively modulates integrin adhesion, thereby facilitating detachment from the substratum and migration to form suprabasal layers. Thus, RhoE integrates two processes essential for keratinocyte differentiation and stratification: regulation of proliferative status and integrin adhesion.

Rho GTPase activation by cell-cell adhesion.

Cell-cell adhesion can occur in a calcium-dependent or calcium-independent manner, depending on the type of receptor involved. Establishment of cell contacts by either type of cell-cell adhesion (calcium-dependent or calcium-independent) has been shown to activate Rho GTPases in different cells. In this chapter, we describe the method used to assess the activation of Rho GTPases by cadherins, the prototype calcium-dependent adhesion receptor in epithelial cells. We cover the optimal cell culture conditions and controls to ensure that the activation of the GTPases is specifically triggered by the formation of cadherin-dependent cell-cell contacts. Controls described herein determine the specificity of activation of Rho proteins with respect to cadherin adhesion and exclude the contribution of other adhesive receptors, calcium-signaling, cell spreading, and migration. Although we focus on cadherin receptors and normal human keratinocytes as our model system, the methods described can be easily adapted to other adhesion receptors and different cell types.

Actin at cell-cell junctions is composed of two dynamic and functional populations.

The ability of epithelial cells to polarize requires cell-cell adhesion mediated by cadherin receptors. During cell-cell contact, the mechanism via which a flat, spread cell shape is changed into a tall, cuboidal epithelial morphology is not known. We found that cadherin-dependent adhesion modulates actin dynamics by triggering changes in actin organization both locally at junctions and within the rest of the cell. Upon induction of cell-cell contacts, two spatial actin populations are distinguishable: junctional actin and peripheral thin bundles. With time, the relative position of these two populations changes and becomes indistinguishable to form a cortical actin ring that is characteristic of mature, fully polarized epithelial cells. Junctional actin and thin actin bundles differ in their actin dynamics and mechanism of formation, and interestingly, have distinct roles during epithelial polarization. Whereas junctional actin stabilizes clustered cadherin receptors at cell-cell contacts, contraction of peripheral actin bundle is essential for an increase in the maximum height at the lateral domain during polarization (cuboidal morphology). Thus, both junctional actin and thin bundles are necessary, and cooperate with each other to generate a polarized epithelial morphology.