Vania M. M. Braga

Cell-cell adhesion and signalling.

Signalling pathways activated by Rho small GTPases have recently been identified that coordinate junction assembly, stability and function, as well as interactions of adhesive complexes with the underlying cortical cytoskeleton. Particularly exciting is the interplay between adherens junctions, activation of Rho proteins and the dynamics of microtubule, actin and intermediate filaments. This interplay has important implications for functional regulation of cell-cell adhesion, and points to a more integrated view of signalling processes.

Rap1 regulates the formation of E-cadherin-based cell-cell contacts

ABSTRACT In epithelial tissues, cells are linked to their neighbors through specialized cell-cell adhesion proteins. E-cadherin is one of the most important membrane proteins for the establishment of intimate cell-cell contacts, but the molecular mechanism by which it is recruited to contact sites is largely unknown. We report here that the cytoplasmic domain of E-cadherin interacts with C3G, a guanine nucleotide exchange factor for Rap1. In epithelial cell cultures, ligation of the extracellular domain of E-cadherin enhances Rap1 activity, which in turn is necessary for the proper targeting of E-cadherin molecules to maturing cell-cell contacts. Furthermore, our data suggest that Cdc42 functions downstream of Rap1 in this process. We conclude that Rap1 plays a vital role in the establishment of E-cadherin-based cell-cell adhesion.

Caspase activation in the terminal differentiation of human epidermal keratinocytes

The epidermis is a multilayered squamous epithelium in which dividing basal cells withdraw from the cell cycle and progressively differentiate as they are displaced toward the skin surface. Eventually, the cells lose their nucleus and other organelles to become flattened squames, which are finally shed from the surface as bags of cross-linked keratin filaments enclosed in a cornified envelope [1]. Although keratinocytes can undergo apoptosis when stimulated by a variety of agents [2], it is not known whether their normal differentiation programme uses any components of the apoptotic biochemical machinery to produce the cornified cell. Differentiating keratinocytes have been reported to share some features with apoptotic cells, such as DNA fragmentation, but these features have not been seen consistently [3]. Apoptosis involves an intracellular proteolytic cascade, mainly mediated by members of the caspase family of cysteine proteases, which cleave one another and various key intracellular target proteins to kill the cell neatly and quickly [4]. Here, we show for the first time that caspases are activated during normal human keratinocyte differentiation and that this activation is apparently required for the normal loss of the nucleus.

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.

Rac Activation upon Cell-Cell Contact Formation Is Dependent on Signaling from the Epidermal Growth Factor Receptor

Cadherins are transmembrane receptors that mediate cell-cell adhesion. They play an essential role in embryonic development and maintenance of tissue architecture. The Rho family small GTPases regulate actin cytoskeletal dynamics in different cell types. The function of two family members, Rho and Rac, is required for the stability of cadherins at cell-cell contacts. Consistent with the published data we have found that Rac is activated upon induction of intercellular adhesion in epithelial cells. This activation is dependent on functional cadherins (Nakagawa, M., Fukata, M., Yamaga, M., Itoh, N., and Kaibuchi, K. (2001) J. Cell Sci. 114, 1829–1838; Noren, N. K., Niessen, C. M., Gumbiner, B. M., and Burridge, K. (2001) J. Biol. Chem. 276, 3305–3308). Here we show for the first time that clustering of cadherins using antibody-coated beads is sufficient to promote Rac activation. In the presence of Latrunculin B, Rac can be partially activated by antibody-clustered cadherins. These results suggest that actin polymerization is not required for initial Rac activation. Contrary to what has been described before, phosphatidylinositol 3-kinases are not involved in Rac activation following cell-cell adhesion in keratinocytes. Interestingly, inhibition of epidermal growth factor receptor signaling efficiently blocks the increased Rac-GTP levels observed after contact formation. We conclude that cadherin-dependent adhesion can activate Rac via epidermal growth factor receptor signaling.

The LIM protein Ajuba influences p130Cas localization and Rac1 activity during cell migration

Cell migration requires extension of lamellipodia that are stabilized by formation of adhesive complexes at the leading edge. Both processes are regulated by signaling proteins recruited to nascent adhesive sites that lead to activation of Rho GTPases. The Ajuba/Zyxin family of LIM proteins are components of cellular adhesive complexes. We show that cells from Ajuba null mice are inhibited in their migration, without associated abnormality in adhesion to extracellular matrix proteins, cell spreading, or integrin activation. Lamellipodia production, or function, is defective and there is a selective reduction in the level and tyrosine phosphorylation of FAK, p130Cas, Crk, and Dock180 at nascent focal complexes. In response to migratory cues Rac activation is blunted in Ajuba null cells, as detected biochemically and by FRET analysis. Ajuba associates with the focal adhesion-targeting domain of p130Cas, and rescue experiments suggest that Ajuba acts upstream of p130Cas to localize p130Cas to nascent adhesive sites in migrating cells thereby leading to the activation of Rac.

Illuminating the functional and structural repertoire of human TBC/RABGAPs

The Tre2–Bub2–Cdc16 (TBC) domain-containing RAB-specific GTPase-activating proteins (TBC/RABGAPs) are characterized by the presence of highly conserved TBC domains and act as negative regulators of RABs. The importance of TBC/RABGAPs in the regulation of specific intracellular trafficking routes is now emerging, as is their role in different diseases. Importantly, TBC/RABGAPs act as key regulatory nodes, integrating signalling between RABs and other small GTPases and ensuring the appropriate retrieval, transport and delivery of different intracellular vesicles.

miR-23b regulates cytoskeletal remodeling, motility and metastasis by directly targeting multiple transcripts

Uncontrolled cell proliferation and cytoskeletal remodeling are responsible for tumor development and ultimately metastasis. A number of studies have implicated microRNAs in the regulation of cancer cell invasion and migration. Here, we show that miR-23b regulates focal adhesion, cell spreading, cell-cell junctions and the formation of lamellipodia in breast cancer (BC), implicating a central role for it in cytoskeletal dynamics. Inhibition of miR-23b, using a specific sponge construct, leads to an increase of cell migration and metastatic spread in vivo, indicating it as a metastatic suppressor microRNA. Clinically, low miR-23b expression correlates with the development of metastases in BC patients. Mechanistically, miR-23b is able to directly inhibit a number of genes implicated in cytoskeletal remodeling in BC cells. Through intracellular signal transduction, growth factors activate the transcription factor AP-1, and we show that this in turn reduces miR-23b levels by direct binding to its promoter, releasing the pro-invasive genes from translational inhibition. In aggregate, miR-23b expression invokes a sophisticated interaction network that co-ordinates a wide range of cellular responses required to alter the cytoskeleton during cancer cell motility.

Armus is a Rac1 effector that inactivates Rab7 and regulates E-cadherin degradation.

BACKGROUND Cell-cell adhesion and intracellular trafficking are regulated by signaling pathways from small GTPases of the Rho, Arf, and Rab subfamilies. How signaling from distinct small GTPases are integrated in a given process is poorly understood. RESULTS We find that a TBC/RabGAP protein, Armus, integrates signaling between Arf6, Rac1, and Rab7 during junction disassembly. Armus binds specifically to activated Rac1 and its C-terminal TBC/RabGAP domain inactivates Rab7. Thus, Armus is a novel Rac1 effector and a bona fide GAP for Rab7 in vitro and in vivo, a unique and previously unreported combination. Arf6 activation efficiently disrupts cell-cell contacts and is known to activate Rac1 and Rab7. Arf6-induced E-cadherin degradation is efficiently blocked by expression of Armus C-terminal domain or after Armus RNAi. Coexpression of Arf6 with dominant-negative Rab7 or Rac1 also inhibits junction disassembly. Importantly, Armus RabGAP expression also prevents EGF-induced scattering in keratinocytes, a process shown here to require Arf6, Rac1, and Rab7 function. To our knowledge, this is the first report to demonstrate a molecular and functional link between Rac1 and Rab7. CONCLUSIONS Our data indicate that active Rac1 recruits Armus to locally inactivate Rab7 and facilitate E-cadherin degradation in lysosomes. Thus, the integration of Rac1 and Rab7 activities by Armus provides an important regulatory node for E-cadherin turnover and stability of cell-cell contacts.

Calcium-Induced Changes in Distribution and Solubility of Cadherins, Integrins and Their Associated Cytoplasmic Proteins in Human Keratinocytes

Studies with cultured human epidermal keratinocytes have shown that stratification, the movement of differentiating cells out of the basal layer, involves changes in cell-extracellular matrix and cell-cell adhesiveness mediated by receptors of the integrin and cadherin families, respectively. Keratinocytes normally lose their integrins when they initiate terminal differentiation. However, when stratification is inhibited by a low concentration of calcium ions in the medium (0.1 mM) or by addition of antibodies to P- and E-cadherin in standard medium (1.8 mM calcium ions), differentiating, involucrin-positive, cells continue to express functional integrins. In order to investigate the mechanism by which cadherins may regulate integrin expression, we have examined the distribution and detergent solubility of the receptors and associated cytoplasmic proteins in keratinocytes grown as a monolayer in low calcium medium or transferred to standard medium to induce stratification. Within 1 hour of raising the concentration of calcium ions, integrins, cadherins, alpha-catenin, beta-catenin, plakoglobin, vinculin and alpha-actinin appeared to accumulate at cell-cell borders, whereas the focal contact proteins, paxillin and talin, did not. The change in distribution was correlated with decreased solubility in 0.5% Triton X-100 of some of the proteins examined, but the integrins, alpha-actinin, paxillin and talin remained completely soluble. Addition of cytochalasin D inhibited both the redistribution of proteins and subsequent stratification of involucrin-positive cells. Cycloheximide treatment allowed protein redistribution and stratification, but involucrin-positive cells continued to express integrins. These results suggest that stratification requires the interactions of cadherins and integrins with the actin cytoskeleton and that the selective loss of integrins from differentiating cells requires de novo protein synthesis.