Activation of the Ca2+ sensing receptor and the PKC/WNK4 downstream signaling cascade induces incorporation of ZO-2 to tight junctions and its separation from 14-3-3.

Zonula occludens-2 (ZO-2) is a tight junction (TJ) cytoplasmic protein, whose localization varies according to cell density and Ca2+ in the media. In cells cultured in low calcium (LC), ZO-2 displays a diffuse cytoplasmic distribution, but activation of the Ca2+ sensing receptor (CaSR) with Gd3+ triggers the appearance of ZO-2 at the cell borders. CaSR downstream signaling involves activation of protein kinase C, which phosphorylates and activates with no lysine kinase-4 that phosphorylates ZO-2 inducing its concentration at TJs. In LC, ZO-2 is protected from degradation by association to 14-3-3 proteins. When monolayers are transferred to normal calcium, the complexes ZO-2/14-3-3ζ and ZO-2/14-3-3σ move to the cell borders and dissociate. The 14-3-3 proteins are then degraded in proteosomes, whereas ZO-2 integrates to TJs. From the plasma membrane residual ZO-2 is endocyted and degradaded in lysosomes. The unique region 2 of ZO-2, and S261 located within a nuclear localization signal, are critical for the interaction with 14-3-3 ζ and σ and for the efficient nuclear importation of ZO-2. These results explain the molecular mechanism through which extracellular Ca2+ triggers the appearance of ZO-2 at TJs in epithelial cells and reveal the novel interaction between ZO-2 and 14-3-3 proteins, which is critical for ZO-2 protection and intracellular traffic.

Quantifying cadherin mechanotransduction machinery assembly/disassembly dynamics using fluorescence covariance analysis.

Quantifying multi-molecular complex assembly in specific cytoplasmic compartments is crucial to understand how cells use assembly/disassembly of these complexes to control function. Currently, biophysical methods like Fluorescence Resonance Energy Transfer and Fluorescence Correlation Spectroscopy provide quantitative measurements of direct protein-protein interactions, while traditional biochemical approaches such as sub-cellular fractionation and immunoprecipitation remain the main approaches used to study multi-protein complex assembly/disassembly dynamics. In this article, we validate and quantify multi-protein adherens junction complex assembly in situ using light microscopy and Fluorescence Covariance Analysis. Utilizing specific fluorescently-labeled protein pairs, we quantified various stages of adherens junction complex assembly, the multiprotein complex regulating epithelial tissue structure and function following de novo cell-cell contact. We demonstrate: minimal cadherin-catenin complex assembly in the perinuclear cytoplasm and subsequent localization to the cell-cell contact zone, assembly of adherens junction complexes, acto-myosin tension-mediated anchoring, and adherens junction maturation following de novo cell-cell contact. Finally applying Fluorescence Covariance Analysis in live cells expressing fluorescently tagged adherens junction complex proteins, we also quantified adherens junction complex assembly dynamics during epithelial monolayer formation.

Cyclophilin A promotes cell migration via the Abl-Crk signaling pathway.

Cyclophilin A (CypA) is overexpressed in a number of human cancer types, but the mechanisms by which the protein promotes oncogenic properties of cells are not understood. Here we demonstrate that CypA binds the CrkII adaptor protein and prevents it from switching to the inhibited state. CrkII influences cell motility and invasion by mediating signaling through its SH2 and SH3 domains. CrkII Tyr221 phosphorylation by the Abl or EGFR kinases induces an inhibited state of CrkII by means of an intramolecular SH2-pTyr221 interaction, causing signaling interruption. We show that the CrkII phosphorylation site constitutes a binding site for CypA. Recruitment of CypA sterically restricts the accessibility of Tyr221 to kinases, thereby suppressing CrkII phosphorylation and promoting the active state. Structural, biophysical and in vivo data show that CypA augments CrkII-mediated signaling. A strong stimulation of cell migration is observed in cancer cells wherein both CypA and CrkII are greatly upregulated.

Balancing spatially regulated ß-actin translation and dynamin-mediated endocytosis is required to assemble functional epithelial monolayers.

Regulating adherens junction complex assembly/disassembly is critical to maintaining epithelial homeostasis in healthy epithelial tissues. Consequently, adherens junction structure and function is often perturbed in clinically advanced tumors of epithelial origin. Some of the most studied factors driving adherens junction complex perturbation in epithelial cancers are transcriptional and epigenetic down-regulation of E-cadherin expression. However, numerous reports demonstrate that post-translational regulatory mechanisms such as endocytosis also regulate early phases of epithelial-mesenchymal transition and metastatic progression. In already assembled healthy epithelia, E-cadherin endocytosis recycles cadherin-catenin complexes to regulate the number of mature adherens junctions found at cell-cell contact sites. However, following de novo epithelial cell-cell contact, endocytosis negatively regulates adherens junction assembly by removing E-cadherin from the cell surface. By contrast, following de novo epithelial cell-cell contact, spatially localized ß-actin translation drives cytoskeletal remodeling and consequently E-cadherin clustering at cell-cell contact sites and therefore positively regulates adherens junction assembly. In this report we demonstrate that dynamin-mediated endocytosis and ß-actin translation-dependent cadherin-catenin complex anchoring oppose each other following epithelial cell-cell contact. Consequently, the final extent of adherens junction assembly depends on which of these processes is dominant following epithelial cell-cell contact. We expressed ß-actin transcripts impaired in their ability to properly localize monomer synthesis (Δ3'UTR) in MDCK cells to perturb actin filament remodeling and anchoring, and demonstrate the resulting defect in adherens junction structure and function is rescued by inhibiting dynamin mediated endocytosis. Therefore, we demonstrate balancing spatially regulated ß-actin translation and dynamin-mediated endocytosis regulates epithelial monolayer structure and barrier function.

CDC42 inhibition suppresses progression of incipient intestinal tumors.

Mutations in the APC or ß-catenin genes are well-established initiators of colorectal cancer, yet modifiers that facilitate the survival and progression of nascent tumor cells are not well defined. Using genetic and pharmacologic approaches in mouse colorectal cancer and human colorectal cancer xenograft models, we show that incipient intestinal tumor cells activate CDC42, an APC-interacting small GTPase, as a crucial step in malignant progression. In the mouse, Cdc42 ablation attenuated the tumorigenicity of mutant intestinal cells carrying single APC or ß-catenin mutations. Similarly, human colorectal cancer with relatively higher levels of CDC42 activity was particularly sensitive to CDC42 blockade. Mechanistic studies suggested that Cdc42 may be activated at different levels, including at the level of transcriptional activation of the stem cell-enriched Rho family exchange factor Arhgef4. Our results indicate that early-stage mutant intestinal epithelial cells must recruit the pleiotropic functions of Cdc42 for malignant progression, suggesting its relevance as a biomarker and therapeutic target for selective colorectal cancer intervention.

The ß-actin mRNA zipcode regulates epithelial adherens junction assembly but not maintenance.

Epithelial cell-cell contact stimulates actin cytoskeleton remodeling to down-regulate branched filament polymerization-driven lamellar protrusion and subsequently to assemble linear actin filaments required for E-cadherin anchoring during adherens junction complex assembly. In this manuscript, we demonstrate that de novo protein synthesis, the ß-actin 3' UTR, and the ß-actin mRNA zipcode are required for epithelial adherens junction complex assembly but not maintenance. Specifically, we demonstrate that perturbing cell-cell contact-localized ß-actin monomer synthesis causes epithelial adherens junction assembly defects. Consequently, inhibiting ß-actin mRNA zipcode/ZBP1 interactions with ß-actin mRNA zipcode antisense oligonucleotides, to intentionally delocalize ß-actin monomer synthesis, is sufficient to perturb adherens junction assembly following epithelial cell-cell contact. Additionally, we demonstrate active RhoA, the signal required to drive zipcode-mediated ß-actin mRNA targeting, is localized at epithelial cell-cell contact sites in a ß-actin mRNA zipcode-dependent manner. Moreover, chemically inhibiting Src kinase activity prevents the local stimulation of ß-actin monomer synthesis at cell-cell contact sites while inhibiting epithelial adherens junction assembly. Together, these data demonstrate that epithelial cell-cell contact stimulates ß-actin mRNA zipcode-mediated monomer synthesis to spatially regulate actin filament remodeling, thereby controlling adherens junction assembly to modulate cell and tissue adhesion.

Mechanisms and cellular roles of local protein synthesis in mammalian cells.

After the export from the nucleus it turns out that all mRNAs are not treated equally. Not only is mRNA subject to translation, but also through RNA-binding proteins and other trans-acting factors, eukaryotic cells interpret codes for spatial sorting within the mRNA sequence. These codes instruct the cytoskeleton and translation apparatus to make decisions about where to transport and when to translate the intended protein product. Signaling pathways decode extra-cellular cues and can modify transport and translation factors in the appropriate cytoplasmic space to achieve translation locally. Identifying regulatory sites on transport factors as well as novel physiological functions for well-known translation factors has provided significant advances in how spatially controlled translation impacts cell function.

Imaging mRNA movement from transcription sites to translation sites.

RNA localization is one mechanism to temporally and spatially restrict protein synthesis to specific subcellular compartments in response to extracellular stimuli. To understand the mechanisms of mRNA localization, a number of methods have been developed to follow the path of these molecules in living cells including direct labeling of target mRNAs, the MS2-GFP system, and molecular beacons. We review advances in these methods with the goal of identifying the particular strengths and weaknesses of the various approaches in their ability to follow the movements of mRNAs from transcription sites to translation sites.

Visualization of mRNA translation in living cells.

The role of mRNA localization is presumably to effect cell asymmetry by synthesizing proteins in specific cellular compartments. However, protein synthesis has never been directly demonstrated at the sites of mRNA localization. To address this, we developed a live cell method for imaging translation of beta-actin mRNA. Constructs coding for beta-actin, containing tetracysteine motifs, were transfected into C2C12 cells, and sites of nascent polypeptide chains were detected using the biarsenial dyes FlAsH and ReAsH, a technique we call translation site imaging. These sites colocalized with beta-actin mRNA at the leading edge of motile myoblasts, confirming that they were translating. beta-Actin mRNA lacking the sequence (zipcode) that localizes the mRNA to the cell periphery, eliminated the translation there. A pulse-chase experiment on living cells showed that the recently synthesized protein correlated spatially with the sites of its translation. Additionally, localization of beta-actin mRNA and translation activity was enhanced at cell contacts and facilitated the formation of intercellular junctions.

Seawi--a sea urchin piwi/argonaute family member is a component of MT-RNP complexes.

The piwi/argonaute family of proteins is involved in key developmental processes such as stem cell maintenance and axis specification through molecular mechanisms that may involve RNA silencing. Here we report on the cloning and characterization of the sea urchin piwi/argonaute family member seawi. Seawi is a major component of microtubule-ribonucleoprotein (MT-RNP) complexes isolated from two different species of sea urchin, Strongylocentrotus purpuratus and Paracentrotus lividus. Seawi co-isolates with purified ribosomes, cosediments with 80S ribosomes in sucrose density gradients, and binds microtubules. Seawi possesses the RNA binding motif common to piwi family members and binds P. lividus bep4 mRNA, a transcript that co-isolates with MT-RNP complexes and whose translation product has been shown to play a role in patterning the animal-vegetal axis. Indirect immunofluorescence studies localized seawi to the cortex of unfertilized eggs within granule-like particles, the mitotic spindle during cell division, and the small micromeres where its levels were enriched during the early cleavage stage. Lastly, we discuss how seawi, as a piwi/argonaute family member, may play a fundamentally important role in sea urchin animal-vegetal axis formation and stem cell maintenance.