TWIST1 interacts with ß/δ-catenins during neural tube development and regulates fate transition in cranial neural crest cells.

Cell fate determination is a necessary and tightly regulated process for producing different cell types and structures during development. Cranial neural crest cells (CNCCs) are unique to vertebrate embryos and emerge from the neural plate borders into multiple cell lineages that differentiate into bone, cartilage, neurons and glial cells. We have previously reported that Irf6 genetically interacts with Twist1 during CNCC-derived tissue formation. Here, we have investigated the mechanistic role of Twist1 and Irf6 at early stages of craniofacial development. Our data indicate that TWIST1 is expressed in endocytic vesicles at the apical surface and interacts with ß/δ-catenins during neural tube closure, and Irf6 is involved in defining neural fold borders by restricting AP2α expression. Twist1 suppresses Irf6 and other epithelial genes in CNCCs during the epithelial-to-mesenchymal transition (EMT) process and cell migration. Conversely, a loss of Twist1 leads to a sustained expression of epithelial and cell adhesion markers in migratory CNCCs. Disruption of TWIST1 phosphorylation in vivo leads to epidermal blebbing, edema, neural tube defects and CNCC-derived structural abnormalities. Altogether, this study describes a previously uncharacterized function of mammalian Twist1 and Irf6 in the neural tube and CNCCs, and provides new target genes for Twist1 that are involved in cytoskeletal remodeling.

Cleavage of the Perlecan-Semaphorin 3A-Plexin A1-Neuropilin-1 (PSPN) Complex by Matrix Metalloproteinase 7/Matrilysin Triggers Prostate Cancer Cell Dyscohesion and Migration.

The Perlecan-Semaphorin 3A-Plexin A1-Neuropilin-1 (PSPN) Complex at the cell surface of prostate cancer (PCa) cells influences cell-cell cohesion and dyscohesion. We investigated matrix metalloproteinase-7/matrilysin (MMP-7)'s ability to digest components of the PSPN Complex in bone metastatic PCa cells using in silico analyses and in vitro experiments. Results demonstrated that in addition to the heparan sulfate proteoglycan, perlecan, all components of the PSPN Complex were degraded by MMP-7. To investigate the functional consequences of PSPN Complex cleavage, we developed a preformed microtumor model to examine initiation of cell dispersion after MMP-7 digestion. We found that while perlecan fully decorated with glycosaminoglycan limited dispersion of PCa microtumors, MMP-7 initiated rapid dyscohesion and migration even with perlecan present. Additionally, we found that a bioactive peptide (PLN4) found in perlecan domain IV in a region subject to digestion by MMP-7 further enhanced cell dispersion along with MMP-7. We found that digestion of the PSPN Complex with MMP-7 destabilized cell-cell junctions in microtumors evidenced by loss of co-registration of E-cadherin and F-actin. We conclude that MMP-7 plays a key functional role in PCa cell transition from a cohesive, indolent phenotype to a dyscohesive, migratory phenotype favoring production of circulating tumor cells and metastasis to bone.

Flipping the Molecular Switch: Influence of Perlecan and Its Modifiers in the Tumor Microenvironment.

The tumor microenvironment (TME) is rich in matrix components, growth factors, cytokines, and enzymatic modifiers that respond to changing conditions, to alter the fundamental properties of the tumor bed. Perlecan/HSPG2, a large, multi-domain heparan sulfate proteoglycan, is concentrated in the reactive stroma that surrounds tumors. Depending on its state in the TME, perlecan can either prevent or promote the progression of cancers to metastatic disease. Breast, prostate, lung, and renal cancers all preferentially metastasize to bone, a dense, perlecan-rich environment that is initially a "hostile" niche for cancer cells. Driven by inflammation, production of perlecan and its enzyme modifiers, which include matrix metalloproteinases (MMPs), sulfatases (SULFs), and heparanase (HPSE), increases in the reactive stroma surrounding growing and invading tumors. MMPs act upon the perlecan core protein, releasing bioactive fragments of the protein, primarily from C-terminal domains IV and V. These fragments influence cell adhesion, invasion, and angiogenesis. Sulfatases and heparanases act directly upon the heparan sulfate chains, releasing growth factors from reservoirs to reach receptors on the cancer cell surface. We propose that perlecan modifiers, by promoting the degradation of the perlecan-rich stroma, "flip the molecular switch" and convert the "hostile" stroma into a welcoming one that supports cancer dissemination and metastasis. Targeted therapies that prevent this molecular conversion of the TME should be considered as potential new therapeutics to limit metastasis.

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.

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.