Formation Of Cell-cell Junctions Research Articles

Transcriptomic profiling of Schlemm’s canal cells reveals a lymphatic-biased identity and three major cell states

Schlemm’s canal (SC) is central in intraocular pressure regulation but requires much characterization. It has distinct inner and outer walls, each composed of Schlemm’s canal endothelial cells (SECs) with different morphologies and functions. Recent transcriptomic studies of the anterior segment added important knowledge, but were limited in power by SEC numbers or did not focus on SC. To gain a more comprehensive understanding of SC biology, we performed bulk RNA sequencing on C57BL/6 J SC, blood vessel, and lymphatic endothelial cells from limbal tissue (~4,500 SECs). We also analyzed mouse limbal tissues by single-cell and single-nucleus RNA sequencing (C57BL/6 J and 129/Sj strains), successfully sequencing 903 individual SECs. Together, these datasets confirm that SC has molecular characteristics of both blood and lymphatic endothelia with a lymphatic phenotype predominating. SECs are enriched in pathways that regulate cell-cell junction formation pointing to the importance of junctions in determining SC fluid permeability. Importantly, and for the first time, our analyses characterize three molecular classes of SECs, molecularly distinguishing inner wall from outer wall SECs and discovering two inner wall cell states that likely result from local environmental differences. Further, and based on ligand and receptor expression patterns, we document key interactions between SECs and cells of the adjacent trabecular meshwork (TM) drainage tissue. Also, we present cell type expression for a collection of human glaucoma genes. These data provide a new molecular foundation that will enable the functional dissection of key homeostatic processes mediated by SECs as well as the development of new glaucoma therapeutics.

Transcriptomic profiling of Schlemm's canal cells reveals a lymphatic-biased identity and three major cell states.

Schlemm's canal (SC) is central in intraocular pressure regulation but requires much characterization. It has distinct inner and outer walls, each composed of Schlemm's canal endothelial cells (SECs) with different morphologies and functions. Recent transcriptomic studies of the anterior segment added important knowledge, but were limited in power by SEC numbers or did not focus on SC. To gain a more comprehensive understanding of SC biology, we performed bulk RNA sequencing on C57BL/6 J SC, blood vessel, and lymphatic endothelial cells from limbal tissue (~4,500 SECs). We also analyzed mouse limbal tissues by single-cell and single-nucleus RNA sequencing (C57BL/6 J and 129/Sj strains), successfully sequencing 903 individual SECs. Together, these datasets confirm that SC has molecular characteristics of both blood and lymphatic endothelia with a lymphatic phenotype predominating. SECs are enriched in pathways that regulate cell-cell junction formation pointing to the importance of junctions in determining SC fluid permeability. Importantly, and for the first time, our analyses characterize three molecular classes of SECs, molecularly distinguishing inner wall from outer wall SECs and discovering two inner wall cell states that likely result from local environmental differences. Further, and based on ligand and receptor expression patterns, we document key interactions between SECs and cells of the adjacent trabecular meshwork (TM) drainage tissue. Also, we present cell type expression for a collection of human glaucoma genes. These data provide a new molecular foundation that will enable the functional dissection of key homeostatic processes mediated by SECs as well as the development of new glaucoma therapeutics.

Transcriptomic profiling of Schlemm's canal cells reveals a lymphatic-biased identity and three major cell states.

Schlemm's canal (SC) is central in intraocular pressure regulation but requires much characterization. It has distinct inner and outer walls, each composed of Schlemm's canal endothelial cells (SECs) with different morphologies and functions. Recent transcriptomic studies of the anterior segment added important knowledge, but were limited in power by SEC numbers or did not focus on SC. To gain a more comprehensive understanding of SC biology, we performed bulk RNA sequencing on C57BL/6J SC, blood vessel, and lymphatic endothelial cells from limbal tissue (~4500 SECs). We also analyzed mouse limbal tissues by single-cell and single-nucleus RNA sequencing (C57BL/6J and 129/Sj strains), successfully sequencing 903 individual SECs. Together, these datasets confirm that SC has molecular characteristics of both blood and lymphatic endothelia with a lymphatic phenotype predominating. SECs are enriched in pathways that regulate cell-cell junction formation pointing to the importance of junctions in determining SC fluid permeability. Importantly, and for the first time, our analyses characterize 3 molecular classes of SECs, molecularly distinguishing inner wall from outer wall SECs and discovering two inner wall cell states that likely result from local environmental differences. Further, and based on ligand and receptor expression patterns, we document key interactions between SECs and cells of the adjacent trabecular meshwork (TM) drainage tissue. Also, we present cell type expression for a collection of human glaucoma genes. These data provide a new molecular foundation that will enable the functional dissection of key homeostatic processes mediated by SECs as well as the development of new glaucoma therapeutics.

Abstract 1554: LIM Domain 7 (LMO7) splice variant influences prostate cancer biology

Abstract LIM Domain 7 (LMO7) has been shown to participate in cell-cell junction formation through interaction with afadin and α-actinin. It has also been reported to collaborate with Rho kinase to regulate actin filament formation and to activate the Rho-myocardin-related transcription factor serum response factor pathway. Additionally, LMO7 itself may function as a transcription factor, regulating the expression of genes involved in Epithelial Mesenchymal Transition (EMT). Alternative splicing of LMO7 has the potential to modulate its function. Previously, we reported LMO7 as a target that is alternatively spliced in prostate cancer (PCa) between self-reported Black and White patients. In addition, by analyzing expression of LMO7 splice variants in The Cancer Genome Atlas (TCGA) data, we previously reported increased expression of a LMO7 splice variant skipping exon 12 in breast, lung and liver cancer compared with paired normal tissues. In the present study, we found that LMO7 is downregulated in metastatic PCa specimens within the Stand Up To Cancer cohort. To investigate the functional impact of expression of the LMO7 splice variant skipping exon 12 in PCa, we utilized CRISPR-Cas9 methodology to delete LMO7 exon 12 in PCa cells and assessed resulting alterations in PCa cell biology. The LMO7 exon 12 deleted PC3 PCa cells exhibited enhanced migratory potential compared with parental PCa cells. An Immunofluorescence (IF) assay showed weak staining of cell-cell junctions in LMO7 exon 12 deleted PCa cells compared with parental PCa cells. Moreover, IF staining showed retardation in f-actin in LMO7 exon 12 deleted PCa cells compared with parental PCa cells. As E-Cadherin is often inhibited during EMT, we assessed the level of E-Cadherin by western blot and real-time polymerase chain reaction (PCR). These analyses revealed that E-Cadherin protein as well as messenger RNA is suppressed in LMO7 exon 12 deleted PCa cells compared with parental PCa cells. These findings suggest a role for skipping of LMO7 exon 12 in disrupting PCa cell-cell junctions, down-regulating E-Cadherin in PCa and enhancing PCa cell migratory potential, consistent with EMT. Citation Format: Muthana Al Abo, Daniel J. George, Zefeng Wang, Steven R. Patierno, Jennifer A. Freedman, Alice Jiang. LIM Domain 7 (LMO7) splice variant influences prostate cancer biology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1554.

Dual role of CASP8AP2/FLASH in regulating epithelial-to-mesenchymal transition plasticity (EMP)

BackgroundEpithelial-to-mesenchymal transition (EMT) is a developmental program that consists of the loss of epithelial features concomitant with the acquisition of mesenchymal features. Activation of EMT in cancer facilitates the acquisition of aggressive traits and cancer invasion. EMT plasticity (EMP), the dynamic transition between multiple hybrid states in which cancer cells display both epithelial and mesenchymal markers, confers survival advantages for cancer cells in constantly changing environments during metastasis. MethodsRNAseq analysis was performed to assess genome-wide transcriptional changes in cancer cells depleted for histone regulators FLASH, NPAT, and SLBP. Quantitative PCR and Western blot were used for the detection of mRNA and protein levels. Computational analysis was performed on distinct sets of genes to determine the epithelial and mesenchymal score in cancer cells and to correlate FLASH expression with EMT markers in the CCLE collection. ResultsWe demonstrate that loss of FLASH in cancer cells gives rise to a hybrid E/M phenotype with high epithelial scores even in the presence of TGFβ, as determined by computational methods using expression of predetermined sets of epithelial and mesenchymal genes. Multiple genes involved in cell-cell junction formation are similarly specifically upregulated in FLASH-depleted cells, suggesting that FLASH acts as a repressor of the epithelial phenotype. Further, FLASH expression in cancer lines is inversely correlated with the epithelial score. Nonetheless, subsets of mesenchymal markers were distinctly up-regulated in FLASH, NPAT, or SLBP-depleted cells. ConclusionsThe ZEB1low/SNAILhigh/E-cadherinhigh phenotype described in FLASH-depleted cancer cells is driving a hybrid E/M phenotype in which epithelial and mesenchymal markers coexist.

Hydrostatic pressure under hypoxia facilitates fabrication of tissue-engineered vascular grafts derived from human vascular smooth muscle cells in vitro.

Biologically compatible vascular grafts are urgently required. The scaffoldless multi-layered vascular wall is considered to offer theoretical advantages, such as facilitating cells to form cell-cell and cell-matrix junctions and natural extracellular matrix networks. Simple methods are desired for fabricating physiological scaffoldless tissue-engineered vascular grafts. Here, we showed that periodic hydrostatic pressurization under hypoxia (HP/HYP) facilitated the fabrication of multi-layered tunica media entirely from human vascular smooth muscle cells. Compared with normoxic atmospheric pressure, HP/HYP increased expression of N-myc downstream-regulated 1 (NDRG1) and the collagen-cross-linking enzyme lysyl oxidase in human umbilical artery smooth muscle cells. HP/HYP increased N-cadherin-mediated cell-cell adhesion via NDRG1, cell-matrix interaction (i.e., clustering of integrin α5β1 and fibronectin), and collagen fibrils. We then fabricated vascular grafts using HP/HYP during repeated cell seeding and obtained 10-layered smooth muscle grafts with tensile rupture strength of 0.218 to 0.396 MPa within 5 weeks. Implanted grafts into the rat aorta were endothelialized after 1 week and patent after 5 months, at which time most implanted cells had been replaced by recipient-derived cells. These results suggest that HP/HYP enables fabrication of scaffoldless human vascular mimetics that have a spatial arrangement of cells and matrices, providing potential clinical applications for cardiovascular diseases. STATEMENT OF SIGNIFICANCE: : Tissue-engineered vascular grafts (TEVGs) are theoretically more biocompatible than prosthetic materials in terms of mechanical properties and recipient cell-mediated tissue reconstruction. Although some promising results have been shown, TEVG fabrication processes are complex, and the ideal method is still desired. We focused on the environment in which the vessels develop in utero and found that mechanical loading combined with hypoxia facilitated formation of cell-cell and cell-matrix junctions and natural extracellular matrix networks in vitro, which resulted in the fabrication of multi-layered tunica media entirely from human umbilical artery smooth muscle cells. These scaffoldless TEVGs, produced using a simple process, were implantable and have potential clinical applications for cardiovascular diseases.

Abstract 1480: ZEB1 dimerization is regulated by acetylation and promotes protein stability, interactions, EMT, and NSCLC metastasis

Abstract Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide due to the ability of cancer cells to metastasize. Therefore, it is essential to expand our current knowledge of the biological processes that contribute to metastasis to guide the discovery of novel therapeutic modalities. The Epithelial-to-mesenchymal transition (EMT) is a mechanism for the acquisition of malignant cells, which causes epithelial cells with specialized cell-cell contacts to gain mesenchymal migratory capacity. High expression of the Zinc finger E-box binding homeobox 1 (ZEB1) transcription factor is correlated with poor clinical outcomes of cancer, including therapeutic resistance and EMT-mediated metastasis. ZEB1 has a calculated molecular weight of 125kDa; however, numerous groups have reported discrepancies in the observed molecular weight (approximately 190-250kDa). This has been attributed to dimerization mediated by post-translational modifications (PTMs). Therefore, we performed mass spectrometry and identified a novel PTM - K811 acetylation - that may regulate ZEB1 dimerization and function. To define the role of ZEB1 acetylation, we generated ZEB1 acetyl mimetic (K811Q) and deficient (K811R) mutant-expressing NSCLC cell lines. We hypothesize that ZEB1 dimerization is regulated by K811 acetylation to promote protein stability and lung adenocarcinoma metastasis. We determined that the K811R ZEB1 (125 kDa) has a shorter half-life wild-type and high ubiquitination rate than (WT) ZEB1 and K811Q ZEB1 (~225 kDa), suggesting that disruption of ZEB1 acetylation hinders its dimerization and protein stability. Further, ZEB1 dimer recruits the nucleosome remodeling and deacetylase (NuRD) complex to bind the promoter of its target genes mir200c-141 and SEMA3F. RNA-sequencing revealed that unlike K811R ZEB1, WT ZEB1 and K811Q ZEB1 downregulate the expression of gene sets involved in tight junction, apical junction, and cell-cell junction formation. We also identified six novel ZEB1 acetylation-dependent gene candidates that function in signaling pathways and epithelial cell maintenance; CBLN3, STAP2, PLEKHG6, LAD1, RNF43, and LLGL2. In vitro 2D migration Boyden chamber, 3D invasion assays, and in vivo studies demonstrated that ZEB1 acetylation promotes the invasive and metastatic potential of the NSCLC cell line. Our findings suggest that K811 acetylation regulates ZEB1 dimerization and stability to promote NuRD interaction and lung adenocarcinoma via EMT. Future studies are focused on assessing changes in gene expression programs and chromatin accessibility that are ZEB1 dimerization dependent and that could contribute to NSCLC metastasis. Citation Format: Mabel Giselle Perez-Oquendo, Roxsan Manshouri, Yanhua Tian, Jared Fradette, Don Gibbons. ZEB1 dimerization is regulated by acetylation and promotes protein stability, interactions, EMT, and NSCLC metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1480.

Nectins and Nectin-like molecules drive vascular development and barrier function.

Angiogenesis, barriergenesis, and immune cell migration are all key physiological events that are dependent on the functional characteristics of the vascular endothelium. The protein family of Nectins and Nectin-like molecules (Necls) is a group of cell adhesion molecules that are widely expressed by different endothelial cell types. The family includes four Nectins (Nectin-1 to -4) and five Necls (Necl-1 to -5) that either interact with each other by forming homo- and heterotypical interactions or bind to ligands expressed within the immune system. Nectin and Necl proteins are mainly described to play a role in cancer immunology and in the development of the nervous system. However, Nectins and Necls are underestimated players in the formation of blood vessels, their barrier properties, and in guiding transendothelial migration of leukocytes. This review summarizes their role in supporting the endothelial barrier through their function in angiogenesis, cell-cell junction formation, and immune cell migration. In addition, this review provides a detailed overview of the expression patterns of Nectins and Necls in the vascular endothelium.

Deficiency of exchange protein directly activated by cAMP (EPAC)-1 in mice augments glucose intolerance, inflammation, and gut dysbiosis associated with Western diet

BackgroundGut microbiota (GM) dysregulation, known as dysbiosis, has been proposed as a crucial driver of obesity associated with “Western” diet (WD) consumption. Gut dysbiosis is associated with increased gut permeability, inflammation, and insulin resistance. However, host metabolic pathways implicated in the pathophysiology of gut dysbiosis are still elusive. Exchange protein directly activated by cAMP (Epac) plays a critical role in cell-cell junction formation and insulin secretion. Here, we used homozygous Epac1-knockout (Epac1–/–), Epac2-knockout (Epac2–/–), and wild-type (WT) mice to investigate the role of Epac proteins in mediating gut dysbiosis, gut permeability, and inflammation after WD feeding.ResultsThe 16S rRNA gene sequencing of fecal DNA showed that the baseline GM of Epac2–/–, but not Epac1–/–, mice was represented by a significantly higher Firmicutes to Bacteroidetes ratio and significant alterations in several taxa compared to WT mice, suggesting that Epac2–/– mice had gut dysbiosis under physiological conditions. However, an 8-week WD led to a similar gut microbiome imbalance in mice regardless of genotype. While Epac1 deficiency modestly exacerbated the WD-induced GM dysbiosis, the WD-fed Epac2–/– mice had a more significant increase in gut permeability than corresponding WT mice. After WD feeding, Epac1–/–, but not Epac2–/–, mice had significantly higher mRNA levels of tumor necrosis factor-alpha (TNF-α) and F4/80 in the epididymal white adipose tissue (EWAT), increased circulating lipocalin-2 protein and more severe glucose intolerance, suggesting greater inflammation and insulin resistance in WD-fed Epac1–/– mice than corresponding WT mice. Consistently, Epac1 protein expression was significantly reduced in the EWAT of WD-fed WT and Epac2–/– mice.ConclusionDespite significantly dysregulated baseline GM and a more pronounced increase in gut permeability upon WD feeding, WD-fed Epac2–/– mice did not exhibit more severe inflammation and glucose intolerance than corresponding WT mice. These findings suggest that the role of gut dysbiosis in mediating WD-associated obesity may be context-dependent. On the contrary, we demonstrate that deficiency of host signaling protein, Epac1, drives inflammation and glucose intolerance which are the hallmarks of WD-induced obesity.2Aidzb_xKQhqE4JPFGVMhLVideo abstract

Rho and Rab Family Small GTPases in the Regulation of Membrane Polarity in Epithelial Cells.

Membrane polarity, defined as the asymmetric distribution of lipids and proteins in the plasma membrane, is a critical prerequisite for the development of multicellular tissues, such as epithelia and endothelia. Membrane polarity is regulated by polarized trafficking of membrane components to specific membrane domains and requires the presence of intramembrane diffusion barriers that prevent the intermixing of asymmetrically distributed membrane components. This intramembrane diffusion barrier is localized at the tight junctions (TJs) in these cells. Both the formation of cell-cell junctions and the polarized traffic of membrane proteins and lipids are regulated by Rho and Rab family small GTPases. In this review article, we will summarize the recent developments in the regulation of apico-basal membrane polarity by polarized membrane traffic and the formation of the intramembrane diffusion barrier in epithelial cells with a particular focus on the role of Rho and Rab family small GTPases.

A commercial ARHGEF17/TEM4 antibody cross-reacts with Nuclear Mitotic Apparatus protein 1 (NuMA).

The Rho family Guanine nucleotide exchange factor (GEF) ARHGEF17 (also known as TEM4) is a large protein with only 3 annotated regions: an N-terminal actin-binding domain, a Rho-specific dbl homology (DH)- pleckstrin homology (PH) type GEF domain and a seven bladed β propeller fold at the C-terminus with unknown function. TEM4 has been implicated in numerous activities that rely on regulation of the cytoskeleton including cell migration, cell-cell junction formation and the spindle assembly checkpoint during mitosis. Here we have assessed the specificity of a TEM4 polyclonal antibody that has been commonly used as a Western blotting and immunocytochemistry probe for TEM4 in mammalian cells. We find that this antibody, in addition to its intended target, cross-reacts with the Nuclear Mitotic Apparatus Protein 1 (NuMA) in Western blotting and immunoprecipitation, and detects NuMA preferentially in immunocytochemistry. This cross-reactivity, with an abundant chromatin- and mitotic spindle-associated factor, is likely to affect the interpretation of experiments that make use of this antibody probe, in particular by immunocytochemistry and immunoprecipitation.

Surface micropatterning for the formation of an in vitro functional endothelial model for cell-based biosensors

Label-free biosensing, such as with surface plasmon resonance (SPR), is a highly efficient method for monitoring the responses of living cells exposed to pharmacological agents and biochemical stimuli in vitro. Conventional cell culture protocols used in cell-based biosensing generally provide little direct control over cell morphologies and phenotypes. Surface micropatterning techniques have been exploited for the controlled immobilization and establishment of well-defined cell morphologies and phenotypes. In this article, surface adhesion micropatterns are used to control the adhesion of endothelial cells within adjacent hexagonal microstructures to promote the emergence of a well-controlled and standardized cell layer phenotype onto SPR sensor surfaces. We show that the formation of cell-cell junctions can be controlled by tuning the inter-cellular spacing in groups of 3 neighbouring cells. Fluorescence microscopy was used to confirm the formation of vascular endothelium cadherin junctions, a structural marker of a functional endothelium. In order to confirm the functionality of the proposed model, the response to thrombin, a modulator of endothelium integrity, was monitored by surface plasmon resonance imaging (SPRI). Experiments demonstrate the potential of the proposed model as a primary biological signal transducer for SPRI-based analysis, with potential applications in cell biology, pharmacology and diagnostic.

Abstract 324: Discovery of novel functional TROP2 antibodies for treatment of epithelial cancers

Abstract Trophoblast cell surface antigen-2 (TROP2) is a membrane-bound protein expressed during development and at lower levels in normal adult tissues. TROP2 has pleiotropic functions including formation of cell-cell junctions by mediating interactions with extracellular and intracellular proteins. In situations of TROP2 deficiency its role in normal tissues appears to be compensated by expression of EpCAM, a highly related family member. In cancer, the levels of TROP2 are increased in many epithelial tumor types. Overexpression of TROP2 confer oncogenic behavior in several in vitro and in vivo models. Both its extracellular and intracellular domains play a role in modulating interactions with ligands and/or receptors and with intracellular signaling partners to result in an oncogenic phenotype. In many solid tumors TROP2 upregulation is associated with increased tumor aggressiveness, metastasis, and decreased survival in large groups of difficult-to-treat cancers, making it an attractive target for cancer therapy. TROP2-based ADCs have been developed and one has been approved for therapeutic use in triple negative breast cancer and advanced bladder cancer. The toxicity associated with the toxin payload limits their use including in combination with chemotherapy in the earlier lines of treatment, and by the development of resistance to the payload. We isolated several anti-TROP2 heavy chain-only antibodies (HCAb) by immunization of a proprietary transgenic HCAb mouse. In this abstract, we describe the properties and activity of several of these antibodies. These antibodies are characterized by potent in vivo antitumor activity in established tumor models in NCG mice in the absence of ADCC, and by the inhibition of tumor cell viability in vitro, directly and in the absence of any effector cells. In NCG mice, which are characterized by a short antibody half-life, anti-TROP2 HCAbs displayed a T1/2 of approximately 3.5 days. These antibodies displayed very potent anti-tumor activity in established tumor xenograft models, causing tumor regression at 2 mg/kg and 80% TGI at 0.5 mg/kg in NCG mice when administered once per week. They also caused tumor regression when dosed once every three weeks in NCG mice and in tumors of very large sizes. Therefore, these antibodies directly target one or more aspects of TROP2 tumor biology that have a profound and long-lasting effect on the cancer cells. Upon glycoengineering, the afucosylated version of the antibodies displayed a strongly enhanced ADCC activity. In vitro they are characterized by different internalization rates which define several non-overlapping epitopes on TROP2. They are characterized by picomolar binding affinity to recombinant human TROP2 as determined by SPR. These antibodies bind with greater affinity to human and cyno but bind poorly to mouse, rat and rabbit. These potent naked TROP2 antibodies represent new opportunities to address TROP2 expressing cancers. Citation Format: Israel Matos, Yahya Ashraf, Hiba Zahreddine, Liying Gong, Amanda Baumholtz, Alex Zhou, Claire Bonfils, Aniel Moya-Torres, Yun Cui, Xiaowei Wang, Richard Wargachuk, Tiffany Cheng, Elijus Undzys, Shugang Yao, Jacynthe Linda Tolouse, Dominic Hou, Gordon Ngan, Luis daCruz, David Young. Discovery of novel functional TROP2 antibodies for treatment of epithelial cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 324.

The CCAAT/Enhancer Binding Protein Beta (cebpb) is essential for the development of enveloping layer (EVL) in zebrafish

Epiboly, the coordinated spreading of the enveloping layer (EVL), the yolk syncytial layer (YSL) and the deep cells (DCs) toward the vegetal pole, is required for gastrulation. Interference with the EVL leads to early developmental failure. However, the genetic factors underlying EVL differentiation are so far unknown. By in-depth analyzing of single-cell sequencing data from 4 h post fertilization zebrafish embryos, we identified 105 genes (FDR<0.05) which showed highly specific EVL expression, among which the transcription factor cebpb and genes (krt18, cldne, cldnb, krt4, krt5) involved in cell-cell junction formation were exclusively expressed in EVL. To explore the role of cebpb in EVL differentiation, we knocked down its expression by morpholino-labeled antisense RNA. The morphant embryos showed disrupted EVL and developmental termination prior to epiboly. In accordance, expression of the EVL specific cell-adhesion molecule genes were significantly reduced. Thus, cebpb is the earliest EVL marker and essential for EVL differentiation in zebrafish. The elucidation of the specific EVL gene set provides us new insights into the molecular mechanisms underlying EVL differentiation.

Overriding native cell coordination enhances external programming of collective cell migration

As collective cell migration is essential in biological processes spanning development, healing, and cancer progression, methods to externally program cell migration are of great value. However, problems can arise if the external commands compete with strong, preexisting collective behaviors in the tissue or system. We investigate this problem by applying a potent external migratory cue-electrical stimulation and electrotaxis-to primary mouse skin monolayers where we can tune cell-cell adhesion strength to modulate endogenous collectivity. Monolayers with high cell-cell adhesion showed strong natural coordination and resisted electrotactic control, with this conflict actively damaging the leading edge of the tissue. However, reducing preexisting coordination in the tissue by specifically inhibiting E-cadherin-dependent cell-cell adhesion, either by disrupting the formation of cell-cell junctions with E-cadherin-specific antibodies or rapidly dismantling E-cadherin junctions with calcium chelators, significantly improved controllability. Finally, we applied this paradigm of weakening existing coordination to improve control and demonstrate accelerated wound closure in vitro. These results are in keeping with those from diverse, noncellular systems and confirm that endogenous collectivity should be considered as a key quantitative design variable when optimizing external control of collective migration.

TRAF2 Is a Novel Ubiquitin E3 Ligase for the Na,K-ATPase β-Subunit That Drives Alveolar Epithelial Dysfunction in Hypercapnia.

Several acute and chronic lung diseases are associated with alveolar hypoventilation leading to accumulation of CO2 (hypercapnia). The β-subunit of the Na,K-ATPase plays a pivotal role in maintaining epithelial integrity by functioning as a cell adhesion molecule and regulating cell surface stability of the catalytic α-subunit of the transporter, thereby, maintaining optimal alveolar fluid balance. Here, we identified the E3 ubiquitin ligase for the Na,K-ATPase β-subunit, which promoted polyubiquitination, subsequent endocytosis and proteasomal degradation of the protein upon exposure of alveolar epithelial cells to elevated CO2 levels, thus impairing alveolar integrity. Ubiquitination of the Na,K-ATPase β-subunit required lysine 5 and 7 and mutating these residues (but not other lysines) prevented trafficking of Na,K-ATPase from the plasma membrane and stabilized the protein upon hypercapnia. Furthermore, ubiquitination of the Na,K-ATPase β-subunit was dependent on prior phosphorylation at serine 11 by protein kinase C (PKC)-ζ. Using a protein microarray, we identified the tumor necrosis factor receptor-associated factor 2 (TRAF2) as the E3 ligase driving ubiquitination of the Na,K-ATPase β-subunit upon hypercapnia. Of note, prevention of Na,K-ATPase β-subunit ubiquitination was necessary and sufficient to restore the formation of cell-cell junctions under hypercapnic conditions. These results suggest that a hypercapnic environment in the lung may lead to persistent epithelial dysfunction in affected patients. As such, the identification of the E3 ligase for the Na,K-ATPase may provide a novel therapeutic target, to be employed in patients with acute or chronic hypercapnic respiratory failure, aiming to restore alveolar epithelial integrity.

Proteomics of purified lamellocytes from Drosophila melanogaster HopTum-l identifies new membrane proteins and networks involved in their functions

In healthy Drosophila melanogaster larvae, plasmatocytes and crystal cells account for 95% and 5% of the hemocytes, respectively. A third type of hemocytes, lamellocytes, are rare, but their number increases after oviposition by parasitoid wasps. The lamellocytes form successive layers around the parasitoid egg, leading to its encapsulation and melanization, and finally the death of this intruder. However, the total number of lamellocytes per larva remains quite low even after parasitoid infestation, making direct biochemical studies difficult. Here, we used the HopTum-l mutant strain that constitutively produces large numbers of lamellocytes to set up a purification method and analyzed their major proteins by 2D gel electrophoresis and their plasma membrane surface proteins by 1D SDS-PAGE after affinity purification. Mass spectrometry identified 430 proteins from 2D spots and 344 affinity-purified proteins from 1D bands, for a total of 639 unique proteins. Known lamellocyte markers such as PPO3 and the myospheroid integrin were among the components identified with specific chaperone proteins. Affinity purification detected other integrins, as well as a wide range of integrin-associated proteins involved in the formation and function of cell-cell junctions. Overall, the newly identified proteins indicate that these cells are highly adapted to the encapsulation process (recognition, motility, adhesion, signaling), but may also have several other physiological functions (such as secretion and internalization of vesicles) under different signaling pathways. These results provide the basis for further in vivo and in vitro studies of lamellocytes, including the development of new markers to identify coexisting populations and their respective origins and functions in Drosophila immunity.

Soy Protein Remnants Digested by Gastro-duodenal Proteases can Alter Microbial Interactions and Intestinal Cholesterol Absorption

Background: Although the food quality of soy protein is known to be as good as that of animal proteins, some soybean proteins are not susceptible to digestion and remain undigested in the intestine. We hypothesized that digestion-resistant soy proteins might interact with the intestinal membrane, microbes, and metabolites, and change the intestinal physiology or the profile of the gut microbiome. Objective: To identify the Protease-Resistant Soy Proteins (PRSPs) and their interaction with intestinal membrane proteins by MS, and to assess the functions of PRSPs in the small intestine. Methods: Soy proteins were sequentially digested with pepsin and pancreatin, and the PRSPs were identified by SDS-PAGE and MS. Intestinal cell membrane proteins interacting with PRSPs were isolated by affinity purification and photo-affinity crosslinking, and identified using MS/MS. Inhibition of cholesterol absorption to lipoprotein-depleted intestinal cells, CaCo-2, and hepatic cells, HepG2, was measured in the presence and absence of PRSPs. FITC-conjugated Gram-positive, Lactobacillus plantarum, and Gram-negative bacteria, Escherichia coli, were incubated with Ca- Co-2 cells in the presence of PRSPs to investigate the regulation of bacterial cell binding to intestinal epithelial cells by PRSPs. Results: MS/MS of PRSPs identified glycinin, β-conglycinin, trypsin inhibitors, lipoxygenase, and sucrose-binding protein. MS analysis also identified the intestinal membrane proteins bound to PRSPs. The functions of the identified interacting proteins included ion transportation, carbohydrate- binding, cytoskeleton formation, hydrolysis, cell-cell junction formation, and cholesterol/steroid- binding. In particular, apolipoprotein E, aminopeptidase N, and Niemann-Pick C1-like protein 1 are known to be involved in cholesterol absorption in the small intestine. The inhibition of cholesterol absorption by CaCo-2 and HepG2 cells by PRSPs confirmed the MS results. Binding of L. plantarum and E. coli to CaCo-2 cells was efficiently inhibited by PRSPs. Conclusion: PRSPs can interact with intestinal membrane proteins, and regulate cholesterol absorption by intestinal epithelial cell and interactions of the gut microbiome. Soy protein in the intestine acts as a nutrient, and triggers changes in intestinal functions by interacting with intestinal cells, microorganisms, and nutrients. These findings will provide valuable new functional information about the effects of soy proteins on human health.

Computational Model of E-cadherin Clustering under Cortical Tension

E-cadherins are adhesion proteins that play a critical role in the formation of cell-cell junctions for several physiological processes, including tissue development and homeostasis. The formation of E-cadherin clusters involves extracellular trans- and cis- associations between cadherin ectodomains and stabilization through intracellular coupling with the contractile actomyosin cortex. The dynamic remodeling of cell-cell junctions largely depends on cortical tension, but previous modeling frameworks did not incorporate this effect. In order to gain insights into the effects of cortical tension on the dynamic properties of E-cadherin clusters, here we developed a computational model based on Brownian dynamics. The model considers individual cadherins as explicit point particles undergoing cycles of lateral diffusion on two parallel surfaces that mimic the membrane of neighboring cells. E-cadherins transit between functional states, including monomers, X- and S- dimers, that are able to selectively bind and unbind other E-cadherins on the same and/or opposite surface(s), and associate with the actomyosin cytoskeleton to stabilize their interactions and transmit tension. Kinetic rates for binding and unbinding are taken from previous experimental measurements, with unbinding rates reproducing catch or slip bonds lifetimes, depending on the E-cadherin functional state. Results from the model show that cortical tension governs the fraction of E-cadherins in clusters and the distribution of cluster size. For low forces (below 10 pN), a large amount of small clusters form, including less than 5 E-cadherins each, with the majority of E-cadherins in clusters. At higher forces, the probability of forming fewer but larger clusters increases, with most E-cadherins not in clusters. These findings support the general idea of force-dependent reinforcement of cell-cell junctions and are consistent with differences in cluster sizes observed between apical and lateral junctions in epithelial tissues.

PLEKHG4B enables actin cytoskeletal remodeling during epithelial cell-cell junction formation.

Cell-cell junction formation requires actin cytoskeletal remodeling. Here, we show that PLEKHG4B, a Rho-guanine nucleotide exchange factor (Rho-GEF), plays a crucial role in epithelial cell-cell junction formation. Knockdown of PLEKHG4B decreased Cdc42 activity and tended to increase RhoA activity in A549 cells. A549 monolayer cells showed 'closed junctions' with closely packed actin bundles along the cell-cell contacts, but PLEKHG4B knockdown suppressed closed junction formation, and PLEKHG4B-knockdown cells exhibited 'open junctions' with split actin bundles located away from the cell-cell boundary. In Ca2+-switch assays, PLEKHG4B knockdown delayed the conversion of open junctions to closed junctions and β-catenin accumulation at cell-cell junctions. Furthermore, PLEKHG4B knockdown abrogated the reduction in myosin activity normally seen in the later stage of junction formation. The aberrant myosin activation and impairments in closed junction formation in PLEKHG4B-knockdown cells were reverted by ROCK inhibition or LARG/PDZ-RhoGEF knockdown. These results suggest that PLEKHG4B enables actin remodeling during epithelial cell-cell junction maturation, probably by reducing myosin activity in the later stage of junction formation, through suppressing LARG/PDZ-RhoGEF and RhoA-ROCK pathway activities. We also showed that annexin A2 participates in PLEKHG4B localization to cell-cell junctions.This article has an associated First Person interview with the first author of the paper.