scholarly journals A Microtiter Assay for Quantifying Protein-Protein Interactions Associated with Cell-Cell Adhesion

2007 ◽  
Vol 12 (5) ◽  
pp. 683-693 ◽  
Author(s):  
Nicholas A. Graham ◽  
Melissa D. Pope ◽  
Tharathorn Rimchala ◽  
Beijing K. Huang ◽  
Anand R. Asthagiri
Keyword(s):  
Protein Complex ◽  
Crucial Role ◽  
Cell Biology ◽  
Transmembrane Protein ◽  
Critical Role ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dramatic Improvement ◽  
E Cadherin ◽  
Cell Cell

Cell-cell adhesions are a hallmark of epithelial tissues, and the disruption of these contacts plays a critical role in both the early and late stages of oncogenesis. The interaction between the transmembrane protein E-cadherin and the intracellular protein β-catenin plays a crucial role in the formation and maintenance of epithelial cell-cell contacts and is known to be downregulated in many cancers. The authors have developed a protein complex enzyme-linked immunosorbent assay (ELISA) that can quantify the amount of β-catenin bound to E-cadherin in unpurified whole-cell lysates with a Z′ factor of 0.74. The quantitative nature of the E-cadherin:β-catenin ELISA represents a dramatic improvement over the low-throughput assays currently used to characterize endogenous E-cadherin:β-catenin complexes. In addition, the protein complex ELISA format is compatible with standard sandwich ELISAs for parallel measurements of total levels of endogenous E-cadherin and β-catenin. In 2 case studies closely related to cancer cell biology, the authors use the protein complex ELISA and traditional sandwich ELISAs to provide a detailed, quantitative picture of the molecular changes occurring within adherens junctions in vivo. Because the E-cadherin: β-catenin protein complex plays a crucial role in oncogenesis, this protein complex ELISA may prove to be a valuable quantitative prognostic marker of tumor progression. ( Journal of Biomolecular Screening 2007:683-693)

Rescuing the N-cadherin knockout by cardiac-specific expression of N- or E-cadherin

Development ◽  
2001 ◽  
Vol 128 (4) ◽  
pp. 459-469
Author(s):  
Y. Luo ◽  
M. Ferreira-Cornwell ◽  
H. Baldwin ◽  
I. Kostetskii ◽  
J. Lenox ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Heart Development ◽  
Critical Role ◽  
Embryonic Cells ◽  
Wild Type ◽  
Specific Expression ◽  
Developmental Defects ◽  
E Cadherin ◽  
Cell Cell

Cell-cell adhesion mediated by some members of the cadherin family is essential for embryonic survival. The N-cadherin-null embryo dies during mid-gestation, with multiple developmental defects. We show that N-cadherin-null embryos expressing cadherins using muscle-specific promoters, alpha- or beta-myosin heavy chain, are partially rescued. Somewhat surprisingly, either N-cadherin or E-cadherin was effective in rescuing the embryos. The rescued embryos exhibited an increased number of somites, branchial arches and the presence of forelimb buds; however, in contrast, brain development was severely impaired. In rescued animals, the aberrant yolk sac morphology seen in N-cadherin-null embryos was corrected, demonstrating that this phenotype was secondary to the cardiac defect. Dye injection studies and analysis of chimeric animals that have both wild-type and N-cadherin-null cells support the conclusion that obstruction of the cardiac outflow tract represents a major defect that is likely to be the primary cause of pericardial swelling seen in null embryos. Although rescued embryos were more developed than null embryos, they were smaller than wild-type embryos, even though the integrity of the cardiovascular system appeared normal. The smaller size of rescued embryos may be due, at least in part, to increased apoptosis observed in tissues not rescued by transgene expression, indicating that N-cadherin-mediated cell adhesion provides an essential survival signal for embryonic cells. Our data provide in vivo evidence that cadherin adhesion is essential for cell survival and for normal heart development. Our data also show that E-cadherin can functionally substitute for N-cadherin during cardiogenesis, suggesting a critical role for cadherin-mediated cell-cell adhesion, but not cadherin family member-specific signaling, at the looping stage of heart development.

scholarly journals Hydroxychloroquine Synergizes With The PI3K Inhibitor BKM120 to Exhibit Antitumor Efficacy Independent of Autophagy

2021 ◽  
Author(s):  
Xin Peng ◽  
Shaolu Zhang ◽  
Wenhui Jiao ◽  
Zhenxing Zhong ◽  
Yuqi Yang ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Cell Biology ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Single Agent ◽  
Antitumor Efficacy ◽  
Antitumor Effects ◽  
Autophagy Inhibitor

Abstract Background: The critical role of phosphoinositide 3-kinase (PI3K) activation in tumor cell biology has prompted massive efforts to develop PI3K inhibitors (PI3Kis) for cancer therapy. However, recent results from clinical trials have shown only a modest therapeutic efficacy of single-agent PI3Kis in solid tumors. Targeting autophagy has controversial context-dependent effects in cancer treatment. As a FDA-approved lysosomotropic agent, hydroxychloroquine (HCQ) has been well tested as an autophagy inhibitor in preclinical models. Here, we elucidated the novel mechanism of HCQ alone or in combination with PI3Ki BKM120 in the treatment of cancer.Methods: The antitumor effects of HCQ and BKM120 on three different types of tumor cells were assessed by in vitro PrestoBlue assay, colony formation assay and in vivo zebrafish and nude mouse xenograft models. The involved molecular mechanisms were investigated by MDC staining, LC3 puncta formation assay, immunofluorescent assay, flow cytometric analysis of apoptosis and ROS, qRT-PCR, Western blot, comet assay, homologous recombination (HR) assay and immunohistochemical staining. Results: HCQ significantly sensitized cancer cells to BKM120 in vitro and in vivo. Interestingly, the sensitization mediated by HCQ could not be phenocopied by treatment with other autophagy inhibitors (Spautin-1, 3-MA and bafilomycin A1) or knockdown of the essential autophagy genes Atg5/Atg7, suggesting that the sensitizing effect might be mediated independent of autophagy status. Mechanistically, HCQ induced ROS production and activated the transcription factor NRF2. In contrast, BKM120 prevented the elimination of ROS by inactivation of NRF2, leading to accumulation of DNA damage. In addition, HCQ activated ATM to enhance HR repair, a high-fidelity repair for DNA double-strand breaks (DSBs) in cells, while BKM120 inhibited HR repair by blocking the phosphorylation of ATM and the expression of BRCA1/2 and Rad51. Conclusions: Our study revealed that HCQ and BKM120 synergistically increased DSBs in tumor cells and therefore augmented apoptosis, resulting in enhanced antitumor efficacy. Our findings provide a new insight into how HCQ exhibits antitumor efficacy and synergizes with PI3Ki BKM120, and warn that one should consider the “off target” effects of HCQ when used as autophagy inhibitor in the clinical treatment of cancer.

Integrin-linked kinase is localized to cell-matrix focal adhesions but not cell-cell adhesion sites and the focal adhesion localization of integrin-linked kinase is regulated by the PINCH-binding ANK repeats

1999 ◽  
Vol 112 (24) ◽  
pp. 4589-4599 ◽  
Author(s):  
F. Li ◽  
Y. Zhang ◽  
C. Wu
Keyword(s):  
Focal Adhesion ◽  
Critical Role ◽  
Focal Adhesions ◽  
Subcellular Compartment ◽  
Cell Matrix ◽  
Integrin Binding Site ◽  
Integrin Linked Kinase ◽  
Cell Cell

Integrin-linked kinase (ILK) is a ubiquitously expressed protein serine/threonine kinase that has been implicated in integrin-, growth factor- and Wnt-signaling pathways. In this study, we show that ILK is a constituent of cell-matrix focal adhesions. ILK was recruited to focal adhesions in all types of cells examined upon adhesion to a variety of extracellular matrix proteins. By contrast, ILK was absent in E-cadherin-mediated cell-cell adherens junctions. In previous studies, we have identified PINCH, a protein consisting of five LIM domains, as an ILK binding protein. We demonstrate in this study that the ILK-PINCH interaction requires the N-terminal-most ANK repeat (ANK1) of ILK and one (the C-terminal) of the two zinc-binding modules within the LIM1 domain of PINCH. The ILK ANK repeats domain, which is capable of interacting with PINCH in vitro, could also form a complex with PINCH in vivo. However, the efficiency of the complex formation or the stability of the complex was markedly reduced in the absence of the C-terminal domain of ILK. The PINCH binding defective ANK1 deletion ILK mutant, unlike the wild-type ILK, was unable to localize and cluster in focal adhesions, suggesting that the interaction with PINCH is necessary for focal adhesion localization and clustering of ILK. The N-terminal ANK repeats domain, however, is not sufficient for mediating focal adhesion localization of ILK, as an ILK mutant containing the ANK repeats domain but lacking the C-terminal integrin binding site failed to localize in focal adhesions. These results suggest that focal adhesions are a major subcellular compartment where ILK functions in intracellular signal transduction, and provide important evidence for a critical role of PINCH and integrins in regulating ILK cellular function.

Contributions of extracellular and intracellular domains of full length and chimeric cadherin molecules to junction assembly in epithelial cells

1998 ◽  
Vol 111 (9) ◽  
pp. 1305-1318 ◽  
Author(s):  
S.M. Norvell ◽  
K.J. Green
Keyword(s):  
Epithelial Cells ◽  
Cell Surface ◽  
Extracellular Domain ◽  
Full Length ◽  
Intracellular Domain ◽  
Epithelial Junctions ◽  
Intracellular Domains ◽  
E Cadherin ◽  
Cell Cell

The integrity of cell-cell junctions in epithelial cells depends on functional interactions of both extracellular and intracellular domains of cadherins with other junction proteins. To examine the roles of the different domains of E-cadherin and desmoglein in epithelial junctions, we stably expressed full length desmoglein 1 and chimeras of E-cadherin and desmoglein 1 in A431 epithelial cells. Full length desmoglein 1 was able to incorporate into or disrupt endogenous desmosomes depending on expression level. Each of the chimeric cadherin molecules exhibited distinct localization patterns at the cell surface. A chimera of the desmoglein 1 extracellular domain and the E-cadherin intracellular domain was distributed diffusely at the cell surface while the reverse chimera, comprising the E-cadherin extracellular domain and the desmoglein 1 intracellular domain, localized in large, sometimes contiguous patches at cell-cell interfaces. Nevertheless, both constructs disrupted desmosome assembly. Expression of constructs containing the desmoglein 1 cytoplasmic domain resulted in approximately a 3-fold decrease in E-cadherin bound to plakoglobin and a 5- to 10-fold reduction in the steady-state levels of the endogenous desmosomal cadherins, desmoglein 2 and desmocollin 2, possibly contributing to the dominant negative effect of the desmoglein 1 tail. In addition, biochemical analysis of protein complexes in the stable lines revealed novel in vivo protein interactions. Complexes containing beta-catenin and desmoglein 1 were identified in cells expressing constructs containing the desmoglein 1 tail. Furthermore, interactions were identified between endogenous E-cadherin and the chimera containing the E-cadherin extracellular domain and the desmoglein 1 intracellular domain providing in vivo evidence for previously predicted lateral interactions of E-cadherin extracellular domains.

LncRNA MALAT1 exhibits positive effects on nucleus pulposus cell biology in vivo and in vitro by sponging miR-503

2020 ◽  
Author(s):  
Hongyu Zheng ◽  
Tingting Wang ◽  
Xiangmin Li ◽  
Wei He ◽  
Zhiqiang Gong ◽  
...  
Keyword(s):  
Nucleus Pulposus ◽  
Disc Degeneration ◽  
Cell Biology ◽  
Mapk Pathway ◽  
Critical Role ◽  
Mapk Signaling ◽  
Positive Effects ◽  
Lncrna Malat1

Abstract Background: Intervertebral disc degeneration (IDD) is characterized by the loss of nucleus pulposus cells (NPCs) and phenotypic abnormalities. Accumulating evidence suggests that long noncoding RNAs (lncRNAs) are involved in the pathogenesis of IDD. In this study, we aimed to investigate the functional effects of lncRNA MALAT1 on NPCs in IDD and the possible mechanism governing these effects. Results: We validated the decreased expression of MALAT1 in the IDD tissues, which was associated with decreased Collagen II and Aggrecan expression. In vitro, overexpressed MALAT1 could attenuate the effect of IL-1β on NPC proliferation, apoptosis, and Aggrecan degradation. In vivo, MALAT1 overexpression attenuated the severity of disc degeneration in IDD model rats. Our molecular study further demonstrated that MALAT1 could sponge miR-503, modulate the expression of miR-503, and activate downstream MAPK signaling pathways. The effects of MALAT1 on NPCs were partially reversed/aggregated by miR-503 mimics/inhibitor treatment. Conclusion: Our data suggested that the MALAT1-miR-503-MAPK pathway plays a critical role in NPCs, which may be a potential strategy for alleviating IDD.

Characterization of monoclonal antibodies recognizing each extracellular loop domain of occludin

2019 ◽  
Vol 166 (4) ◽  
pp. 297-308 ◽  
Author(s):  
Yoshimi Shimizu ◽  
Yoshitaka Shirasago ◽  
Takeru Suzuki ◽  
Tomoyuki Hata ◽  
Masuo Kondoh ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Transmembrane Protein ◽  
Site Directed Mutagenesis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Intact Cells ◽  
Flow Cytometric ◽  
Junction Protein ◽  
Fcm Analysis

Abstract The tight junction protein occludin (OCLN) is a four-pass transmembrane protein with two extracellular loops (ELs), and also functions as a co-receptor for hepatitis C virus (HCV). Recently, we reported the establishment of monoclonal antibodies (mAbs) recognizing each intact EL domain of OCLN that can strongly prevent HCV infection in vitro and in vivo, and these mAbs were applicable for flow cytometric (FCM) analysis, immunocytochemistry (ICC) and cell-based enzyme-linked immunosorbent assay. In the present study, we further examined the application of these anti-OCLN mAbs and characterized their binding properties. All four mAbs were available for immunoprecipitation. The three first EL (EL1)-recognizing mAbs were applicable for immunoblotting, but the second EL (EL2)-recognizing one was not. Using site-directed mutagenesis, we also determined residues of OCLN critical for recognition by each mAb. Our findings showed that the small loop between two cysteines of the EL2 domain is essential for the binding to one EL2-recognizing mAb and that the recognition regions by three EL1-recognizing mAbs overlap, but are not the same sites of EL1. To obtain a deeper understanding of OCLN biology and its potential as a therapeutic target, specific mAbs to detect or target OCLN in intact cells should be powerful tools for future studies.

009. KALLIKREIN-RELATED PROTEASES AS NOVEL THERAPEUTIC TARGETS IN PROSTATE AND OVARIAN CANCER

2009 ◽  
Vol 21 (9) ◽  
pp. 6
Author(s):  
J. A. Clements ◽  
Y. Dong ◽  
D. Loessner ◽  
O. Tan ◽  
S. Sieh ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cancer Progression ◽  
Cell Biology ◽  
Critical Role ◽  
Bone Matrix ◽  
Therapeutic Targets ◽  
Fibrillar Structure ◽  
Pilot Studies ◽  
Novel Therapeutic

The kallikrein-related (KLKs) peptidases are implicated in prostate and ovarian cancer invasion/metastasis via activation of growth factors, proteases and extracellular matrix degradation involved in. In our published work, we used cell biology approaches to show novel associations of KLK peptidases with processes indicative of metastasis and the potential of our novel sunflower trypsin inhibitor scaffold-engineered KLK4 inhibitor. Our current studies are directed towards discovering the precise KLK target proteins/substrates and the subsequent signalling pathways involved in these events in order to determine their therapeutic target potential. In this regard, we are using novel tissue engineered biomimetic 3D gel matrices to better mimic the in vivo micro-environment of prostate cancer cells especially in bone metastasis and peritoneal invasion in ovarian cancer. Pilot studies show that PC3 cells cultured on an osteoblast-derived bone matrix undergo an EMT-like change but remain dispersed on the cell surface. In contrast, LNCaP cells cluster aligning with the fibrillar structure as they invade into the bone matrix as typically seen in vivo. KLK4 proteolysis of the osteoblast-derived bone matrix has identified additional novel substrates. In addition, we are exploring the cell biology that underlies the reported high KLK4 or KLK7 levels associated with poorer outcome in women with epithelial ovarian cancer (EOC). Of note, KLK4 or KLK7 transfected SKOV3 EOC cells have increased chemoresistance to taxol and/or cisplastin suggesting a mechanism for this poor outcome. Furthermore, KLK7 transfected SKOV-3 cells form multicellular aggregates (MCA) in agarose suspension (a process indicative of peritoneal tumour cell spread seen in ascites fluid clinically) which can be reversed by a KLK7 blocking antibody indicating the critical role played by KLK7 in this event. These new paradigms are providing novel information on the role of KLK peptidases in prostate and ovarian cancer progression and their potential as novel therapeutic targets.

Identification of three human type-II classic cadherins and frequent heterophilic interactions between different subclasses of type-II classic cadherins

2000 ◽  
Vol 349 (1) ◽  
pp. 159-167 ◽  
Author(s):  
Yutaka SHIMOYAMA ◽  
Gozoh TSUJIMOTO ◽  
Masaki KITAJIMA ◽  
Michiya NATORI
Keyword(s):  
In Vitro Study ◽  
Cell Aggregation ◽  
Type Ii ◽  
Mixed Cell ◽  
Aggregation Assay ◽  
Human Type ◽  
E Cadherin ◽  
Cell Cell

We identified three novel human type-II classic cadherins, cadherin-7, -9 and -10, by cDNA cloning and sequencing, and confirmed that they interact with catenins and function in cell-cell adhesion as do other classic cadherins. Cell-cell binding activities of the eight human type-II classic cadherins, including the three new molecules, were evaluated by long-term cell-aggregation experiments using mouse L fibroblast clones transfected with the individual cadherins. The experiments indicated that all the type-II cadherins appeared to possess similar binding strength, which was virtually equivalent to that of E-cadherin. We next examined the binding specificities of the type-II cadherins using the mixed cell-aggregation assay. Although all of the type-II cadherins exhibited binding specificities distinct from that of E-cadherin, heterophilic interactions ranging from incomplete to complete were frequently observed among them. The combinations of cadherin-6 and -9, cadherin-7 and -14, cadherin-8 and -11, and cadherin-9 and -10 interacted in a complete manner, and in particular cadherin-7 and -14, and cadherin-8 and -11 showed an indistinguishable binding specificity against other cadherin subclasses, at least in this assay system. Although these data were obtained from an in vitro study, they should be useful for understanding cadherin-mediated mechanisms of development, morphogenesis and cell-cell interactions in vivo.

scholarly journals Biogenesis of Polarized Epithelial Cells During Kidney Development In Situ: Roles of E-Cadherin–mediated Cell–Cell Adhesion and Membrane Cytoskeleton Organization

1998 ◽  
Vol 9 (11) ◽  
pp. 3161-3177 ◽  
Author(s):  
Peter A. Piepenhagen ◽  
W. James Nelson
Keyword(s):  
Cell Adhesion ◽  
Epithelial Cells ◽  
Kidney Development ◽  
Lateral Membrane ◽  
Membrane Cytoskeleton ◽  
Triton X ◽  
E Cadherin ◽  
Cell Cell

Organization of proteins into structurally and functionally distinct plasma membrane domains is an essential characteristic of polarized epithelial cells. Based on studies with cultured kidney cells, we have hypothesized that a mechanism for restricting Na/K-ATPase to the basal-lateral membrane involves E-cadherin–mediated cell–cell adhesion and integration of Na/K-ATPase into the Triton X-100–insoluble ankyrin- and spectrin-based membrane cytoskeleton. In this study, we examined the relevance of these in vitro observations to the generation of epithelial cell polarity in vivo during mouse kidney development. Using differential detergent extraction, immunoblotting, and immunofluorescence histochemistry, we demonstrate the following. First, expression of the 220-kDa splice variant of ankyrin-3 correlates with the development of resistance to Triton X-100 extraction for Na/K-ATPase, E-cadherin, and catenins and precedes maximal accumulation of Na/K-ATPase. Second, expression of the 190-kDa slice variant of ankyrin-3 correlates with maximal accumulation of Na/K-ATPase. Third, Na/K-ATPase, ankyrin-3, and fodrin specifically colocalize at the basal-lateral plasma membrane of all epithelial cells in which they are expressed and during all stages of nephrogenesis. Fourth, the relative immunofluorescence staining intensities of Na/K-ATPase, ankyrin-3, and fodrin become more similar during development until they are essentially identical in adult kidney. Thus, renal epithelial cells in vivo regulate the accumulation of E-cadherin–mediated adherens junctions, the membrane cytoskeleton, and Na/K-ATPase through sequential protein expression and assembly on the basal-lateral membrane. These results are consistent with a mechanism in which generation and maintenance of polarized distributions of these proteins in vivo and in vitro involve cell–cell adhesion, assembly of the membrane cytoskeleton complex, and concomitant integration and retention of Na/K-ATPase in this complex.

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