scholarly journals Quantitative proximity proteomics resolves the epithelial apical-lateral border and uncovers a vertebrate marginal zone defined by the polarity protein Pals1

2019 ◽  
Author(s):  
Benedict Tan ◽  
Suat Peng ◽  
Sara Sandin ◽  
Jayantha Gunaratne ◽  
Walter Hunziker ◽  
...  
Keyword(s):  
Cell Polarity ◽  
Marginal Zone ◽  
Hippo Pathway ◽  
Molecular Organization ◽  
Junctional Complex ◽  
Lateral Border ◽  
Spatially Resolved ◽  
Apical Junctional Complex ◽  
Membrane Compartment ◽  
Associated Proteins

AbstractEpithelial apico-basal polarity is established through the asymmetric cortical distribution of the Par, Crumbs and Scribble polarity modules. Apical (Par and Crumbs) and basolateral (Scribble) polarity modules overlap at the apical-lateral border, which, in mammals, is defined by the apical junctional complex (AJC). The AJC is composed of tight junctions (TJ) and adherens junctions (AJ) and plays fundamental roles in epithelial morphogenesis and plasticity. However, the molecular composition and precise sub-junctional organization of the AJC and its associated polarity regulators are still not well defined. Here we used the peroxidase APEX2 for quantitative proximity proteomics (QPP) and electron microscopy (EM) imaging to generate a nanometer-scale spatio-molecular map of the apical-lateral border in fully polarized MDCK-II cells. Using Par3 and Pals1 as surrogates for QPP we present a spatially resolved network of ∼800 junction-associated proteins. The network dissects TJ and AJ components and provides strong evidence that TJ are composed of distinct apical and basal subdomains. Moreover, we find that Pals1 and its binding partners PatJ, Lin7c and Crumbs3 define a hitherto unidentified membrane compartment apical of TJ, which we coin the vertebrate marginal zone (VMZ). The VMZ is physically associated with HOMER scaffolding proteins, regulators of apical exocytosis, and membrane-proximal HIPPO pathway proteins. Taken together our work defines the spatial and molecular organization of the apical-lateral border in fully polarized mammalian epithelial cells, reveals an intriguing molecular and spatial conservation of invertebrate and vertebrate cell polarity protein domains, and provides a comprehensive resource of potentially novel regulators of cell polarity and the mammalian AJC.

The spatial organization of apical junctional complex-associated proteins in feline and human corneal endothelium

1999 ◽  
Vol 18 (1) ◽  
pp. 10-19 ◽  
Author(s):  
W.M. Matthew Petroll ◽  
J.K.W. Joseph Hsu ◽  
J. Jacquelyn Bean ◽  
H.D. Dwight Cavanagh ◽  
J.V. James Center
Keyword(s):  
Corneal Endothelium ◽  
Spatial Organization ◽  
Junctional Complex ◽  
Human Corneal Endothelium ◽  
Apical Junctional Complex ◽  
Associated Proteins

scholarly journals Proliferation-independent regulation of organ size by Fgf/Notch signaling

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Agnė Kozlovskaja-Gumbrienė ◽  
Ren Yi ◽  
Richard Alexander ◽  
Andy Aman ◽  
Ryan Jiskra ◽  
...  
Keyword(s):  
Cell Migration ◽  
Cell Adhesion ◽  
Notch Signaling ◽  
Hippo Pathway ◽  
Organ Size ◽  
Junctional Complex ◽  
Lateral Line System ◽  
Fgf Signaling ◽  
Line System ◽  
Apical Junctional Complex

Organ morphogenesis depends on the precise orchestration of cell migration, cell shape changes and cell adhesion. We demonstrate that Notch signaling is an integral part of the Wnt and Fgf signaling feedback loop coordinating cell migration and the self-organization of rosette-shaped sensory organs in the zebrafish lateral line system. We show that Notch signaling acts downstream of Fgf signaling to not only inhibit hair cell differentiation but also to induce and maintain stable epithelial rosettes. Ectopic Notch expression causes a significant increase in organ size independently of proliferation and the Hippo pathway. Transplantation and RNASeq analyses revealed that Notch signaling induces apical junctional complex genes that regulate cell adhesion and apical constriction. Our analysis also demonstrates that in the absence of patterning cues normally provided by a Wnt/Fgf signaling system, rosettes still self-organize in the presence of Notch signaling.

scholarly journals Planar cell polarity induces local microtubule bundling for coordinated ciliary beating

2021 ◽  
Vol 220 (7) ◽  
Author(s):  
Shogo Nakayama ◽  
Tomoki Yano ◽  
Toshinori Namba ◽  
Satoshi Konishi ◽  
Maki Takagishi ◽  
...  
Keyword(s):  
Cell Polarity ◽  
Single Molecule ◽  
Molecular Basis ◽  
Planar Cell Polarity ◽  
Tissue Level ◽  
Junctional Complex ◽  
Ciliary Beating ◽  
Voltage Electron ◽  
Apical Junctional Complex

Multiciliated cells (MCCs) in tracheas generate mucociliary clearance through coordinated ciliary beating. Apical microtubules (MTs) play a crucial role in this process by organizing the planar cell polarity (PCP)–dependent orientation of ciliary basal bodies (BBs), for which the underlying molecular basis remains elusive. Herein, we found that the deficiency of Daple, a dishevelled-associating protein, in tracheal MCCs impaired the planar polarized apical MTs without affecting the core PCP proteins, causing significant defects in the BB orientation at the cell level but not the tissue level. Using live-cell imaging and ultra-high voltage electron microscope tomography, we found that the apical MTs accumulated and were stabilized by side-by-side association with one side of the apical junctional complex, to which Daple was localized. In vitro binding and single-molecule imaging revealed that Daple directly bound to, bundled, and stabilized MTs through its dimerization. These features convey a PCP-related molecular basis for the polarization of apical MTs, which coordinate ciliary beating in tracheal MCCs.

Angiomotin to regulate prostate cancer cell proliferation by signaling through Hippo-YAP pathway.

2017 ◽  
Vol 35 (6_suppl) ◽  
pp. e559-e559
Author(s):  
Pengfei Shen ◽  
Hao Zeng ◽  
Angelica Ortiz ◽  
Chien-Jui Cheng ◽  
Yu-Chen Lee ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cell Proliferation ◽  
Nuclear Translocation ◽  
Hippo Pathway ◽  
Neurofibromatosis Type ◽  
Tumor Promoter ◽  
Migration And Invasion ◽  
Junctional Complex ◽  
Chi Square ◽  
Apical Junctional Complex

e559 Background: Angiomotin (AMOT) is a family of proteins found to be a component of the apical junctional complex of vertebrate epithelial cells and is recently found to play important roles in neurofibromatosis type 2 (NF-2). Whether AMOT plays a role in prostate cancer (PCa) is unknown. Methods: Purified GST-AMOTp80 was used as immunogen for antibody generation. Real-time PCR, western blot and immunohistochemistry were used to identify the expression of AMOT. To study the function of AMOT, retroviral vector were constructed, also shRNA was used to knockdown AMOT in cells. Cell migration and invasion assays were performed by using transwell chambers. Nuclear and cytoplasmic protein fractions were prepared by using NE-PER reagents (Pierce). The SPSS 19.0 software was used for statistical analysis. Chi-square test and t test were used for the comparisons between groups. Results: AMOT is expressed as two isoforms, AMOTp80 and AMOTp130, which has a 409 aa N-terminal domain that is absent in AMOTp80. Both AMOTp80 and AMOTp130 are expressed in LNCaP and C4-2B4, but at a low to undetectable level in PC3 cells. Further study showed that AMOTp130 and AMOTp80 have distinct functions in PCa cells. We found that AMOTp80 functioned as a tumor promoter by enhancing PCa cell proliferation while AMOTp130 did not. Mechanistic studies showed that AMOTp80 signaled through the Hippo pathway by promoting the nuclear translocation of YAP, resulting in an increased expression of YAP target protein BMP4. Moreover, inhibition of BMP receptor activity by LDN-193189 abrogates AMOTp80-mediated cell proliferation. Conclusions: Together, this study reveals a novel mechanism whereby the AMOTp80-Merlin-MST1-LATS-YAP-BMP4 pathway leads to AMOTp80-induced tumor cell proliferation.

scholarly journals ZO-2 Is a Master Regulator of Gene Expression, Cell Proliferation, Cytoarchitecture, and Cell Size

2019 ◽  
Vol 20 (17) ◽  
pp. 4128 ◽  
Author(s):  
Lorenza González-Mariscal ◽  
Helios Gallego-Gutiérrez ◽  
Laura González-González ◽  
Christian Hernández-Guzmán
Keyword(s):  
Cell Proliferation ◽  
Cultured Cells ◽  
Hippo Pathway ◽  
Junctional Complex ◽  
Rho Proteins ◽  
Downstream Signaling ◽  
Calcium Sensing ◽  
Apical Junctional Complex ◽  
Regulator Protein ◽  
The Hippo Pathway

ZO-2 is a cytoplasmic protein of tight junctions (TJs). Here, we describe ZO-2 involvement in the formation of the apical junctional complex during early development and in TJ biogenesis in epithelial cultured cells. ZO-2 acts as a scaffold for the polymerization of claudins at TJs and plays a unique role in the blood–testis barrier, as well as at TJs of the human liver and the inner ear. ZO-2 movement between the cytoplasm and nucleus is regulated by nuclear localization and exportation signals and post-translation modifications, while ZO-2 arrival at the cell border is triggered by activation of calcium sensing receptors and corresponding downstream signaling. Depending on its location, ZO-2 associates with junctional proteins and the actomyosin cytoskeleton or a variety of nuclear proteins, playing a role as a transcriptional repressor that leads to inhibition of cell proliferation and transformation. ZO-2 regulates cell architecture through modulation of Rho proteins and its absence induces hypertrophy due to inactivation of the Hippo pathway and activation of mTOR and S6K. The interaction of ZO-2 with viral oncoproteins and kinases and its silencing in diverse carcinomas reinforce the view of ZO-2 as a tumor regulator protein.

GENETIC SUSCEPTIBILITY TO IBD OPERATES VIA CONTROL OF APICAL JUNCTIONAL COMPLEXES

2021 ◽  
Vol 27 (Supplement_1) ◽  
pp. S30-S30
Author(s):  
Isabelle Hébert-Milette ◽  
Chloé Lévesque ◽  
Guy Charron ◽  
John Rioux
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Intestinal Inflammation ◽  
Protein Localization ◽  
Junctional Complex ◽  
Epithelial Permeability ◽  
3D Cultures ◽  
Apical Junctional Complex ◽  
Junction Protein ◽  
The Impact

Abstract Introduction Intestinal permeability is increased in unaffected 1st degree relatives of patients with inflammatory bowel disease (IBD), and is considered a risk factor for the development of IBD, likely increasing the interactions between intestinal microorganisms and the immune system. We recently reported that C1orf106, a gene located within a genomic region associated with IBD, regulates epithelial permeability. We further demonstrated that a rare coding variant within C1orf106 (p.Y333F) decreases protein stability and that lower levels of C1orf106 protein leads altered stability of adherens junctions (AJ) and to an increase in epithelial permeability. Hypothesis In addition to altering AJ, we believe that C1orf106 is also involved in the regulation of tight junction (TJ) formation, which also impacts epithelial permeability. Objectives The objectives of the project are to (a) validate the impact of C1orf106 on tight junctions and (b) verify the impact of C1orf106 IBD-associated variants on intestinal barrier integrity. Results We observed that knocking down the expression of C1orf106 in Caco-2 cells leads to a number of phenotypes in human epithelial monolayer (2D) and spheroid (3D) cultures that are associated with alterations in TJs. Specifically, when studying the dynamic reformation of TJ in 2D cultures after transient withdrawal of calcium, which is required for TJ stability, we observed that lower levels of C1orf106 resulted in (1) decreased recovery of barrier function as measured by transepithelial electrical resistance (TEER); (2) an alteration of tight junction protein localization; and (3) thickening of the circumferential actin belt. Moreover, in 3D cultures, we observed an altered spheroid formation associated with impaired epithelial polarization. In addition, our preliminary studies of human induced pluripotent stem cell (hiPSC)-derived epithelial cultures support that Y333F heterozygotes also have altered structure and function of their tight junctions. Conclusion Our observations indicate an important role of C1orf106 in apical junctional complex (AJC) formation likely mediated by a regulation of the circumferential actin belt. This can affect other functions of AJC, like the establishment of cell polarity. AJC formation is important for epithelial repair after an injury and its dysregulation impairs the formation of an impermeable epithelial barrier, which likely facilitates the passage of microorganisms and the induction and maintenance of intestinal inflammation.

scholarly journals DAAM1 stabilizes epithelial junctions by restraining WAVE complex–dependent lateral membrane motility

2016 ◽  
Vol 215 (4) ◽  
pp. 559-573 ◽  
Author(s):  
Tamako Nishimura ◽  
Shoko Ito ◽  
Hiroko Saito ◽  
Sylvain Hiver ◽  
Kenta Shigetomi ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Junctional Complex ◽  
Lateral Membrane ◽  
Apical Junctional Complex ◽  
Cell Layers ◽  
Epithelial Junctions ◽  
Membrane Contacts ◽  
Epithelial Layers ◽  
Actin Cables ◽  
Wave Complex

Epithelial junctions comprise two subdomains, the apical junctional complex (AJC) and the adjacent lateral membrane contacts (LCs), that span the majority of the junction. The AJC is lined with circumferential actin cables, whereas the LCs are associated with less-organized actin filaments whose roles are elusive. We found that DAAM1, a formin family actin regulator, accumulated at the LCs, and its depletion caused dispersion of actin filaments at these sites while hardly affecting circumferential actin cables. DAAM1 loss enhanced the motility of LC-forming membranes, leading to their invasion of neighboring cell layers, as well as disruption of polarized epithelial layers. We found that components of the WAVE complex and its downstream targets were required for the elevation of LC motility caused by DAAM1 loss. These findings suggest that the LC membranes are motile by nature because of the WAVE complex, but DAAM1-mediated actin regulation normally restrains this motility, thereby stabilizing epithelial architecture, and that DAAM1 loss evokes invasive abilities of epithelial cells.

scholarly journals Automated analysis of filopodial length and spatially resolved protein concentration via adaptive shape tracking

2016 ◽  
Vol 27 (22) ◽  
pp. 3616-3626 ◽  
Author(s):  
Tanumoy Saha ◽  
Isabel Rathmann ◽  
Abhiyan Viplav ◽  
Sadhana Panzade ◽  
Isabell Begemann ◽  
...  
Keyword(s):  
Protein Concentration ◽  
Growth Dynamics ◽  
Automated Analysis ◽  
Cell Types ◽  
Control Mechanisms ◽  
Spatially Resolved ◽  
Novel Approach ◽  
Associated Proteins

Filopodia are dynamic, actin-rich structures that transiently form on a variety of cell types. To understand the underlying control mechanisms requires precise monitoring of localization and concentration of individual regulatory and structural proteins as filopodia elongate and subsequently retract. Although several methods exist that analyze changes in filopodial shape, a software solution to reliably correlate growth dynamics with spatially resolved protein concentration along the filopodium independent of bending, lateral shift, or tilting is missing. Here we introduce a novel approach based on the convex-hull algorithm for parallel analysis of growth dynamics and relative spatiotemporal protein concentration along flexible filopodial protrusions. Detailed in silico tests using various geometries confirm that our technique accurately tracks growth dynamics and relative protein concentration along the filopodial length for a broad range of signal distributions. To validate our technique in living cells, we measure filopodial dynamics and quantify spatiotemporal localization of filopodia-associated proteins during the filopodial extension–retraction cycle in a variety of cell types in vitro and in vivo. Together these results show that the technique is suitable for simultaneous analysis of growth dynamics and spatiotemporal protein enrichment along filopodia. To allow readily application by other laboratories, we share source code and instructions for software handling.

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