scholarly journals Protein exchange is reduced in calcium-independent epithelial junctions

2020 ◽  
Vol 219 (6) ◽  
Author(s):  
Emily I. Bartle ◽  
Tejeshwar C. Rao ◽  
Reena R. Beggs ◽  
William F. Dean ◽  
Tara M. Urner ◽  
...  
Keyword(s):  
Fluorescence Polarization ◽  
Cell Junctions ◽  
Polarization Microscopy ◽  
Cardiac Tissues ◽  
Calcium Dependent ◽  
Rapid Control ◽  
Ordered Array ◽  
Epithelial Junctions ◽  
Inside Out ◽  
Protein Exchange

Desmosomes are cell–cell junctions that provide mechanical integrity to epithelial and cardiac tissues. Desmosomes have two distinct adhesive states, calcium-dependent and hyperadhesive, which balance tissue plasticity and strength. A highly ordered array of cadherins in the adhesive interface is hypothesized to drive hyperadhesion, but how desmosome structure confers adhesive state is still elusive. We employed fluorescence polarization microscopy to show that cadherin order is not required for hyperadhesion induced by pharmacologic and genetic approaches. FRAP experiments in cells treated with the PKCα inhibitor Gö6976 revealed that cadherins, plakoglobin, and desmoplakin have significantly reduced exchange in and out of hyperadhesive desmosomes. To test whether this was a result of enhanced keratin association, we used the desmoplakin mutant S2849G, which conferred reduced protein exchange. We propose that inside-out regulation of protein exchange modulates adhesive function, whereby proteins are “locked in” to hyperadhesive desmosomes while protein exchange confers plasticity on calcium-dependent desmosomes, thereby providing rapid control of adhesion.

scholarly journals Cadherin Organization in Desmosomes Probed using Fluorescence Polarization Microscopy

2020 ◽  
Vol 118 (3) ◽  
pp. 308a-309a
Author(s):  
William F. Dean ◽  
Emily I. Bartle ◽  
Alexa L. Mattheyses
Keyword(s):  
Fluorescence Polarization ◽  
Polarization Microscopy ◽  
Fluorescence Polarization Microscopy

scholarly journals Formin-mediated actin polymerization at cell–cell junctions stabilizes E-cadherin and maintains monolayer integrity during wound repair

2016 ◽  
Vol 27 (18) ◽  
pp. 2844-2856 ◽  
Author(s):  
Megha Vaman Rao ◽  
Ronen Zaidel-Bar
Keyword(s):  
Cell Adhesion ◽  
Wound Repair ◽  
Actin Polymerization ◽  
Cell Junctions ◽  
Epithelial Tissue ◽  
Collective Cell Migration ◽  
Epithelial Junctions ◽  
Major Factors ◽  
E Cadherin ◽  
Cell Cell

Cadherin-mediated cell–cell adhesion is required for epithelial tissue integrity in homeostasis, during development, and in tissue repair. E-cadherin stability depends on F-actin, but the mechanisms regulating actin polymerization at cell–cell junctions remain poorly understood. Here we investigated a role for formin-mediated actin polymerization at cell–cell junctions. We identify mDia1 and Fmnl3 as major factors enhancing actin polymerization and stabilizing E-cadherin at epithelial junctions. Fmnl3 localizes to adherens junctions downstream of Src and Cdc42 and its depletion leads to a reduction in F-actin and E-cadherin at junctions and a weakening of cell–cell adhesion. Of importance, Fmnl3 expression is up-regulated and junctional localization increases during collective cell migration. Depletion of Fmnl3 or mDia1 in migrating monolayers results in dissociation of leader cells and impaired wound repair. In summary, our results show that formin activity at epithelial cell–cell junctions is important for adhesion and the maintenance of epithelial cohesion during dynamic processes, such as wound repair.

scholarly journals αvβ3 Integrins and Pyk2 Mediate Insulin-Like Growth Factor I Activation of Src and Mitogen-Activated Protein Kinase in 3T3-L1 Cells

2005 ◽  
Vol 19 (7) ◽  
pp. 1859-1867 ◽  
Author(s):  
Hiroko Sekimoto ◽  
Jodi Eipper-Mains ◽  
Sunthorn Pond-Tor ◽  
Charlotte M. Boney
Keyword(s):  
Binding Site ◽  
Mapk Pathway ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Immunofluorescent Staining ◽  
Calcium Dependent ◽  
Tyrosine Kinase 2 ◽  
Igf I ◽  
Αvβ3 Integrins ◽  
Inside Out

Abstract IGF-I stimulates cell growth through interaction of the IGF receptor with multiprotein signaling complexes. However, the mechanisms of IGF-I receptor-mediated signaling are not completely understood. We have previously shown that IGF-I-stimulated 3T3-L1 cell proliferation is dependent on Src activation of the ERK-1/2 MAPK pathway. We hypothesized that IGF-I activation of the MAPK pathway is mediated through integrin activation of Src-containing signaling complexes. The disintegrin echistatin decreased IGF-I phosphorylation of Src and MAPK, and blocking antibodies to αv and β3 integrin subunits inhibited IGF-I activation of MAPK, suggesting that αvβ3 integrins mediate IGF-I mitogenic signaling. IGF-I increased ligand binding to αvβ3 as detected by immunofluorescent staining of ligand-induced binding site antibody and stimulated phosphorylation of the β3 subunit, consistent with inside-out activation of αvβ3 integrins. IGF-I increased tyrosine phosphorylation of the focal adhesion kinase (FAK) Pyk2 (calcium-dependent proline-rich tyrosine kinase-2) to a much greater extent than FAK, and increased association of Src with Pyk2 but not FAK. The intracellular calcium chelator BAPTA prevented IGF-I phosphorylation of Pyk2, Src, and MAPK, suggesting that IGF-I activation of Pyk2 is calcium dependent. Transient transfection with a dominant-negative Pyk2, which lacks the autophosphorylation and Src binding site, decreased IGF-I activation of MAPK, but no inhibition was seen with transfected wild-type Pyk2. These results indicate that IGF-I signaling to MAPK is dependent on inside-out activation of αvβ3 integrins and integrin-facilitated multiprotein complex formation involving Pyk2 activation and association with Src.

scholarly journals Measuring the Orientation of Single Proteins Interacting with DNA using Fluorescence Polarization Microscopy

2017 ◽  
Vol 112 (3) ◽  
pp. 169a
Author(s):  
Emil Marklund ◽  
Elias Amselem ◽  
Kalle Kipper ◽  
Magnus Johansson ◽  
Sebastian Deindl ◽  
...  
Keyword(s):  
Fluorescence Polarization ◽  
Polarization Microscopy ◽  
Fluorescence Polarization Microscopy

scholarly journals An RPTPα/Src family kinase/Rap1 signaling module recruits myosin IIB to support contractile tension at apical E-cadherin junctions

2015 ◽  
Vol 26 (7) ◽  
pp. 1249-1262 ◽  
Author(s):  
Guillermo A. Gomez ◽  
Robert W. McLachlan ◽  
Selwin K. Wu ◽  
Benjamin J. Caldwell ◽  
Elliott Moussa ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Tyrosine Phosphatase ◽  
Myosin Ii ◽  
Active Role ◽  
Cell Junctions ◽  
Actin Assembly ◽  
Src Family Kinase ◽  
Epithelial Junctions ◽  
E Cadherin ◽  
Cell Cell

Cell–cell adhesion couples the contractile cortices of epithelial cells together, generating tension to support a range of morphogenetic processes. E-cadherin adhesion plays an active role in generating junctional tension by promoting actin assembly and cortical signaling pathways that regulate myosin II. Multiple myosin II paralogues accumulate at mammalian epithelial cell–cell junctions. Earlier, we found that myosin IIA responds to Rho-ROCK signaling to support junctional tension in MCF-7 cells. Although myosin IIB is also found at the zonula adherens (ZA) in these cells, its role in junctional contractility and its mode of regulation are less well understood. We now demonstrate that myosin IIB contributes to tension at the epithelial ZA. Further, we identify a receptor type-protein tyrosine phosphatase alpha–Src family kinase–Rap1 pathway as responsible for recruiting myosin IIB to the ZA and supporting contractile tension. Overall these findings reinforce the concept that orthogonal E-cadherin–based signaling pathways recruit distinct myosin II paralogues to generate the contractile apparatus at apical epithelial junctions.

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