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1.
J Cell Biol ; 219(6)2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32399559

RESUMO

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.


Assuntos
Cálcio/metabolismo , Adesão Celular , Desmogleína 3/metabolismo , Desmoplaquinas/metabolismo , Desmossomos/metabolismo , Queratinócitos/metabolismo , Caderinas/genética , Caderinas/metabolismo , Cálcio/farmacologia , Carbazóis/farmacologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Linhagem Celular , Desmogleína 3/genética , Desmoplaquinas/genética , Desmossomos/efeitos dos fármacos , Desmossomos/ultraestrutura , Humanos , Queratinócitos/efeitos dos fármacos , Microscopia Eletrônica , Microscopia de Fluorescência , Mutação , Fosforilação , Ligação Proteica/genética , Proteína Quinase C-alfa/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , gama Catenina/genética , gama Catenina/metabolismo
2.
Nat Commun ; 10(1): 4507, 2019 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-31628308

RESUMO

Podosomes are ubiquitous cellular structures important to diverse processes including cell invasion, migration, bone resorption, and immune surveillance. Structurally, podosomes consist of a protrusive actin core surrounded by adhesion proteins. Although podosome protrusion forces have been quantified, the magnitude, spatial distribution, and orientation of the opposing tensile forces remain poorly characterized. Here we use DNA nanotechnology to create probes that measure and manipulate podosome tensile forces with molecular piconewton (pN) resolution. Specifically, Molecular Tension-Fluorescence Lifetime Imaging Microscopy (MT-FLIM) produces maps of the cellular adhesive landscape, revealing ring-like tensile forces surrounding podosome cores. Photocleavable adhesion ligands, breakable DNA force probes, and pharmacological inhibition demonstrate local mechanical coupling between integrin tension and actin protrusion. Thus, podosomes use pN integrin forces to sense and respond to substrate mechanics. This work deepens our understanding of podosome mechanotransduction and contributes tools that are widely applicable for studying receptor mechanics at dynamic interfaces.


Assuntos
Fenômenos Biomecânicos/fisiologia , DNA/metabolismo , Mecanotransdução Celular/fisiologia , Nanotecnologia/métodos , Podossomos/fisiologia , Actinas/metabolismo , Animais , Adesão Celular , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Transferência Ressonante de Energia de Fluorescência , Humanos , Integrinas/metabolismo , Camundongos , Microscopia de Fluorescência/métodos , Células NIH 3T3 , Podossomos/metabolismo
3.
Tissue Barriers ; 6(1): e1404189, 2018 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-29420122

RESUMO

Cell junctions are critical for cell adhesion and communication in epithelial tissues. It is evident that the cellular distribution, size, and architecture of cell junctions play a vital role in regulating function. These details of junction architecture have been challenging to elucidate in part due to the complexity and size of cell junctions. A major challenge in understanding these features is attaining high resolution spatial information with molecular specificity. Fluorescence microscopy allows localization of specific proteins to junctions, but with a resolution on the same scale as junction size, rendering internal protein organization unobtainable. Super-resolution microscopy provides a bridge between fluorescence microscopy and nanoscale approaches, utilizing fluorescent tags to reveal protein organization below the resolution limit. Here we provide a brief introduction to super-resolution microscopy and discuss novel findings into the organization, structure and function of epithelial cell junctions.


Assuntos
Junções Aderentes/metabolismo , Células Epiteliais/metabolismo , Microscopia de Fluorescência/métodos , Junções Íntimas/metabolismo , Humanos
4.
Biophys J ; 113(11): 2519-2529, 2017 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-29212005

RESUMO

Desmosomes are macromolecular cell-cell junctions that provide adhesive strength in epithelial tissue. Desmosome function is inseparably linked to structure, and it is hypothesized that the arrangement, or order, of desmosomal cadherins in the intercellular space is critical for adhesive strength. However, due to desmosome size, molecular complexity, and dynamics, the role that order plays in adhesion is challenging to study. Herein, we present an excitation resolved fluorescence polarization microscopy approach to measure the spatiotemporal dynamics of order and disorder of the desmosomal cadherin desmoglein 3 (Dsg3) in living cells. Simulations were used to establish order factor as a robust metric for quantifying the spatiotemporal dynamics of order and disorder. Order factor measurements in keratinocytes showed the Dsg3 extracellular domain is ordered at the individual desmosome, single cell, and cell population levels compared to a series of disordered controls. Desmosomal adhesion is Ca2+ dependent, and reduction of extracellular Ca2+ leads to a loss of adhesion measured by dispase fragmentation assay (λ = 15.1 min). Live cell imaging revealed Dsg3 order decreased more rapidly (λ = 5.5 min), indicating that cadherin order is not required for adhesion. Our results suggest that rapid disordering of cadherins can communicate a change in extracellular Ca2+ concentration to the cell, leading to a downstream loss of adhesion. Fluorescence polarization is an effective bridge between protein structure and complex dynamics and the approach presented here is broadly applicable to studying order in macromolecular structures.


Assuntos
Desmogleína 3/metabolismo , Desmossomos/metabolismo , Sobrevivência Celular , Desmogleína 3/química , Humanos , Queratinócitos/citologia , Microscopia de Fluorescência , Microscopia de Polarização , Modelos Moleculares , Conformação Proteica
5.
J Cell Sci ; 129(15): 2897-904, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27505428

RESUMO

Desmosomes are macromolecular junctions responsible for providing strong cell-cell adhesion. Because of their size and molecular complexity, the precise ultrastructural organization of desmosomes is challenging to study. Here, we used direct stochastic optical reconstruction microscopy (dSTORM) to resolve individual plaque pairs for inner and outer dense plaque proteins. Analysis methods based on desmosomal mirror symmetry were developed to measure plaque-to-plaque distances and create an integrated map. We quantified the organization of desmoglein 3, plakoglobin and desmoplakin (N-terminal, rod and C-terminal domains) in primary human keratinocytes. Longer desmosome lengths correlated with increasing plaque-to-plaque distance, suggesting that desmoplakin is arranged with its long axis at an angle within the plaque. We next examined whether plaque organization changed in different adhesive states. Plaque-to-plaque distance for the desmoplakin rod and C-terminal domains decreased in PKP-1-mediated hyperadhesive desmosomes, suggesting that protein reorganization correlates with function. Finally, in human epidermis we found a difference in plaque-to-plaque distance for the desmoplakin C-terminal domain, but not the desmoplakin rod domain or plakoglobin, between basal and suprabasal cells. Our data reveal the molecular organization of desmosomes in cultured keratinocytes and skin as defined by dSTORM.


Assuntos
Desmossomos/metabolismo , Microscopia/métodos , Fenômenos Ópticos , Adesão Celular , Humanos , Masculino , Placofilinas/metabolismo , Pele/metabolismo , Processos Estocásticos
6.
Steroids ; 77(7): 774-9, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22504555

RESUMO

The endogenous neurosteroids, pregnenolone sulfate (PS) and 3α-hydroxy-5ß-pregnan-20-one sulfate (PREGAS), have been shown to differentially regulate the ionotropic glutamate receptor (iGluR) family of ligand-gated ion channels. Upon binding to these receptors, PREGAS decreases current flow through the channels. Upon binding to non-NMDA or NMDA receptors containing an GluN2C or GluN2D subunit, PS also decreases current flow through the channels, however, upon binding to NMDA receptors containing an GluN2A or GluN2B subunit, flow through the channels increases. To begin to understand this differential regulation, we have cloned the S1S2 and amino terminal domains (ATD) of the NMDA GluN2B and GluN2D and AMPA GluA2 subunits. Here we present results that show that PS and PREGAS bind to different sites in the ATD of the GluA2 subunit, which when combined with previous results from our lab, now identifies two binding domains for each neurosteroid. We also show both neurosteroids bind only to the ATD of the GluN2D subunit, suggesting that this binding is distinct from that of the AMPA GluA2 subunit, with both leading to iGluR inhibition. Finally, we provide evidence that both PS and PREGAS bind to the S1S2 domain of the NMDA GluN2B subunit. Neurosteroid binding to the S1S2 domain of NMDA subunits responsible for potentiation of iGluRs and to the ATD of NMDA subunits responsible for inhibition of iGluRs, provides an interesting option for therapeutic design.


Assuntos
Neurotransmissores/metabolismo , Pregnanolona/análogos & derivados , Pregnenolona/metabolismo , Receptores Ionotrópicos de Glutamato/metabolismo , Sítios de Ligação , Clonagem Molecular , Modelos Moleculares , Pregnanolona/metabolismo , Receptores Ionotrópicos de Glutamato/química , Receptores Ionotrópicos de Glutamato/genética , Espectrometria de Fluorescência
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