Protein exchange is reduced in calcium-independent epithelial junctions

Bartle, Emily I.; Rao, Tejeshwar C.; Beggs, Reena R.; Dean, William F.; Urner, Tara; Kowalczyk, Andrew P.; Mattheyses, Alexa L.

doi:10.1083/jcb.201906153

Cited by 25 publications

(45 citation statements)

References 62 publications

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“…The correlation between membrane mobility of desmosomal cadherins and desmosome maturation was recently shown 31 . In line with this concept, fluorescence recovery after photo‐bleaching (FRAP) at the interfaces of two adjacent cells revealed increased mobility of Dsg2‐GFP in Dp ko cells (Figure 5E).…”

Section: Resultssupporting

confidence: 78%

Clustering of desmosomal cadherins by desmoplakin is essential for cell‐cell adhesion

et al. 2021

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AIM Desmoplakin (Dp) is a crucial component of the desmosome, a supramolecular cell junction complex anchoring intermediate filaments. The mechanisms how Dp modulates cell‐cell adhesion are only partially understood. Here, we studied the impact of Dp on the function of desmosomal adhesion molecules, desmosome turnover and intercellular adhesion. METHODS CRISPR/Cas9 was used for gene editing of human keratinocytes which were characterized by Western blot and immunostaining. Desmosomal ultrastructure and function were assessed by electron microscopy and cell adhesion assays. Single molecule binding properties and localization of desmosomal cadherins were studied by atomic force microscopy and super‐resolution imaging. RESULTS Knockout (ko) of Dp impaired cell cohesion to drastically higher extents as ko of another desmosomal protein, plakoglobin (Pg). In contrast to Pg ko, desmosomes were completely absent in Dp ko. Binding properties of the desmosomal adhesion molecules desmocollin (Dsc) 3 and desmoglein (Dsg) 3 remained unaltered under loss of Dp. Dp was required for assembling desmosomal cadherins into large clusters, as Dsg2 and Dsc3, adhesion molecules primarily localized within desmosomes, were redistributed into small puncta in the cell membrane of Dp ko cells. Additional silencing of desmosomal cadherins in Dp ko did not further increase loss of intercellular adhesion. CONCLUSION Our data demonstrate that Dp is essential for desmosome formation but does not influence intercellular adhesion on the level of individual cadherin binding properties. Rather, macro‐clustering of desmosomal adhesion molecules through Dp is crucial. These results may help to better understand severe diseases which are caused by Dp dysfunction.

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Section: Resultssupporting

confidence: 78%

Clustering of desmosomal cadherins by desmoplakin is essential for cell‐cell adhesion

et al. 2021

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“…We suggest that this stabilisation of DP makes a significant contribution to the gaining of hyper-adhesion. This view is consistent with data showing that a phospho-null DP mutant with enhanced keratin binding properties leads to an increased association of the desmosomal plaque with keratin filaments, resulting in decreased protein exchange and increased desmosome stability (Albrecht et al, 2015; Bartle et al, 2020). It was proposed that inhibition of PKC inhibitors or downregulation of PKCα may promote DP-keratin integration leading to the onset of hyper-adhesion (Bartle et al, 2020).…”

Section: Discussionsupporting

confidence: 91%

“…The already low mobile fractions almost halved for the DCs, PG and DP, and decreased by 15% for Pkp2a. A similar result was found recently when hyper-adhesion was pharmacologically induced with Gö6976, an inhibitor of conventional PKC isoforms, though Pkp was not included in that study (Bartle et al, 2020). The disadvantage of using such an inhibitor is that it may block multiple cellular events, any one of which may directly or indirectly affect the mobility of desmosomal components.…”

Section: Discussionsupporting

confidence: 85%

“…Desmosomes are undoubtedly highly stable structures, stability being key to their function, and it is becoming clear that the protein exchange rates within desmosomes mirror their stability as has been shown for Dsg2, Dsg3, Dsc2, DP and PG (Bartle et al, 2020; Foote et al, 2013; Gloushankova et al, 2003; Lowndes et al, 2014; Moch et al, 2019; Vielmuth et al, 2018; Windoffer et al, 2002). Nevertheless they are also dynamic, with a life cycle of four phases: de novo assembly from their component molecules, a weakly-adhesive calcium-dependent phase compatible with cell migration, a strongly adhesive calcium-independent phase in tissue homeostasis, and desmosome removal or breakdown as epithelia become activated by relevant signalling.…”

Section: Introductionmentioning

confidence: 97%

“…It has been suggested that hyperadhesion is associated with an ordered arrangement of the extracellular domains of the desmosomal cadherins, indicated by the presence of an electron-dense midline present halfway between the desmosomal cell membranes, and that this may result in Ca 2+ being locked into the structure (Garrod et al, 2005). It has been reported that this ordered arrangement of extracellular domains of genetically modified Dsg3 is lost upon removal of extracellular Ca 2+ , both from pharmacologically-induced hyper-adhesive and calcium-dependent desmosomes, suggesting the ordering of DCs is not the only factor involved in desmosomal adhesion (Bartle et al, 2020;Bartle et al, 2017). Whilst tissue integrity depends upon the maintenance of stable cell-cell junctions, cells must retain the ability to free themselves from their neighbours in order to move or to get rid of damaged cells.…”

Section: Introductionmentioning

confidence: 99%

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Desmosomal dualism: the core is stable while plakophilin is dynamic

Huppert

Liebl

et al. 2021

Preprint

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Desmosomes, strong cell-cell junctions of epithelia and cardiac muscle, link intermediate filaments to cell membranes and mechanically integrate cells across tissues, dissipating mechanical stress. They comprise 5 major protein classes - desmocollins and desmogleins (the desmosomal cadherins), plakoglobin, plakophilins and desmoplakin - whose individual contribution to the structure and turnover of desmosomes is poorly understood. Using live-cell imaging together with FRAP and FLAP we show that desmosomes consist of two contrasting protein fractions or modules: a very stable desmosomal core of desmosomal cadherins and plakoglobin, and a highly mobile plakophilin. As desmosomes mature from calcium-dependence to calcium-independent hyper-adhesion, core stability increases, but Pkp2a remains highly mobile. Desmoplakin is initially mobile but stabilises with hyper-adhesion. We show that desmosome down-regulation during growth factor-induced cell scattering proceeds by internalisation of whole desmosomes, which still retain a stable core and highly mobile Pkp2a. This molecular mobility of Pkp2a suggests a transient and probably regulatory role for Pkp2a in the desmosome.

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Cytoskeletal anchorage of different Dsg3 pools revealed by combination of hybrid STED/SMFS-AFM

Fuchs

Radeva

Spindler

et al. 2023

Cell. Mol. Life Sci.

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Desmoglein 3 (Dsg3) is a desmosomal cadherin mediating cell adhesion within desmosomes and is the antigen of the autoimmune blistering skin disease pemphigus vulgaris. Therefore, understanding of the complex desmosome turnover process is of high biomedical relevance. Recently, super resolution microscopy was used to characterize desmosome composition and turnover. However, studies were limited because adhesion measurements on living cells were not possible in parallel. Before desmosomal cadherins are incorporated into nascent desmosomes, they are not bound to intermediate filaments but were suggested to be associated with the actin cytoskeleton. However, direct proof that adhesion of a pool of desmosomal cadherins is dependent on actin is missing. Here, we applied single-molecule force spectroscopy measurements with the novel single molecule hybrid-technique STED/SMFS-AFM to investigate the cytoskeletal anchorage of Dsg3 on living keratinocytes for the first time. By application of pharmacological agents we discriminated two different Dsg3 pools, only one of which is anchored to actin filaments. We applied the actin polymerization inhibitor Latrunculin B to modify the actin cytoskeleton and the PKCα activator PMA to modulate intermediate filament anchorage. On the cellular surface Dsg3 adhesion was actin-dependent. In contrast, at cell–cell contacts, Dsg3 adhesion was independent from actin but rather is regulated by PKC which is well established to control desmosome turn-over via intermediate filament anchorage. Taken together, using the novel STED/SMFS-AFM technique, we demonstrated the existence of two Dsg3 pools with different cytoskeletal anchorage mechanisms.

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Protein exchange is reduced in calcium-independent epithelial junctions

Cited by 25 publications

References 62 publications

Clustering of desmosomal cadherins by desmoplakin is essential for cell‐cell adhesion

Clustering of desmosomal cadherins by desmoplakin is essential for cell‐cell adhesion

Desmosomal dualism: the core is stable while plakophilin is dynamic

Cytoskeletal anchorage of different Dsg3 pools revealed by combination of hybrid STED/SMFS-AFM

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