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

Abstract

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.

Dsg3 epitope-specific signalling in pemphigus

“…It is likely that extradesmosomal Dsg complexes are degraded in response to AK23, since Dsg3, E-Cadherin and Pg containing complexes were reported to be regulated in a Src-dependent manner ( 48 , 70 , 71 ). This would impact adhesion via alterations in the actin cytoskeleton which was reported to stabilize extradesmosomal Dsg3 binding events ( 72 ).…”

Plakophilin-3 Binds the Membrane and Filamentous Actin without Bundling F-Actin

“…The available data also show that desmosome assembly starts with desmosomal cadherins and desmoplakin clustering. The formation of these initial clusters relies on the actin cytoskeleton but not on intermediate filaments, which anchor matured desmosomes [33][34][35][36][37]. Finally, plakophilins, even though they interact with all known desmosomal proteins and intermediate filaments [3,38], can be recruited to the cell cortex without these interactions [7,12,37].…”

“…It failed to bundle the actin filaments in low-speed conditions, corroborating our colocalization results obtained with A431 cells. Additional studies are needed to understand whether the direct interaction of plakophilin-3 with F-actin is involved in the mechanisms of desmosome assembly and the reported anchorage of different desmosomal proteins to the actin cytoskeleton [35,36].…”

ARHGAP32 as a novel RhoGAP interacting with desmoplakin is required for desmosomal organization and assembly

“…The actin cytoskeleton is reported to be involved in the assembly and turnover of desmosomal components (Fuchs et al, 2023; Godsel et al, 2010; Hiermaier et al, 2021; Moch et al, 2022). The relocation of DSP to the desmosome during desmosome assembly can be accelerated by active RhoA, but the maturation of desmosome plaques is hindered by persistent activation of RhoA (Godsel et al, 2010).…”