The molecular architecture of hemidesmosomes as revealed by super-resolution microscopy

Abstract

Hemidesmosomes have been extensively studied with immunofluorescence microscopy, but owing to its limited resolution, the precise organization of hemidesmosomes remains poorly understood. We studied hemidesmosome organization in cultured keratinocytes with two-and three-color super-resolution microscopy. We observed that, in the cell periphery, nascent hemidesmosomes are associated with individual keratin filaments and that β4 integrin (also known as ITGB4) is distributed along, rather than under, keratin filaments. By applying innovative methods to quantify molecular distances, we demonstrate that the hemidesmosomal plaque protein plectin interacts simultaneously and asymmetrically with β4 integrin and keratin. Furthermore, we show that BP180 (BPAG2, also known as collagen XVII) and BP230 (BPAG1e, an epithelial splice variant of dystonin) are characteristically arranged within hemidesmosomes with BP180 surrounding a central core of BP230 molecules. In skin cross-sections, hemidesmosomes of variable sizes could be distinguished with BP230 and plectin occupying a position in between β4 integrin and BP180, and the intermediate filament system. In conclusion, our data provide a detailed view of the molecular architecture of hemidesmosomes in cultured keratinocytes and skin.

Hemidesmosomes modulate force generation via focal adhesions

“…According to an established method (Nahidiazar et al, 2015), cells for superresolution microscopy were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100, blocked with PBS containing 5% BSA (Sigma-Aldrich), and subsequently incubated with primary and secondary antibodies with washing steps in between. Imaging was performed with a Leica SR-GSD microscope (Leica Microsystems) equipped with 405-nm/30-mW, 488-nm/300-mW, and 642-nm/500-mW lasers, with samples immersed in OxEA buffer (Nahidiazar et al, 2016).…”

Recessive mutation in tetraspanin CD151 causes Kindler syndrome-like epidermolysis bullosa with multi-systemic manifestations including nephropathy

“…Tetraspanin CD151 is widely distributed in different tissues, including epithelium, endothelium, muscle, renal glomeruli and proximal and distal tubules [15]. This protein is also expressed in the epidermis and has been shown to be a component of the hemidesmosomes [4,5]. Tetraspanin CD151 is an endogenous component of basement membranes in the skin, in hemidesmosomes and focal adhesions, where it forms stable laminin-binding complexes with α3β1 and α6β4 integrins, as well as in the kidneys where it interacts with α3β1 and α6β1 integrins [16].…”

“…While most basement membranes universally consist of the principal components, such as type IV collagen, laminin-111 and heparin sulfate proteoglycans, certain basement membranes have characteristic protein components in a restricted tissue distribution critical for their specialized tissue-specific function. For example, in the skin, hemidesmosomes, critical attachment complexes, consist of type XVII collagen, α6β4 integrin and tetraspanin CD151 [4,5]. The hemidesmosomes connect the intermediate filament network, consisting of keratins 5 and 14 in the intracellular milieu of basal keratinocytes, to anchoring filaments, extracellular filamentous structures which traverse lamina lucida and consist primarily of laminin-332.…”

Protein exchange is reduced in calcium-independent epithelial junctions

“…Cell junctions represent a class of plasma membraneassociated macromolecular complexes with roles in adhesion, force transmission, and electrical connections (Garcia et al, 2018;Goodenough and Paul, 2009;Parsons et al, 2010). To perform these myriad functions, cell junctions have complex architectures that are key in signal integration and dynamic regulation (Bertocchi et al, 2017;Kanchanawong et al, 2010;Kaufmann et al, 2012;Mehta et al, 2016;Nahidiazar et al, 2015;Stahley et al, 2016). Epithelial cells have two similar yet distinct adhesive junctions that span neighboring cells: desmosomes and adherens junctions.…”