Mechanisms regulating desmoglein clustering and hyper-adhesion of desmosomes

Fuchs, Michael Tobias (2022): Mechanisms regulating desmoglein clustering and hyper-adhesion of desmosomes. Dissertation, LMU München: Fakultät für Biologie

Abstract

Desmosomes provide strong intercellular adhesion and therefore are essential in tissues exposed to constant mechanical stress, e.g. the epidermis or the heart. The molecular composition of desmosomes consists of several protein families, including intercellular adhesion molecules and plaque proteins. Desmosomal cadherins represent the adhesion molecules and are a group of proteins comprising four desmogleins (Dsg1-4) and three desmocollins (Dsc1-3) isoforms. These are transmembrane proteins which interact in a Ca2+-dependent homophilic or heterophilic manner, thereby mediating strong intercellular adhesion with their neighboring cell counterparts. Within the desmosome, plaque proteins serve as linkers of desmosomal cadherins to the intermediate filament cytoskeleton. The plaque proteins consist of armadillo family proteins, plakoglobin (PG) and plakophilins (Pkps), and desmoplakin (DP), a protein of the plakin family. Pkps act as scaffolds in order to mediate adhesion and signaling and further regulate desmosomal turnover and assembly. They exist in three different isoforms (Pkp1-3) which show a tissue-specific expression. Further, Pkps show a wide ranging role in the cell-biological context and their physiological significance is evident in diseases, e.g. mutations in Pkp1 lead to ectodermal dysplasia skin fragility syndrome. However, their role in regulating Dsg binding properties as well as Dsg clustering is not yet elucidated. Murine keratinocytes lacking either Pkp1 or Pkp3 were compared to wild type (wt) cells to characterize the role of Pkp1 and Pkp3 in the regulation of Dsg1- and Dsg3-binding properties and contribution to Dsg3 clustering. Dsg1 and Dsg3 are of particular interest since they are the main targets of autoantibodies in the autoimmune skin blistering disease pemphigus vulgaris (PV). In the first part of the thesis, the roles of Pkp1 and Pkp3 in desmoglein clustering and adhesion were investigated. Characterization of Pkp1- or 3-deficient cell lines revealed reduced cell cohesion and especially in Pkp1-deficient cells loss of intercellular adhesion is strong. Force mapping measurements, performed with atomic force microscopy (AFM), revealed reduced binding frequency of Dsg1 and Dsg3 at the cell borders and displayed low membrane availability of Dsg1 and Dsg3 in Pkp1- or Pkp3-deficient cell lines. This indicates that both proteins are important for proper membrane availability of desmogleins. The single molecule-binding properties of the remaining membrane-bound desmogleins showed Pkp-dependent changes. However, because their numbers were low, presumably these altered binding properties of the remaining cadherins have no strong effect on overall adhesion. Extracellular crosslinking revealed that Pkp1 but not Pkp3 is required for Dsg3 clustering. Overexpression of Dsg3 rescued the reduced number of Dsg3 clusters in Pkp3- but not in Pkp1-deficient cells, as shown by AFM and stimulated emission depletion (STED) microscopy experiments. This demonstrates that Pkp1 and Pkp3 regulate the membrane availability of Dsgs. Furthermore, these data demonstrate that Pkp1 but not Pkp3 is required for the clustering of Dsg3. This novel function of Pkp1 appears to be essential for proper intercellular adhesion. In the second part of the thesis, regulation of desmosomal hyper-adhesion was studied. An interesting property of desmosomes is their ability to occur in two adhesive states, a weaker and a stronger one. Desmosomes in the stronger adhesive state become independent from extracellular Ca2+ and are called hyper-adhesive. However, the roles of Pkps and Dsgs in desmosomal hyper-adhesion is not fully elucidated yet. To address this unsolved issue, the aforementioned cell culture model, a murine keratinocyte Dsg3 knockout cell line and an ex-vivo skin model were used. Murine keratinocytes acquire the hyper-adhesive state 72 hours after exposure to high Ca2+, whereas Pkp- and Dsg3-deficient cell lines fail to reach this state during this time line. AFM force mappings revealed that the hyper-adhesive state correlates with increased Dsg3 single molecule binding strength and prolonged interaction lifetime. Both parameters were unchanged in the Pkp-deficient cells. In parallel, during differentiation, i.e. during the acquisition of hyper-adhesion, the Dsg3 clusters become Ca2+-independent. In contrast, deficiency of Pkp1 prevented Ca2+-independency of Dsg3 clusters whereas lack of Pkp3 led to a reduced amount of Ca2+-independent Dsg3 clusters. Interestingly, Dsg1 clusters remained the same during the investigated time period. In accordance, Dsg1 single molecule binding properties were not altered. This shows that acquisition of hyper-adhesion may not be a state acquired by the entire desmosome but rather reveals an isoform-dependent regulation by Pkps. Ca2+ chelation of ex-vivo human skin samples showed further isoform-specific differences in membrane localization between desmosomal cadherins. Immunostaining for Dsg3 at the cell membrane appeared to be more resistant to Ca2+ chelation compared to Dsg1 staining. This demonstrates that desmosomal cadherins have different roles in the acquisition of hyper-adhesion and that Pkp1-mediated clustering of desmogleins is required for the acquisition of the hyper-adhesive state of desmosomes. In summary, this project revealed that Pkp1 and Pkp3 are important for membrane availability of desmogleins. However, for Pkp3 this phenotype is differentiation dependent and decreases over time. Pkp1, in contrast to Pkp3, plays a crucial role in the clustering of desmogleins. This Pkp1-mediated clustering contributes to acquisition of hyper-adhesion and correlates with altered single molecule binding properties such as binding strength and interaction-lifetime of Dsg3 when becoming hyper-adhesive. Thus, development of the hyper-adhesive state is paralleled by alterations of binding properties of specific Dsg isoforms rather than by the entire desmosome. In a side-project, keratin-dependent regulation of desmoglein-binding was characterized. Keratin filament detachment from the desmosomal plaque occurs as one of the pathomechanism of PV. We found that p38 mitogen-activated protein kinases (p38MAPK) signaling is regulated by keratins. Moreover, this signaling is essential for the regulation of cell adhesion and involves both keratin-dependent and independent mechanisms. Using fluorescence recovery after photobleaching (FRAP) experiments it was observed that desmosomal cadherin uncoupling from the cytoskeleton resulted in higher Dsg3 mobility in the plasma membrane. The studies presented demonstrate that Pkp1-mediated regulation of desmosomal clustering is important for desmosomal adhesion and contributes to hyper-adhesion of desmosomes.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Desmosome, Plakophilins, Hyper-adhesion, desmosomal adhesion
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 9. Juni 2022
• 1. Bericht­erstatter:in: Leonhardt, Heinrich