Chronic wounds represent a severe socio-economic burden and a key area of unmet clinical need. PKCα is ubiquitous in the skin, particularly the epidermis and functions in numerous pathways that are fundamental to wound repair. By utilising a global PKCα-/- mouse we have identified PKCα-regulated processes both in unwounded skin and during wound healing. PKCα-/- mice display considerably delayed wound healing with a dramatic reduction in re-epithelialisation. By analysing the ultrastructure of the epidermis, I have shown that this delay directly correlates with a failure of wound edge desmosomes to switch to a their adhesive properties. A major risk factor for the development of chronic wounds is age. Crucially, this delay in modulating cell adhesion is conserved in human chronic wounds and aged murine skin. Furthermore, manipulation of PKCα using an inducible bitransgenic mouse containing epidermal specific constitutively active PKCα can accelerate the modulation of desmosome adhesion and subsequently improve re-epithelialisation. Global gene expression analysis of PKCα-/- skin and wounds revealed further defects. Upon wounding, we observed a failure to correctly regulate expression of key collagen and Wnt signalling genes that are essential for correct and timely wound healing. Finally, intrinsic gene expression changes were identified in the skin of PKCα-/- mice, specifically a downregulation of multiple extracellular matrix genes. Of note was the downregulation of small leucine-rich proteoglycans which led to alterations to dermal collagen structure and skin tensile strength. These changes render the PKCα-/- skin susceptible to breaking and wound development. To conclude, we have identified multiple roles for PKCα intrinsically in the skin and also during cutaneous wound healing. Importantly, these intrinsic changes appear to predispose PKCα-/- skin to the development of cutaneous wounds and altered wound-specific processes that manifest in a delayed healing phenotype.

<p>Microtubules often adopt non-centrosomal arrays in differentiated tissues, where they are important for providing structure to the cell and maintaining polarity. Although the formation and organization of centrosomal arrays has been well-characterized, little is known about how microtubules form non-centrosomal arrays.</p><p>In the mouse epidermis, centrosomes in differentiated cells lose their microtubule-anchoring ability through the loss of proteins from the centrosome. Instead, microtubules are organized around the cell cortex. The cell-cell adhesion protein desmoplakin is required for this organization. Our model is that desmoplakin recruits microtubule-anchoring proteins like ninein to the desmosome, where they subsequently recruit and organize microtubules.</p><p>To test this model, we confirmed that the microtubule-binding proteins Lis1, Ndel1, and CLIP170 are recruited by desmoplakin to the cell cortex. Furthermore, by creating an epidermis-specific conditional Lis1 knockout mouse, I found that Lis1 is required for cortical microtubule organization. Surprisingly, however, Lis1 is also required for desmosome stability. This work reveals essential desmosome-associated components that control cortical microtubule organization and unexpected roles for centrosomal proteins in epidermal function.</p><p>Although Lis1 is required for microtubule organization, it is not sufficient. I created a culture-based system to determine what other factors may be required for cortical organization for microtubules. My work reveals that stabilization of the microtubules is sufficient to induce their cortical organization. Functionally, cortical microtubules are important for increasing the mechanical integrity of cell sheets by engaging adherens junctions. In turn, tight junction activity is increased. Therefore, I propose that cortical microtubules in the epidermis are important in forming a robust barrier by cooperatively strengthening each cell-cell junction.</p><p>To determine whether desmosomes play similar roles in simple epithelia as stratified epithelia, I examined intestinal epithelial-specific conditional desmoplakin conditional knockout mice. Unexpectedly, I found that desmoplakin is not required for cell-cell adhesion and tissue integrity in the small intestine. Furthermore, it does not organize intermediate filaments. Desmoplakin is required, however, for proper microvillus architecture. </p><p>Overall, my studies highlight novel tissue-specific roles for desmosomes, in particular desmoplakin, in organizing and integrating different cytoskeletal networks. How desmoplakin's function is regulated in each tissue will be a new interesting area of research.</p> / Dissertation

The desmosome is one of the major cell adhesion junctions found in the epithelia, heart, and hair follicle. Described as a “rivet” that hold cells together, it provides these tissues with the integrity to withstand the tremendous forces they face in everyday life. Defects in this junction can lead to devastating diseases where patients are susceptible to skin infections and cardiovascular defects. Limited treatments exist for diseases of the desmosome, and strategies do not target all symptoms. Therefore, delineating the function and regulation of desmosomes is of paramount importance for the development of prevention and treatment strategies. The Xenopus laevis has been utilized for the study of embryonic development and tissue movements. This study takes advantage of the frog model to study a key desmosomal protein, desmoplakin (Dsp), in the epidermal development of the embryo. First, Xenopus embryonic epidermis has junctional desmosomes as early as the blastula stages. Desmosomes numbers per junction increase as the embryo develops. Dsp is present in many epidermally-derived structures in the embryo at varying levels. Xenopus embryos deficient in desmoplakin have phenotypic defects in epidermal structures and the heart, mimicking mammalian models. Embryos with reduced Dsp exhibit an increased susceptibility to epidermal damage under applied mechanical forces. Assays also reveal a potential role for desmosomes in radial intercalation, a process through which cells move from the inner to the outer epidermal layers. Embryos with reduced Dsp exhibit a slight reduction in intercalation and defects in intercalating cell types, including multiciliated cells and small secretory cells. Finally, c-Jun N-terminal kinase (JNK) may have a potential role in the regulation of desmosome assembly and adhesion. Embryos with deficient Dsp display a partial recovery of mechanical integrity when treated with a JNK inhibitor.

The Exocyst is a hetero-octameric complex involved in tethering of post-Golgi vesicle transport to sites of membrane expansion. In budding yeast, the Exocyst targets vesicles to bud site resulting in bud emergence and abscission of the daughter cell. Mammalian Exocyst is recruited to developing lateral membranes after cadherin mediated adhesion and then is segregated to adherens junctional complexes (AJC). In polarized epithelia, the Exocyst is required for basal-lateral transport of LDL receptor. Additional Exocyst subunit localizations and functions have been identified. It is not known whether these supplementary roles can be attributed to the Exocyst or other unidentified Exocyst subcomplexes. Sec3, an Exocyst subunit, is hypothesized to be a landmark of polarization in yeast. In polarized epithelia, GFP tagged Sec3 remained cytosolic in polarized epithelia unlike Sec6/8. Sec3-GFP was recruited to lateral membranes only after dual over expression of heterologous GLYT1. Little is known about endogenous mammalian Sec3. Our work suggests Sec3 defines an Exocyst subcomplex that is required for desmosome integrity. Sec3 and additional subunits (Sec6, Sec8, Sec15, Exo70, and Exo84) were present at desmosomes, but Sec3 failed to localize to AJC. Only antibodies to Sec6 and Sec8 labeled AJC. Reduction of Sec3 protein expression resulted in the impairment of desmosome morphology and function with no detrimental effect on adherens junctions. These data suggest the existence of functionally different Exocyst subcomplexes. Sec3-exocyst recruited minus-end directed microtubule motor KifC3 to desmosomes. KifC3 was previously shown to be recruited with a microtubule anchoring complex to basal-lateral membrane. This suggests Sec3 may recruit KifC3 to organize microtubules at desmosomes. This would establish a pathway to efficiently transport newly synthesized basal-lateral cargo. These results suggest a novel mechanism of the Exocyst to regulate post-Golgi vesicular transport and intercellular adhesion.

Cardiomyopathies represent a heterogeneous group of diseases of the heart muscle that often lead to progressive heart failure with high morbidity and mortality. In a significant and increasing percentage of the patient population, cardiomyopathies have been associated with hereditary mutations in genes encoding critical cellular components that make up the cytoarchitecture of cardiac muscle cells, or cardiomyocytes. While specific mutations have been linked to different classes of cardiomyopathies, it is however not well understood how these mutations cause cytostructural abnormalities that ultimately lead to dysfunction of cardiomyocytes. To gain insights into the pathogenesis of inherited cardiomyopathies, we focus in this thesis on a particular set of mutations in the cardiac cytoskeleton and desmosomes that are associated with dilated and arrhythmogenic cardiomyopathies, and probe their pathogenic mechanisms using cardiomyocytes derived from human induced pluripotent stem cells and bioengineered culture platforms. In part one, we describe the mechanical and molecular basis for the assembly of sarcomeres, the fundamental contractile units within cardiomyocytes, and reveal how mutations in titin (TTN) abolish this process by disrupting cell-matrix interaction and impairing diastolic force generation, a hallmark of dilated cardiomyopathy. In the second part of this thesis, we reveal that plakophilin-2 (PKP2) mutations that are associated with arrhythmogenic cardiomyopathy lead to impaired systolic function by destabilizing cell-cell junctions and in turn disrupting sarcomere stability and organization. Together, our studies establish a deeper understanding of how cell-matrix and cell-cell interactions contribute to the organization and function of cardiomyocytes and how disruption of these interactions by pathogenic mutations lead to cardiac dysfunction. / 2022-05-18T00:00:00Z

La cardiomyopathie/dysplasie arythmogène du ventricule droit (ARVC/D) est un désordre d’origine génétique caractérisé par le remplacement du myocarde par du tissus fibro-adipeux dans le ventricule droit. Ce désordre est responsable d’un grand pourcentage de mort subite, spécialement chez les plus jeunes. ARVC/D est difficile à diagnostiquer avec les outils cliniques actuels. Elle est causée en grande majorité par des mutations dans les protéines desmosomales. ARVC/D a donc des implications d’une grande importance chez les membres de la famille, qui peuvent sans le savoir, être aussi à risque de mort subite. Dans le but d’améliorer le diagnostique, un nouvel outil, le test génétique, est de plus en plus utilisé. Hypothèses: Dans le but d’évaluer la valeur du test génétique en complément du test clinique classique chez ARVC/D nous avons effectué une investigation clinique et génétique chez 23 cas-index atteints. Méthodes: Les cas-index sont diagnostiqué après une mort subite dans la famille ou après un examen clinique poussé pour arythmies. Le diagnostique d’ARVC/D a été fait avec les outils cliniques selon les critères. L’analyse génétique des protéines desmosomales associées à la maladie a été effectuée en séquençant leurs exons ainsi que les régions introniques nécessaires à l’épissage alternatif. Résultats: Le diagnostique clinique était clair dans 18/23 et incertain dans 5/23 des individus. Nous avons identifié 15 différentes mutations chez 10 cas-index. 64% des mutations n’avaient jamais été décrites. De plus, nous avons observé la présence de double ou triple mutant dans 40% des cas-index positifs. Les individus avec mutations sont plus jeunes et ont plus de symptômes que les individus sans mutation. Conclusion: Les tests génétiques sont positifs dans 43% des patients avec ARVC/D. L’utilisation de la technologie génétique basée sur l’identification de mutations connues a une valeur limitée vu le haut pourcentage des mutations nouvelles dans la maladie. La présence de double, même de triple mutant n’est pas associé avec un phénotype plus sévère, mais renforce l’idée de la nécessité d’un test génétique pour tous les gènes. Le test génétique est un outil fort utile à ajouter aux tests cliniques pour le diagnostique des patients qui ne remplissent pas tous les critères cliniques de la maladie. Mots clés: génétique, ARVC/D, mort subite, desmosome / Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is a genetic disorder characterized by the presence of fibro-fatty replacement of the myocardium in the right ventricle. The disease is thought responsible for an important percentage of sudden cardiac death in the young. Hence the disease is usually difficult to diagnose with present clinical tools. ARVC/D is it caused in greater part by mutations in desmosomal proteins. The diagnosis of the genetic carriers bears important implications in family members, who unknowingly may be at risk for sudden death. In order to improve the diagnosis, a new tool, genetic testing, is increasingly being used. Hypothesis: In order to assess the value of genetic testing in complementing clinical testing in ARVC/D, we undertook the project to collect and perform clinical and genetic investigation in 23 probands with the disease. Methods: The probands were usually identified either after the death of a family member or after their clinical investigation for arrhythmias. The diagnosis of ARVC was made with clinical tools according to accepted criteria. Genetic analysis of desmosomal proteins previously associated with the disease was performed by sequencing the exons and intron-exon boundaries. Results: The clinical diagnosis was clear in 18/23 and suspicious in 5/23 individuals. We identified 15 different mutations in 10 probands. 64% of the mutations were not previously described. Interestingly we also observed the presence of double or triple mutants in 40% of the positive individuals. Individuals with mutations were younger and had more symptoms than individuals with no mutation. Conclusion: Genetic testing is useful in 43% of patients with ARVC. The use of mutation-based genetic technology has a very limited value due to the high percentage of previously unknown mutations in this disease. The presence of double and even triple mutants is not associated with a more severe phenotype but it indicates the need to have genetic testing performed for all genes for familial screening. Genetic testing is a useful tool to add to the clinical testing for the diagnosis of patients who do not completely fulfill the clinical criteria for the disease. Key words: genetic, ARVC/D, sudden death, desmosome

No description available.

No description available.

Les pemphigus sont des maladies auto-immunes spécifiques d'organe qui affectent la peau et les muqueuses. Ils sont caractérisés par la production d'autoanticorps pathogènes dirigés contre des protéines du desmosome, plus particulièrement, les desmogléines qui permettent l'adhésion entre eux des kératinocytes de l'épiderme. Au cours des pemphigus superficiels (PS), la réponse auto-immune est dirigée contre la desmogléine 1 (Dsg1). Chez les malades atteints de PS, des lymphocytes T autoréactifs vis à vis de la Dsg1 sont présents et interviennent dans la production d'autoanticorps anti-Dsg1. Cet autoantigène est aussi la cible de la réponse auto-immune au cours d'autres formes de pemphigus tels que les pemphigus vulgaire et paranéoplasique et par conséquent, semble jouer un rôle clé dans ces maladies. Depuis plus d'une décennie, le rôle de l'autoantigène lui-même dans l'initiation, la propagation et la pérennisation de la réponse auto-immune a été conforté par de nombreux arguments expérimentaux. En nous appuyant sur ce concept, nous avons entrepris d'étudier l'intervention de la Dsg1 dans les mécanismes physiopathologiques qui concourent au développement des pemphigus. Dans un premier temps, nous avons démontré l'expression dans l'épiderme humain, d'une isoforme tronquée de la Dsg1 générée par épissage alternatif des transcrits DSG1. Cette Dsg1 soluble est porteuse d'une séquence peptidique spécifique qui se fixe avec une forte affinité à certaines molécules HLA de classe II de susceptibilité au PS en particulier, à la molécule DRB1*0102. Ce peptide est par ailleurs capable d'induire la prolifération de cellules mononuclées du sang périphérique chez 50% des malades atteints de la forme sporadique de PS. Ces patients expriment des allèles HLA de classe II associés à la maladie et deux d'entre eux sont porteurs de la molécule DRB1*0102. Ainsi, la modification de la Dsg1 par épissage alternatif pourrait-elle intervenir dans la rupture de la tolérance au niveau du compartiment T chez les individus prédisposés génétiquement par l'expression de certains allèles HLA de classe II et in fine, conduire à l'initiation d'une réponse auto-immune B dirigée contre la Dsg1. En second lieu, nous avons observé une diversification de la réponse anticorps chez des souris normales immunisées avec la région extracellulaire recombinante de la Dsg1. Les animaux immunisés produisent non seulement des IgG dirigées contre la Dsg1 mais aussi, contre d'autres protéines de l'épiderme. Nous avons dérivé cinq anticorps monoclonaux à partir des splénocytes isolés de ces souris et montré que trois d'entre eux sont dirigés spécifiquement contre la Dsg1. Les deux autres, 10A1 et CK1, ne réagissent pas avec la Dsg1 mais reconnaissent des protéines épidermiques de plus haut poids moléculaire, compatible avec ceux des autoantigènes de la famille des plakines spécifiquement reconnus par les anticorps au cours du pemphigus paranéoplasique, e.g. l'envoplakine et la périplakine. Grâce à une analyse protéomique ciblée combinant l'immunocriblage d'une carte protéique 2D d'épiderme humain et la spectrométrie de masse MALDI-ToF, nous avons montré que la protéine cible du 10A1 est l'envoplakine, et que le CK1 reconnaît à la fois l'envoplakine et la périplakine. Ainsi, en accord ce modèle expérimental murin, la réponse B anti-Dsg1 pourrait-elle gouverner la réponse vis à vis d'autres protéines du desmosome en particulier, les plakines, mimant de ce fait la diversité de la réponse auto-immune B observée au cours du pemphigus paranéoplasique. Enfin, nous démontrons par des analyses en PCR que l'ARNm de la Dsg1 est exprimé dans le thymus humain normal, que son expression augmente avec l'âge et de ce fait, que l'absence de l'expression thymique de cette autoantigène ne constitue pas l'origine de la rupture de la tolérance qui concoure au développement des PS. Nos résultats mettent en exergue le rôle la Dsg1 dans le processus auto-immun au cours des pemphigus, d'une part, à la phase d'initiation, avec l'intervention de cet autoantigène modifié par épissage alternatif dans la réponse lymphocytaire T et d'autre part, à la phase de propagation, avec la diversification de la réponse anticorps vis à vis d'autres antigènes desmosomiaux induite chez des souris normales immunisées avec cet autoantigène.

Desmosomes are intercellular junctions that anchor intermediate filaments to the sites of intercellular contacts. They are critical for maintaining the integrity of tissues that experience constant mechanical and structural stresses, like the skin and heart. Perturbation of desmosomal adhesion can lead to devastating epidermal and myocardial diseases. However, little is known about the regulators of desmosomes and the role of desmosomes in cell signalling events. Recent work has suggested that iASPP, an inhibitor of the p53 family of proteins, localises at the intercalated discs where desmosomes reside. However, its role at the desmosomes has remained elusive. Thus, in this thesis, it was investigated whether iASPP is a dual function protein that links desmosome adhesion to gene expression and if desmosome-related diseases develop in the absence of iASPP. iASPP was found to be a novel regulator of desmosomes, co-localising with them by physically interacting with the desmosomal components desmoplakin and K5 intermediate filaments. Loss of iASPP resulted in increased phosphorylation and solubilisation of desmoplakin, leading to the formation of K5 aggregates. This culminated in disrupted intercellular adhesion and enhanced cellular migration. Consistent with the role of iASPP in the maintenance of desmosomal adhesion integrity, focal palmoplantar keratoderma was observed in iASPP-deficient mice — a disorder often associated with desmosome dysfunction. This was accompanied by disrupted intracellular signalling, as exemplified by the disrupted expression of differentiation markers; an increase in the thickness of cell layers expressing differentiation marker K1 was noted, and K5 and K6 cells were ectopically expressed throughout the diseased palmoplantar epidermis. Impaired intercellular adhesion and migration had consequences for wound healing, as iASPP-deficient mice exhibited delayed wound closure. Furthermore, defects in eyelid closure in iASPP-deficient mice were found to be due to increased apoptosis. The localisation of apoptotic cells at the leading edge of the eyelid epidermis implied that apoptosis might have occurred due to a loss of cell-matrix or cell-cell contact, i.e. anoikis. Taken together, these results suggest that iASPP is involved in pathological (palmoplantar keratoderma), physiological (wound healing) and developmental processes (embryonic eyelid closure) through its regulation of desmosomes and their dynamics. Therefore, iASPP represents a new candidate gene in cutaneous disorders and could be implicated in a variety of epidermal and myocardial diseases.