Browsing by Subject "Desmosome"

Desmosomes are a class of cell-cell adhesions whose primary physiological function is to maintain the mechanical integrity of tissues. Morphologically, desmosomes are very distinct protein complexes. They are dense pairs of plaques that straddle the interface between two plasma membranes of adjacent cells. In the extracellular space between each plaque are series of desmosome receptors that mediate the cell-cell attachment events and drive cellular cohesion throughout tissues. Emanating from the intracellular face of desmosomes are networks of intermediate filaments to which desmosome plaques are anchored. It is through this molecular chain that desmosomes traditionally bear mechanical load and promote tissue integrity. Molecular roles of the core components sufficient for completing this chain have been well described. However, insights from desmosome pathologies, genetic interactions, and biochemical fractionation have introduced perspectives that strongly suggest that desmosome composition and function extends beyond what is currently known. It is within this general context that I began my exploration into desmosome biology and employed global approaches towards uncovering a novel proteome that revealed unexpected interactions important for tissue function.

First, we used targeted proximity labeling approaches to elaborate the desmosome proteome in epidermal keratinocytes. Quantitative mass spectrometry analysis uncovered a diverse array of new constituents with broad molecular functions. We validated a number of novel desmosome-associated proteins and found that many are membrane proximal proteins that show a dependence on functional desmosomes for their cortical localization. We further explored the mechanism of localization and function of two adaptor proteins enriched in the desmosome proteome, Crk and CrkL. Epidermal deletion of both Crk and CrkL resulted in perinatal lethality with defects in desmosome morphology and keratin organization, thus demonstrating the utility of this dataset in identifying novel proteins required for desmosome-dependent epidermal integrity.

Desmosomes role in mechanical integrity is paramount in the epidermis as skin-blistering pathologies represent one of the primary hallmarks of diseases caused by desmosome disruption. Pemphigus Vulgaris (PV) is a series of autoimmune syndromes where patients produce autoantibodies that target desmosome receptors, desmoglein 3 and desmoglein 1. PV is characterized by weakening cell adhesions that result in widespread epidermal cell separations. While pathologies that characterize PV have been long documented, molecular mechanisms that drive initial desmosome disruption are still not fully understood. We sought to address initial molecular signaling events induced by PV antibodies using targeted proteomic analysis to identify early phosphorylation events. We performed time course phosphoproteomic analysis on keratinocytes early after exposure to PV antibody, AK23. With this approach we observed large coverage of the keratinocyte phosphoproteome that included significant shifts of abundant phosphorylation events after AK23 treatment. To identify the functional relevance of novel phosphorylation events, we followed the localization of constructs containing tagged protein candidates harboring phosphomimetic or non-phosphorylatable mutations. Altogether, these results provide an initial analysis of global phosphorylation events that occur in keratinocytes during the early stages of PV.

Finally, we sought to understand what was an unusual enrichment of RNA-binding proteins in our desmosome proteome. Amongst enriched protein classes were regulators of protein translation. Using immunofluorescent approaches, we were able to visualize distinct desmosome localization of representative members of protein translation machinery as well as assembled ribosomes. Considering this unusual abundance, I was interested in finding out the types of RNAs associated with these desmosome-associated RBPs. To do this we combined our proteomic approach with RNA sequencing and developed new techniques to enrich for specific RNA subpopulations localized at desmosomes. By probing for candidates of interest from this list I was able to observe enrichment of specific transcripts at desmosomes, suggesting an intimate link between RNA regulation and cell-cell adhesion. Taken together, these studies represent the first comprehensive proteomic analyses of desmosome adhesions and highlight novel desmosome interactions that may broaden mechanistic roles for desmosomes in epithelial biology.

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.

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.

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.

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.

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.

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.