Keratin-dependent regulation of desmosome composition and actin organization

哺乳动物表皮是一种多层上皮，通过连接到细胞骨架角蛋白的桥粒将角质形成细胞结合在一起，保护身体免受物理和化学损伤。表皮分化、创面愈合和屏障形成需要不断重塑桥粒和角蛋白细胞骨架，将立方体角质形成细胞转化为扁平角质形成屏障，并转化为迁移性角质形成细胞，促进创面修复。这种重塑需要角蛋白和桥粒体蛋白的分化特异性表达。它受生长因子和机械力的调节，需要与肌动蛋白细胞骨架进行串扰。关于角蛋白同型调节桥粒组成和粘附强度的机制以及这些变化如何与肌动蛋白细胞骨架和粘附连接协调，我们知之甚少。在第一个资助期间，我们发现角蛋白同型组成可以调节桥粒组成和粘接强度。K14的表达导致桥粒稳定，Dsg1水平高，桥粒蛋白c端磷酸化水平低，而K17的表达降低了Dsg1的mRNA和蛋白水平，增加了Dsg3，并增加了c端桥粒蛋白磷酸化水平。总的来说，这些变化削弱了细胞间的粘附。此外，K17的表达与角化细胞皮层肌动蛋白组织的减少相一致，这与角蛋白缺陷小鼠皮肤中的观察结果相似。在皮肤中，肌动蛋白组织的改变与角化细胞在屏障形成过程中扁平化的失败相一致。在第二个资助期，我们希望验证角蛋白同型通过与桥丝蛋白和亲血小板蛋白的直接相互作用以及通过翻译后蛋白修饰间接调节桥丝体组成和粘附强度的假设。为此，我们将重点关注选择的蛋白质结构域的磷酸化。此外，我们想要研究角蛋白是否通过Dsg1影响皮质肌动蛋白组织，从而在表皮分化过程中介导角化细胞变平。除了转基因小鼠外，我们还将利用我们的小鼠角化细胞小组来验证我们的假设，该小组重新表达角蛋白同型和变体。我们期望我们的项目提供了角蛋白家族成员如何通过与桥粒体蛋白相互作用调节细胞间粘附的理解。

The mammalian epidermis is a multilayered epithelium that protects the body against physical and chemical insults by virtue of keratinocytes that are held together by desmosomes connected to cytoskeletal keratins. Epidermal differentiation, wound healing and barrier formation require constant remodeling of desmosomes and the keratin cytoskeleton to transform cuboidal keratinocytes into flat corneocytes for barrier formation and into migratory keratinocytes to promote wound repair. This remodeling requires differentiation-specific expression of isotypes of keratins and desmosomal proteins. It is regulated by growth factors and mechanical force and requires cross-talk to the actin cytoskeleton. Little is known about mechanisms by which keratin isotypes regulate desmosome composition and adhesive strength and how these changes are coordinated with the actin cytoskeleton and adherens junctions.During the first funding period, we have found that keratin isotype composition serves as a regulator of desmosome composition and adhesive strength. Whereas K14 expression led to stable desmosomes with high levels of Dsg1 and low C-terminal phosphorylation of desmoplakin, expression of K17 reduced Dsg1 at mRNA and protein levels and increased Dsg3, in addition to increasing C-terminal desmoplakin phosphorylation. Collectively, these changes weakened intercellular adhesion. Further, K17 expression coincided with decreased cortical actin organization in keratinocytes, similar to observations in the skin of keratin-deficient mice. In the skin, altered actin organization coincided with failure of keratinocytes to flatten during barrier formation. In the second funding period, we want to test the hypothesis that keratin isotypes regulate desmosome composition and adhesive strength through direct protein interactions with desmoplakin and plakophilin, and indirectly, through posttranslational protein modifications. To that end, we will focus on phosphorylation of select protein domains. Further, we want to examine whether keratins, via Dsg1, affect cortical actin organization and thereby mediate keratinocyte flattening during epidermal differentiation. We will test our hypothesis by exploiting our panel of mouse keratinocytes re-expressing keratin isotypes and variants, in addition to transgenic mice.We expect that our project provides an understanding how members of the keratin family regulate intercellular adhesion through interaction with desmosomal proteins.