Desmosome-cytoskeleton dynamics

桥粒细胞间粘附及其相关的角蛋白中间丝是上皮分化的标志，对上皮的力学和功能有着深远的贡献。桥粒和角蛋白丝之间的交联决定了上皮在不同功能背景下的可塑性。重点是在前一个项目阶段的结果，角蛋白组织的桥粒。我们发现，相当令人惊讶的是，新生桥粒作为拉长角蛋白丝的成核位点。随后的角蛋白细丝成束和桥粒融合也被协调，产生稳定的跨细胞角蛋白-桥粒支架，其呈“边缘和辐条”排列，由桥粒间皮质角蛋白细丝束（边缘）和连接桥粒与核周角蛋白笼（辐条）的放射状角蛋白细丝束组成。我们的观察进一步表明，角蛋白-桥粒支架的形态发生和维持是由肌动蛋白细胞骨架指导的。我们现在想研究这种关系，重点是质膜下角蛋白强化肌动蛋白丰富的细胞皮质作为一个主要的机械组成部分的上皮细胞与潜在的功能mechanosensing。基于角蛋白-桥粒支架与肌动球蛋白皮质的动态相互作用对于上皮组织完整性和组织重组很重要的假设，我们将追求三个主要目标：（i）研究在选择性灭活每种主要成分后，皮质桥粒相关的基于肌动球蛋白和角蛋白的细胞骨架的形态发生和维持，（ii）研究桥粒分裂和角蛋白介导的桥粒解离对肌动蛋白细胞骨架的分布模式和功能的影响，以及（iii）研究角蛋白-桥粒动力学和上皮片迁移的功能。我们将使用荧光报告监测细胞骨架动力学和桥粒成分的高分辨率延时荧光显微镜在培养的上皮细胞上涂布的载玻片或专门定制的缩微。将采用遗传和药理学方法来抑制和调节特定组分。我们的目标是有助于理解角蛋白桥粒支架作为上皮可塑性功能单位的性质和作用。

Desmosomal intercellular adhesions and their associated keratin intermediate filaments are hallmarks of epithelial differentiation with profound contributions to epithelial mechanics and function. Crosstalk between desmosomes and keratin filaments determines epithelial plasticity in different functional contexts. Emphasis was on the consequences of desmosomes for keratin organization during the previous project phase. We found, quite surprisingly, that nascent desmosomes serve as nucleation sites for elongating keratin filaments. Subsequent keratin filament bundling and desmosome fusion were also co-ordinated generating a stable transcellular keratin-desmosome scaffold in a "rim-and-spoke" arrangement consisting of interdesmosomal cortical keratin filament bundles (rim) and radial keratin filament bundles connecting desmosomes with the perinuclear keratin cage (spokes). Our observations further suggested that the morphogenesis and maintenance of the keratin-desmosome scaffold is guided by the actin cytoskeleton. We now want to investigate this relationship with a focus on the subplasmalemmal keratin-enforced actin-rich cell cortex as a major mechanical component of epithelial cells with potential functions in mechanosensing. Based on the hypothesis that dynamic interaction of the keratin-desmosome scaffold with the actomyosin cortex is important for epithelial tissue integrity and tissue re-organization, we will pursue three major aims: (i) Investigate the morphogenesis and maintenance of the cortical desmosome-associated actomyosin- and keratin-based cytoskeleton after selectively inactivating each major component, (ii) investigate the consequences of desmosome splitting and keratin filament-desmosome dissociation on distribution patterns and function of the actin cytoskeleton, and (iii) investigate keratin-desmosome dynamics and function in epithelial sheet migration. We will use fluorescent reporters to monitor cytoskeletal dynamics and desmosomal components by high-resolution time-lapse fluorescence microscopy in cultured epithelial cells on coated glass slides or specifically-tailored microprints. Genetic and pharmacologic approaches will be employed to inactivate and modulate specific components. Our goal is to contribute to the understanding of the properties and the role of the keratin-desmosome scaffold as a functional unit in epithelial plasticity.