Forces and signal transduction by desmosomal cadherins

桥粒钙粘蛋白（DCs）是一种位于桥粒内部的跨膜粘附分子，也存在于桥粒外的膜中。在桥粒内，dc在细胞内与斑块蛋白亲血小板蛋白和血小板红蛋白偶联。桥蛋白将斑块连接到中间丝细胞骨架上。这种结构为遭受高度机械应力的组织（如表皮或心肌）提供细胞间粘连强度，这些组织可以从DC功能受损引起的严重疾病中得出结论。然而，到目前为止，胶质细胞的粘附特性还没有被表征，并且内皮细胞外的胶质细胞在膜中的功能在很大程度上是未知的。最近的证据表明，树突状细胞除了具有粘附功能外，还通过以粘附依赖的方式调节细胞内信号传导来塑造细胞行为。在这里，esmosomal dc可能起关键作用。为了进一步了解这些功能，该提案旨在通过生物物理和生化方法在活角质形成细胞中表征dc的粘附和信号特性。我们将使用原子力显微镜（AFM）和光学捕获方法评估(i)在活的角质形成细胞中特定DC亚型的分布、结合伙伴和结合特征，以及（ii）确定这些参数是否依赖于细胞质斑块的锚定。在这里，我们将应用缺乏血小板蛋白、桥蛋白和角蛋白细丝的小鼠角化细胞。为了研究DC以粘附依赖的方式引发的外-内信号，我们将（iii）使用FRET探针和生化分析来测量p38MAPK、RhoA、PKC和Src在干扰特定DC同工异构体与抗体或肽的结合后的活性。由于已知这些分子也可调节角质形成细胞内聚，因此我们最终将（iv）评估通过这些信号通路的内向外信号如何改变dc的分布和结合特性。综上所述，这些实验将为特定dc的功能及其作为细胞内信号传导受体和靶点的作用提供新的见解。

Desmosomal cadherins (DCs) are transmembrane adhesion molecules located inside desmosomes and also found extradesmosomal in the membrane. Within desmosomes, DCs are coupled intracellularly to the plaque proteins plakophilin and plakoglobin. Desmoplakin connects the plaque to the intermediate filament cytoskeleton. This construct provides intercellular adhesive strength to tissues subjected to high degrees of mechanical stress such as the epidermis or the heart muscle which can be concluded from severe diseases arising from impaired DC function. However, the adhesive properties of DCs have not been characterized so far and the functions of extradesmosomal DCs in the membrane are largely unknown. Recent evidence demonstrates that DCs, in addition to their adhesive function, shape cell behavior via modulating intracellular signaling in an adhesion-dependent manner. Here, the extradesmosomal DCs may play a crucial role. To gain further insights into these functions, the proposal aims to characterize the adhesive and the signaling properties of DCs via biophysical and biochemical approaches in living keratinocytes. We will evaluate (i) the distribution, binding partners and binding characteristics of specific DC isoforms in living keratinocytes using atomic force microscopy (AFM) and optical trapping approaches and (ii) determine whether these parameters are dependent on the anchorage to the cytoplasmic plaque. Here we will apply murine keratinocytes deficient for plakoglobin, desmoplakin and keratin filaments. To investigate outside-in-signaling elicited by DCs in an adhesion-dependent manner, we will (iii) use FRET probes and biochemical assays to measure the activity of p38MAPK, RhoA, PKC and Src after interference with the binding of specific DC isoforms with antibodies or peptides. Because these molecules are also known to modulate keratinocyte cohesion, we finally will (iv) evaluate how the distribution and binding properties of DCs are altered by inside-out signaling via these signaling pathways. Taken together, these experiments will provide novel insights into the function of specific DCs and their roles as receptors and targets of intracellular signaling.