Desmoplakin Assembly and Function in Epidermis

桥粒斑蛋白在表皮中的组装和功能

• 批准号: 7393195
• 负责人: Kathleen Janee Green
• 金额: $ 42.54万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-02-20 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7393195
• 关键词: ATP2A2; Actins; Adhesives; Adult; Affect; Animal Model; Appearance; Automobile Driving; Behavior; Binding; Biochemical; Ca(2+)-Transporting ATPase; Cadherins; Calcium; Cells; Collaborations; Congenital Heart Defects; Crystallization; Cytoskeleton; Data; Defect; Desmosomes; Disease; Elements; Embryo; Endoplasmic Reticulum; Engineering; Epidermis; Epithelium; Exhibits; Facility Construction Funding Category; Family Dasypodidae; Family member; Funding; Goals; Homeostasis; Human; Image Analysis; In Vitro; Intercellular Junctions; Intermediate Filaments; Keratosis Follicularis; Kinetics; Lead; Life; Mechanics; Mediating; Membrane; Molecular; Mus; Mutation; Myosin ATPase; Patients; Phase; Positioning Attribute; Process; Property; Purpose; RNA Interference; Regulation; Relative (related person); Research Design; Research Personnel; Resolution; Role; Signal Transduction; Skin; Specific qualifier value; Staging; Structure; Takeda brand of pioglitazone hydrochloride; Testing; Thapsigargin; Time; Tissues; Translating; Work; Wound Healing; base; cell motility; cell type; cellular imaging; desmoplakin; human disease; in vivo; insight; keratinocyte; knock-down; mutant; particle; polypeptide; research study; scaffold; skin disorder; spatial relationship; trafficking

DESCRIPTION (provided by applicant): Desmoplakin (DP) is the most abundant component of desmosomes, intercellular junctions required for maintaining tissue integrity in embryos and adults. Loss of DP results in early embryonic lethality in mice, and DP mutations in humans cause skin and heart defects. Work performed during the last funding period supports our hypothesis that DP forms a multi-functional scaffold where differentiation-specific components converge to couple intermediate filaments (IF) to the membrane. Based on our live cell imaging analysis, we now propose that the proper construction of the DP-IF scaffold requires that interaction among DP and its partners be precisely coordinated spatially and temporally during junction assembly. In the next period we will continue to elucidate critical structural features of the DP-IF scaffolding, to investigate the molecular and regulatory mechanisms driving its assembly, and to determine how defects in assembly due to engineered or naturally occurring DP human mutations lead to structural and functional defects in vitro and in vivo. Our specific goals are: 1. To determine how sequences within the DP C-terminus specify interactions with different IF by testing the effects of engineered and naturally occurring DP mutations in vitro and in cells on binding to cell type-specific IF; 2. To determine how DP collaborates with the armadillo family member PKP2 in assembly and regulation of the DP-IF scaffold by a) investigating the biochemical, temporal and spatial relationship of DP and PKP2 during junction assembly, b) identifying DP or PKP2 mutants that uncouple DP-PKP2 or PKP2-actin, and c) examining effects of these mutants and RNAi-mediated PKP2 knock down on desmosome assembly and function ; 3. To investigate how intracellular calcium stores and downstream signaling regulate DP trafficking to desmosomes by analyzing DP assembly behavior in keratinocytes in which ATP2A2/SERCA2 is impaired pharmacologically or by Darier's disease mutations or RNAi-mediated knock down; and 4. To define how engineered and naturally occurring DP mutations that regulate DP-IF interactions affect desmosome assembly and maturation, adhesive strength and wound healing in vitro and in an animal model. These experiments will help us to understand how DP mutations and defects in desmosome assembly and structure lead to human skin disease, and also to appreciate why carefully regulating desmosome assembly is important for proper epidermal wound healing.

描述（由申请人提供）：脱莫普拉金（DP）是脱骨体的最丰富成分，是维持胚胎和成人组织完整性所需的细胞间连接。 DP的丧失导致小鼠早期胚胎致死性，而人类的DP突变会导致皮肤和心脏缺陷。在上一期间进行的工作支持了我们的假设，即DP形成了多功能支架，其中分化特异性的成分会融合到膜中间丝（如果）与膜。基于我们的实时细胞成像分析，我们现在建议DP-IF脚手架的正确构造要求DP及其伴侣之间的相互作用在交界装配过程中在空间和时间上进行精确协调。在下一个时期，我们将继续阐明DP-IF脚手架的关键结构特征，研究驱动其组装的分子和调节机制，并确定由于工程或自然发生的DP人类突变导致的组装缺陷如何导致体内和体内的结构和功能缺陷。我们的具体目标是：1。确定DP C末端中的序列如何通过测试工程和天然发生的DP突变在体外和细胞与细胞类型特异性的结合的效果来指定不同的相互作用； 2。为了确定DP如何与ARMADILLO家族成员PKP2合作，并通过a）调查连接装配过程中DP和PKP2的生物化学，时间和空间关系的DP-IF支架，b）识别DP或PKP2突变的dp-pkp2或PKP2或PKP2或PKP2-cp2-cp2-cp2-cpp2-cpp2-cpp2-cpp2-cpp2-cp2-cp2- RNAi介导的PKP2击倒了脱骨体组装和功能； 3。为了研究细胞内钙存储和下游信号如何通过分析角质细胞中的DP组装行为来调节DP运输到脱粒体，其中ATP2A2/SERCA2在药理学上受损或Darier疾病突变或RNAi介导的敲击; 4。定义调节DP- IF相互作用的工程和自然发生的DP突变如何影响脱骨体组装和成熟，粘合强度和体外和动物模型中的伤口愈合。这些实验将有助于我们了解脱骨组装和结构中的DP突变和缺陷导致人体皮肤病，并欣赏为什么仔细调节脱骨体组装对于适当的表皮伤口愈合很重要。