Regulation of cardiac nuclear pore structure and function

心脏核孔结构和功能的调节

• 批准号: 7260258
• 负责人: Carmen Militza Terzic
• 金额: $ 37.78万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-15 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7260258
• 关键词: Address; Affect; Architecture; Atomic Force Microscopy; Binding; Calcium; Calcium Binding; Cardiac; Cardiac Myocytes; Cations; Cell Differentiation process; Cells; Chromosome Mapping; Communication; Competence; Confocal Microscopy; Congenital Abnormality; Congenital Heart Defects; Data; Defect; Dependence; Derivation procedure; Development; Disruption; Electrons; Embryo; Event; Fluorescence Resonance Energy Transfer; Foundations; Functional disorder; Genes; Genetic; Genetic Information Processing Pathway; Genomics; Homeostasis; Immunofluorescence Immunologic; Individual; Knock-out; Lasers; Length; Link; Maps; Mediating; Metabolism; Microanatomy; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Myofibrillogenesis; Nuclear; Nuclear Envelope; Nuclear Pore; Nuclear Pore Complex; Nuclear Translocation; Null Lymphocytes; Numbers; Pathway interactions; Phenotype; Pilot Projects; Population; Proteins; Proteomics; Regulation; Research Personnel; Reticulin; Role; Sarcoplasmic Reticulum; Scanning; Secure; Site-Directed Mutagenesis; Sorting - Cell Movement; Stem cells; Structure; Testing; Transcriptional Regulation; Transfection; Trefoil Motif; Ventricular; Western Blotting; Work; base; calreticulin; cardiogenesis; comparative; defined contribution; genetic regulatory protein; immunocytochemistry; insight; knockout gene; mutant; novel; nucleocytoplasmic transport; programs; protein function; restoration; trafficking; transcription factor

DESCRIPTION (provided by applicant): Transport across the nuclear envelope is vital in the processing of genetic information during cell differentiation. Deficiency in the nuclear localization of transcription factors has been related to congenital abnormalities including cardiac defects. A case in point is the lethal defect in heart development described recently in embryos lacking calreticulin, a multifunctional calcium-binding chaperone protein. However, the mechanisms responsible for calreticulin-mediated regulation of nuclear function and the contribution of calreticulin in cardiogenesis is poorly understood. To address this question, we established a stem cell- based model of calreticulin gene knockout (crt-/-) and obtained preliminary data suggesting a contribution of calreticulin in securing the structural and functional competence of nuclear pores. In Aim #1, the requirement for calreticulin in nuclear translocation of cardiac transcription factors will be determined by laser confocal microscopy, and the microanatomy of nuclear pores will be evaluated by mapping the architecture and composition of individual nuclear pore complexes using atomic force microscopy and immunocytochemistry. Proteomic analysis will be performed to define the molecular basis of the calreticulin effect. To further define the mechanism underlying regulation of pore structure and function, in Aim #2, we will evaluate the contribution of calreticulin in determining the calcium filling state of the nuclear cisterna by fluorescence resonance energy transfer-based probing. In addition, the gene profile of wildtype versus crt-/- stem cell- derived cardiomyocytes will be established by genomic scans to map genes affected by calreticulin deficiency including genes involved in the regulation of the nuclear transport machinery, calcium dependent pathways and cellular metabolism. In Aim #3, rescue of nuclear pore competence and restoration of cardiogenesis will be tested by transfection of calreticulin constructs that recapitulate specialized functions of this protein. This proposal will thus provide novel mechanistic insight into molecular events associated with nucleocytoplasmic communication critical for cardiac differentiation. As abnormal function of the nuclear envelope has been linked to a growing number of cardiac congenital defects, this proposal will establish a framework for targeted strategies aimed at supporting reparative cardiogenesis.

描述（由申请人提供）：跨核包膜的运输对于细胞分化过程中遗传信息的处理至关重要。转录因子的核定位缺乏与先天性异常有关，包括心脏缺陷。一个很好的例子是最近在缺乏钙网蛋白（一种多功能钙结合伴侣蛋白）的胚胎中描述的心脏发育缺陷。但是，对钙蛋白介导的核功能调节的调节和钙网蛋白在心脏病发生的贡献的机制知之甚少。为了解决这个问题，我们建立了基于干细胞的钙网蛋白基因敲除（CRT - / - ）的模型，并获得了初步数据，这表明钙网蛋白在确保核孔的结构和功能能力方面有贡献。在AIM＃1中，将通过激光共聚焦显微镜确定心脏转录因子核易位的需求，并通过绘制使用原子力显微镜和免疫细胞化学的单个核孔复合物的结构和组成来评估核孔的微分裂。将进行蛋白质组学分析以定义钙网蛋白作用的分子基础。为了进一步定义孔结构和功能调节的基础机制，在AIM＃2中，我们将评估钙网蛋白在确定核库斯特纳的钙填充状态下通过荧光共振能量传递探测的贡献。此外，基因组扫描将建立野生型与CRT - / - 干细胞衍生的心肌细胞的基因谱，以绘制受钙网蛋白缺乏症影响的基因，包括涉及调节核转运机械，钙依赖性途径和细胞代谢的基因。在AIM＃3中，将通过转染钙蛋白构建体的转染来测试核孔的能力和心脏病的恢复，从而概括了该蛋白质的专业功能。因此，该提案将提供对与心脏分化至关重要的核质沟通相关的分子事件的新机械洞察力。由于核包络的异常功能与越来越多的心脏先天性缺陷有关，该提案将建立旨在支持修复性心脏病的目标策略的框架。