Nematode UNC-98 functions in focal adhensions and nuclei

线虫 UNC-98 在粘着点和细胞核中发挥作用

• 批准号: 6968518
• 负责人: GUY Martin BENIAN
• 金额: $ 22.4万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6968518
• 关键词: Caenorhabditis elegans; alleles; chimeric proteins; fluorescence recovery after photobleaching; focal adhesion kinase; gene expression; gene mutation; green fluorescent proteins; helminth genetics; homeostasis; molecular assembly /self assembly; muscle proteins; myofibrils; phenotype; polymerase chain reaction; protein localization; protein protein interaction; sarcomeres

DESCRIPTION (provided by applicant): The long-term goal is to understand the mechanism by which myofibrils assemble from their components, and how these precise structures are maintained in the face of repeated muscle activity. This goal has relevance to many types of human muscle disease, including those of skeletal and cardiac muscle. This proposal outlines studies of three genes in C. elegans, unc-98, unc-96 and unc-97 which are required for proper myofibril assembly and/or maintenance. UNC-98 is a novel 310 residue polypeptide consisting of 4 C2H2 Zn fingers and predicted NLS and NES sequences. By use of UNC-98 antibodies and UNC-98:GFP fusions, UNC-98 resides at M-lines, dense bodies (Z line analogs) and muscle cell nuclei. UNC-98 interacts with UNC-97 (PINCH in mammals), a LIM domain protein required for muscle focal adhesion assembly. Like UNC-98, UNC-97:GFP localizes to dense bodies, M-lines and nuclei. It is hypothesized that UNC-98 and UNC-97 function in muscle focal adhesion homeostasis, in which these proteins when localized to the adhesion sites monitor myofibril structure or activity, and travel to the nucleus to affect gene expression, unc-96 has a similar mutant phenotype to unc-98, interacts with unc-98 genetically, and may protect the sarcomere from breakdown resulting from normal muscle usage. We have determined that UNC-96 is a novel 418 aa protein and by 2 hybrid interacts with two conserved LIM domain proteins that also interact with UNC-97. Goals include: (1) for UNC-98, prove that its nuclear localization is important for its function, show that it can move from myofibrils into the nucleus, determine whether it can influence the expression of other genes, and whether it interacts with paramyosin in thick filaments; (2) for UNC-96, find out where it is localized in the sarcomere, provide further evidence that it interacts with paramyosin, UNC-98 and two LIM domain proteins; (3) for UNC-97 study its dynamics, whether it influences gene expression, whether its nuclear localization depends on UNC-98, and whether it indeed interacts with the two LIM domain proteins.

描述（由申请人提供）：长期目标是了解肌原纤维从其组件组装的机制，以及面对重复的肌肉活动如何维持这些精确的结构。这一目标与许多类型的人类肌肉疾病相关，包括骨骼肌和心肌疾病。该提案概述了对秀丽隐杆线虫中三个基因（unc-98、unc-96 和 unc-97）的研究，这些基因是正确的肌原纤维组装和/或维持所必需的。 UNC-98 是一种新型 310 个残基多肽，由 4 个 C2H2 Zn 指和预测的 NLS 和 NES 序列组成。通过使用 UNC-98 抗体和 UNC-98:GFP 融合体，UNC-98 驻留在 M 线、致密体（Z 线类似物）和肌肉细胞核上。 UNC-98 与 UNC-97（哺乳动物中的 PINCH）相互作用，UNC-97 是肌肉粘着斑组装所需的 LIM 结构域蛋白。与 UNC-98 一样，UNC-97:GFP 定位于致密体、M 线和细胞核。据推测，UNC-98和UNC-97在肌肉粘着斑稳态中发挥作用，其中这些蛋白质当定位于粘着位点时监测肌原纤维的结构或活性，并进入细胞核影响基因表达，unc-96具有与unc-98相似的突变表型，与unc-98在遗传上相互作用，并且可以保护肌节免受正常肌肉使用导致的破坏。我们已确定 UNC-96 是一种新型 418 个氨基酸蛋白，并通过 2 个杂交与两个保守的 LIM 结构域蛋白相互作用，而这两个蛋白也与 UNC-97 相互作用。目标包括：（1）对于UNC-98，证明其核定位对其功能很重要，表明它可以从肌原纤维移动到细胞核，确定它是否可以影响其他基因的表达，以及是否与粗丝中的副肌球蛋白相互作用； (2) 对于UNC-96，找出其在肌节中的定位，提供其与副肌球蛋白、UNC-98和两个LIM结构域蛋白相互作用的进一步证据； (3)对于UNC-97，研究其动态，是否影响基因表达，其核定位是否依赖于UNC-98，以及是否确实与两个LIM结构域蛋白相互作用。