Nematode UNC-98 functions in focal adhensions and nuclei

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，以及这些精确的结构如何在面对重复的肌肉活动时保持。这一目标与许多类型的人类肌肉疾病有关，包括骨骼肌和心肌疾病。本提案概述了秀丽隐杆线虫中三个基因unc-98、unc-96和unc-97的研究，这三个基因是肌原纤维正常组装和/或维持所必需的。UNC-98是一种新的310残基多肽，由4个C2H2锌指组成，预测了NLS和NES序列。通过使用UNC-98抗体和UNC-98:GFP融合，UNC-98存在于m细胞系、致密体（Z细胞系类似物）和肌细胞核中。UNC-98与UNC-97（哺乳动物中的PINCH）相互作用，后者是肌肉局灶黏附组装所需的LIM结构域蛋白。像UNC-98一样，UNC-97:GFP定位于致密体、m线和核。假设UNC-98和UNC-97在肌肉局灶黏附稳态中起作用，其中这些蛋白定位于黏附位点时监测肌原纤维的结构或活性，并前往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基因相互作用，并可能保护肌节免受正常肌肉使用导致的破坏。我们已经确定UNC-96是一个新的418aa蛋白，并且通过2杂交与两个保守的LIM结构域蛋白相互作用，这些蛋白也与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的重要性，表明其可以从肌原纤维进入细胞核，确定其是否可以影响其他基因的表达，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，进一步证明其与副肌球蛋白、UNC-98和两种LIM结构域蛋白相互作用；(3)针对UNC-97研究其动力学，是否影响基因表达，其核定位是否依赖于UNC-98，是否确实与两种LIM结构域蛋白相互作用。

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.