Nematode UNC-98 functions in focal adhensions and nuclei

描述(申请人提供)：长期目标是了解肌原纤维从其组成部分组装的机制，以及如何在反复肌肉活动的情况下保持这些精确的结构。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。这项建议概述了线虫的三个基因UNC-98、UNC-96和UNC-97的研究，这些基因是正确组装和/或维护肌原纤维所必需的。UNC-98是一个新的310个残基的多肽，由4个C2H2锌指和预测的NLS和NES序列组成。通过使用UNC-98抗体和UNC-98：GFP融合，UNC-98位于M线、致密小体(Z线类似物)和肌细胞核。UNC-98与UNC-97(哺乳动物中的夹点)相互作用，UNC-97是肌肉局部黏附组装所需的LIM结构域蛋白。与UNC-98一样，UNC-97：GFP定位于致密小体、M线和细胞核。据推测，UNC-98和UNC-97在肌肉局部黏附动态平衡中发挥作用，当这些蛋白质定位于黏附部位时，这些蛋白质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，在基因上与UNC-98相互作用，并可能保护肌节免受正常肌肉使用造成的破坏。我们已经确定UNC-96是一个新的418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，这两个蛋白也与UNC-97相互作用。目标包括：(1)对于UNC-98，证明其核定位对于其功能至关重要，证明其能够从肌原纤维进入细胞核，确定其是否能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，找出其在肌小球中的定位位置，为其与副肌球蛋白、UNC-98和两个LIM结构域蛋白的相互作用提供进一步的证据；(3)对于UNC-97，研究其动力学，是否影响基因表达，其核定位是否取决于UNC-98，以及它是否确实与两个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.