Nematode UNC-98 functions in focal adhensions and nuclei

描述(申请人提供)：对于肌原纤维是如何组装的，或者在反复肌肉活动的情况下肌原纤维是如何保持的，人们知之甚少。这项建议概述了线虫的三个基因的研究，UNC-98，UNC-96和UNC-97，这是正确的肌原纤维组织所必需的。UNC-98是一种新的310个残基的多肽，由四个C2H2锌指组成，并预测了NLS和NES序列。通过使用UNC-98抗体和UNC-98：：GFP融合，UNC-98驻留在M系、肌肉细胞核，并可能驻留在致密小体(Z线类似物)。通过双杂交实验，UNC-98与UNC-97(哺乳动物中的夹点)相互作用，UNC-97是蠕虫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已被证明定位于肌肉致密小体、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，监控肌肉的活动，并移动到细胞核影响转录。由于它们相似的突变表型、遗传交互作用以及UNC-96活性对UNC-98正确定位的要求，因此假设UNC-96和UNC-98相互作用或共同作用。目标包括：(1)研究UNC-98，包括定位核和附着结构所需的区域，确定UNC-98是否从肌原纤维移动到细胞核，表征新的UNC-98突变等位基因，表征几个可疑的伙伴，寻找新的UNC-98伙伴，并确定UNC-98是否影响其他基因的表达；(2)确定UNC-96蛋白的性质、其细胞内位置和分子伙伴；(3)研究UNC-97，包括确定UNC-97是否从肌原纤维移动到细胞核，核定位是否依赖于UNC-98，用抗体定位UNC-97，寻找UNC-97‘S与几个已知和新的蛋白质相互作用的进一步证据，分离UNC-97的遗传修饰物，以及确定UNC-97是否影响其他基因的表达。

DESCRIPTION (provided by applicant): Relatively little is known about how myofibrils are assembled, or how the myofibrils are maintained in the face of repeated muscle activity. This proposal outlines studies of three genes in C. elegans, unc-98, unc-96 and unc-97, which are required for proper myofibril organization. UNC-98 is a novel 310 residue polypeptide consisting of four 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, muscle cell nuclei, and probably at dense bodies (Z line analogs). By 2-hybrid experiments, UNC-98 interacts with UNC-97 (PINCH in mammals), a LIM domain protein required for worm muscle focal adhesion assembly. Like UNC-98, UNC-97::GFP had been shown to localize to muscle 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 muscle activity, and travel to the nucleus to affect transcription. Because of their similar mutant phenotypes, genetic interaction, and requirement of unc-96 activity for proper UNC-98 localization, it is hypothesized that UNC-96 and UNC-98 interact or work together. Goals include: (1) study UNC-98 including mapping regions required for nuclear vs. attachment structure localization, to determine whether, UNC-98 moves from myofibrils to the nucleus, to characterize new unc-98 mutant alleles, to characterize several suspected partners and to identify new partners for UNC-98, and to determine whether UNC-98 influences the expression of other genes; (2) to determine the nature of the UNC-96 protein, its intracellular location and molecular partners; and (3) to study UNC-97 including to determine whether UNC-97 moves from myofibrils to nucleus, whether nuclear localization depends on UNC-98, localize UNC-97 with antibodies, seek further evidence for UNC-97's interaction with several known and new proteins, isolate genetic modifiers of unc-97,and to determine whether UNC-97 influences the expression of other genes.