Nematode UNC-98 functions in focal adhensions and nuclei

描述（由申请人提供）：关于肌原纤维如何组装或肌原纤维如何在重复的肌肉活动中保持相对较少。该建议概述了C. elegans、UNC-98、UNC-96和UNC-97，它们是适当的肌原纤维组织所需的。NLS-98是一个新的310个氨基酸残基的多肽，由4个C2 H2锌指和预测的NLS和内斯序列组成。通过使用β-98抗体和β-98：：GFP融合体，β-98位于M线、肌细胞核，并且可能位于致密体（Z线类似物）。通过双杂交实验，PINCH-98与PINCH-97（哺乳动物中的PINCH）相互作用，PINCH-97是蠕虫肌肉粘着斑组装所需的LIM结构域蛋白。与GFP-98一样，GFP-97：：GFP已被证明定位于肌肉致密体、M线和细胞核。据推测，E10 -98和E10 -97在肌肉局灶性粘附稳态中起作用，其中这些蛋白质当定位于粘附位点时监测肌肉活动，并行进到细胞核以影响转录。由于它们相似的突变体表型、遗传相互作用和unc-96活性对正确的nc-98定位的要求，推测nc-96和nc-98相互作用或一起工作。目标包括：（1）研究UNC-98，包括绘制核与附着结构定位所需的区域，以确定UNC-98是否从肌原纤维移动到核，表征新的UNC-98突变等位基因，表征几个可疑的配偶体并鉴定UNC-98的新配偶体，并确定UNC-98是否影响其他基因的表达;（2）确定P1996蛋白的性质、细胞内定位和分子伴侣;（3）研究肌钙蛋白-97，包括确定肌钙蛋白-97是否从肌原纤维移动到细胞核，核定位是否依赖于肌钙蛋白-98，用抗体定位肌钙蛋白-97，进一步寻找unc-97与几种已知和新的蛋白质相互作用的证据，分离unc-97的遗传修饰因子，并确定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.