我们研究了指导横纹肌中高度有序的肌动蛋白-肌球蛋白细胞骨架形成和维持的机制。秀丽隐杆线虫的UNC-82激酶与哺乳动物激酶ARK5/NUAK1和SNARK/NUAK2是直系同源的。UNC-82定位到M线,并且需要适当地组织粗丝,但其底物和作用机制尚不清楚。在UNC-82催化结构域中具有错义突变的三个突变体的抗体染色显示出肌肉结构比在空的unc-82(0)中更少混乱,但包含在unc-82中没有发现的独特的异位积累(0)这些积累包含副肌球蛋白和肌球蛋白B,但是缺乏肌球蛋白A和肌球蛋白A相关蛋白,以及整合素相关复合物的蛋白质。荧光标记错义突变蛋白UNC-82E424K在野生型中正常定位;然而,在UNC-82(0)中,标记的蛋白质是在异位积累中发现的,我们还显示用最近合成的副肌球蛋白标记。在五个影响肌肉的突变体中,将野生型UNC-82::GFP募集到不同蛋白质组成的聚集体中,表明UNC-82和副肌球蛋白的共定位不需要UNC-96,UNC-98/ZnF,UNC-89/obscurin,CSN-5肌球蛋白A,或肌球蛋白B单独。副肌球蛋白突变体的剂量效应表明UNC-82作为复合体的一部分,其中它与副肌球蛋白的化学计量关系至关重要。UNC-82剂量在没有副肌球蛋白的情况下影响肌肉组织,我们提供的证据表明,UNC-98/ZnF与肌球蛋白A的相互作用独立于UNC-82,并且UNC-82在UNC-98/ZnF的上游起作用,该通路在粗丝组装期间组织副肌球蛋白。
We study the mechanisms that guide the formation and maintenance of the highly ordered actin-myosin cytoskeleton in striated muscle. The UNC-82 kinase of Caenorhabditis elegans is orthologous to mammalian kinases ARK5/NUAK1 and SNARK/NUAK2. UNC-82 localizes to the M-line, and is required for proper organization of thick filaments, but its substrate and mechanism of action are unknown. Antibody staining of three mutants with missense mutations in the UNC-82 catalytic domain revealed muscle structure that is less disorganized than in the null unc-82(0), but contained distinctive ectopic accumulations not found in unc-82(0) These accumulations contain paramyosin and myosin B, but lack myosin A and myosin A-associated proteins, as well as proteins of the integrin-associated complex. Fluorescently tagged missense mutant protein UNC-82 E424K localized normally in wild type; however, in unc-82(0), the tagged protein was found in the ectopic accumulations, which we also show to label with recently synthesized paramyosin. Recruitment of wild-type UNC-82::GFP to aggregates of differing protein composition in five muscle-affecting mutants revealed that colocalization of UNC-82 and paramyosin does not require UNC-96, UNC-98/ZnF, UNC-89/obscurin, CSN-5, myosin A, or myosin B individually. Dosage effects in paramyosin mutants suggest that UNC-82 acts as part of a complex, in which its stoichiometric relationship with paramyosin is critical. UNC-82 dosage affects muscle organization in the absence of paramyosin, perhaps through myosin B. We present evidence that the interaction of UNC-98/ZnF with myosin A is independent of UNC-82, and that UNC-82 acts upstream of UNC-98/ZnF in a pathway that organizes paramyosin during thick filament assembly.