Abstract:
The α-actinin proteins are a highly conserved family of actin crosslinkers that mediate interactions between several cytoskeletal and sarcomeric proteins. Nonsarcomeric α-actinin-1 and α-actinin-4 crosslink actin filaments in the cytoskeleton, while sarcomeric α-actinin-2 and α-actinin-3 serve a crucial role in anchoring actin filaments to the muscle Z-line. To assess the difference in turnover dynamics and structure/function properties between the α-actinin isoforms at the sarcomeric Z-line, we used Fluorescence Recovery After Photobleaching (FRAP) in primary myofiber cultures. We found that the recovery kinetics of these proteins followed three distinct patterns: α-actinin-2/α-actinin-3 had the slowest turn over, α-actinin-1 recovered to an intermediate degree, and α-actinin-4 had the fastest recovery. Interestingly, the isoforms\' patterns of recovery were reversed at adhesion plaques in fibroblasts. This disparity suggests that the different α-actinin isoforms have unique association kinetics in myofibers and that nonmuscle isoform interactions are more dynamic at the sarcomeric Z-line. Protein domain-specific investigations using α-actinin-2/4 chimeric proteins showed that differential dynamics between sarcomeric and nonmuscle isoforms are regulated by cooperative interactions between the N-terminal actin-binding domain, the spectrin-like linker region and the C-terminal calmodulin-like EF hand domain. Together, these findings demonstrate that α-actinin isoforms are unique in binding dynamics at the Z-line and suggest differentially evolved interactive and Z-line association capabilities of each functional domain.
摘要:
α-肌动蛋白蛋白是肌动蛋白交联剂的高度保守家族,可介导几种细胞骨架和肌节蛋白之间的相互作用。细胞骨架中的非肌节α-肌动蛋白1和α-肌动蛋白4交联肌动蛋白丝,而肌节α-肌动蛋白-2和α-肌动蛋白-3在将肌动蛋白丝锚定到肌肉Z线上中起着至关重要的作用。为了评估肌节Z线α-肌动蛋白同工型之间的周转动力学和结构/功能特性的差异,我们在原代肌纤维培养物中使用光漂白后荧光恢复(FRAP)。我们发现这些蛋白质的回收动力学遵循三种不同的模式:α-肌动蛋白2/α-肌动蛋白3的翻转最慢,α-肌动蛋白-1恢复到中等程度,α-肌动蛋白-4恢复最快。有趣的是,同工型的恢复模式在成纤维细胞的粘连斑块上逆转。这种差异表明,不同的α-肌动蛋白同工型在肌纤维中具有独特的缔合动力学,并且非肌肉同工型相互作用在肌节Z线上更加动态。使用α-肌动蛋白2/4嵌合蛋白进行的蛋白质结构域特异性研究表明,肌节和非肌肉同工型之间的差异动力学受N末端肌动蛋白结合结构域之间的协同相互作用调节,光谱蛋白样接头区和C末端钙调蛋白样EF手结构域。一起,这些发现表明α-肌动蛋白同种型在Z线的结合动力学中是独特的,并表明每个功能域的差异进化的相互作用和Z线关联能力。