Lewis Raymond A, Yamashiro Sawako, Gokhin David S, Fowler Velia M
Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California.
Cytoskeleton (Hoboken). 2014 Jul;71(7):395-411. doi: 10.1002/cm.21179. Epub 2014 Jul 2.
Tropomodulins (Tmods) interact with tropomyosins (TMs) via two TM-binding sites and cap the pointed ends of TM-coated actin filaments. To study the functional interplay between TM binding and TM-actin filament capping by Tmods, we introduced disabling mutations into the first, second, or both TM-binding sites of full-length Tmod1 (Tmod1-L27G, Tmod1-I131D, and Tmod1-L27G/I131D, respectively) and full-length Tmod3 (Tmod3-L29G, Tmod3-L134D, and Tmod3-L29G/L134D, respectively). Tmod1 and Tmod3 showed somewhat different TM-binding site utilization, but nearly all TM binding was abolished in Tmod1-L27G/I131D and Tmod3-L29G/L134D. Disruption of Tmod-TM binding had a modest effect on Tmod1's ability and no effect on Tmod3's ability to stabilize TM-actin pointed ends against latrunculin A-induced depolymerization. However, disruption of Tmod-TM binding did significantly impair the ability of Tmod3 to reduce elongation rates at pointed ends with α/βTM, albeit less so with TM5NM1, and not at all with TM5b. For Tmod1, disruption of Tmod-TM binding only slightly impaired its ability to reduce elongation rates with α/βTM and TM5NM1, but not at all with TM5b. Thus, Tmod-TM binding has a greater influence on Tmods' ability to inhibit subunit association as compared to dissociation from TM-actin pointed ends, particularly for α/βTM, with Tmod3's activity being more dependent on TM binding than Tmod1's activity. Nevertheless, disruption of Tmod1-TM binding precluded Tmod1 targeting to thin filament pointed ends in cardiac myocytes, suggesting that the functional effects of Tmod-TM binding on TM-coated actin filament capping can be significantly modulated by the in vivo conformation of the pointed end or other factors in the intracellular environment.
原肌球蛋白调节蛋白（Tmods）通过两个原肌球蛋白（TMs）结合位点与原肌球蛋白相互作用，并封闭TM包被的肌动蛋白丝的尖端。为了研究Tmods的TM结合与TM-肌动蛋白丝封闭之间的功能相互作用，我们在全长Tmod1（分别为Tmod1-L27G、Tmod1-I131D和Tmod1-L27G/I131D）和全长Tmod3（分别为Tmod3-L29G、Tmod3-L134D和Tmod3-L29G/L134D）的第一个、第二个或两个TM结合位点引入了失活突变。Tmod1和Tmod3显示出略有不同的TM结合位点利用情况，但几乎所有的TM结合在Tmod1-L27G/I131D和Tmod3-L29G/L134D中都被消除。Tmod-TM结合的破坏对Tmod1稳定TM-肌动蛋白尖端抵抗拉沙里菌素A诱导的解聚的能力有适度影响，对Tmod3的这种能力没有影响。然而，Tmod-TM结合的破坏确实显著损害了Tmod3降低α/βTM存在时尖端伸长率的能力，尽管在TM5NM1存在时损害较小，而在TM5b存在时则完全没有影响。对于Tmod1，Tmod-TM结合的破坏仅轻微损害其在α/βTM和TM5NM1存在时降低伸长率的能力，而在TM5b存在时则完全没有影响。因此，与从TM-肌动蛋白尖端解离相比，Tmod-TM结合对Tmods抑制亚基结合能力的影响更大，特别是对于α/βTM，Tmod3的活性比Tmod1的活性更依赖于TM结合。然而，Tmod1-TM结合的破坏阻止了Tmod1靶向心肌细胞中的细肌丝尖端，这表明Tmod-TM结合对TM包被的肌动蛋白丝封闭的功能影响可被尖端的体内构象或细胞内环境中的其他因素显著调节。
