Sato Osamu, Komatsu Satoshi, Sakai Tsuyoshi, Tsukasaki Yoshikazu, Tanaka Ryosuke, Mizutani Takeomi, Watanabe Tomonobu M, Ikebe Reiko, Ikebe Mitsuo
From the Department of Cellular and Molecular Biology, University of Texas Health Science Center at Tyler, Tyler, Texas 75708.
Department of Pharmacology, University of Illinois College of Medicine, Chicago, Illinois 60612.
J Biol Chem. 2017 Jun 30;292(26):10950-10960. doi: 10.1074/jbc.M116.765966. Epub 2017 May 15.
Human myosin VIIa (MYO7A) is an actin-linked motor protein associated with human Usher syndrome (USH) type 1B, which causes human congenital hearing and visual loss. Although it has been thought that the role of human myosin VIIa is critical for USH1 protein tethering with actin and transportation along actin bundles in inner-ear hair cells, myosin VIIa's motor function remains unclear. Here, we studied the motor function of the tail-truncated human myosin VIIa dimer (HM7AΔTail/LZ) at the single-molecule level. We found that the HM7AΔTail/LZ moves processively on single actin filaments with a step size of 35 nm. Dwell-time distribution analysis indicated an average waiting time of 3.4 s, yielding ∼0.3 s for the mechanical turnover rate; hence, the velocity of HM7AΔTail/LZ was extremely slow, at 11 nm·s We also examined HM7AΔTail/LZ movement on various actin structures in demembranated cells. HM7AΔTail/LZ showed unidirectional movement on actin structures at cell edges, such as lamellipodia and filopodia. However, HM7AΔTail/LZ frequently missed steps on actin tracks and exhibited bidirectional movement at stress fibers, which was not observed with tail-truncated myosin Va. These results suggest that the movement of the human myosin VIIa motor protein is more efficient on lamellipodial and filopodial actin tracks than on stress fibers, which are composed of actin filaments with different polarity, and that the actin structures influence the characteristics of cargo transportation by human myosin VIIa. In conclusion, myosin VIIa movement appears to be suitable for translocating USH1 proteins on stereocilia actin bundles in inner-ear hair cells.
人类肌球蛋白VIIa（MYO7A）是一种与肌动蛋白相连的运动蛋白，与人类1B型遗传性耳聋-视网膜色素变性综合征（USH）相关，该综合征会导致人类先天性听力和视力丧失。尽管人们一直认为人类肌球蛋白VIIa在USH1蛋白与肌动蛋白的连接以及在内耳毛细胞中沿肌动蛋白束运输方面起着关键作用，但其运动功能仍不清楚。在这里，我们在单分子水平上研究了截尾的人类肌球蛋白VIIa二聚体（HM7AΔTail/LZ）的运动功能。我们发现HM7AΔTail/LZ在单根肌动蛋白丝上进行连续移动，步长为35纳米。驻留时间分布分析表明平均等待时间为3.4秒，机械转换速率约为0.3秒；因此，HM7AΔTail/LZ的速度极慢，为11纳米·秒。我们还研究了HM7AΔTail/LZ在去膜细胞中各种肌动蛋白结构上的运动。HM7AΔTail/LZ在细胞边缘的肌动蛋白结构（如片状伪足和丝状伪足）上表现出单向运动。然而，HM7AΔTail/LZ在肌动蛋白轨道上经常错过步移，并在应力纤维上表现出双向运动，而截尾的肌球蛋白Va则未观察到这种情况。这些结果表明，人类肌球蛋白VIIa运动蛋白在片状伪足和丝状伪足的肌动蛋白轨道上的运动比在由具有不同极性的肌动蛋白丝组成的应力纤维上更有效，并且肌动蛋白结构会影响人类肌球蛋白VIIa的货物运输特性。总之，肌球蛋白VIIa的运动似乎适合在内耳毛细胞的静纤毛肌动蛋白束上转运USH1蛋白。
