Spire2 and Rab11a synergistically activate myosin-5b motor function
Spire2 and Rab11a synergistically activate myosin-5b motor function
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DOI：
10.1016/j.bbrc.2024.149653
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发表时间：
2024-02-16
影响因子：
3.1
通讯作者：
Ji，Huan-Hong
中科院分区：
文献类型：
--
作者：
Yao，Lin -Lin;Hou，Wei-Dong;Ji，Huan-Hong
最近在一些细胞系统中发现了沿细胞质肌动蛋白网络的细胞囊泡远距离运输。在中期小鼠卵母细胞中，运动蛋白Myo5b （Myo5b）和肌动蛋白成核因子Spire被募集到Rab11a阳性的囊泡膜上，形成Myo5b/Spire/Rab11a的三联体，驱动囊泡远距离运输到卵母细胞皮层。然而，Myo5b/Spire/Rab11a复合体的分子间调控机制尚不清楚。在本研究中，我们表达并纯化了Myo5b、Spire2和Rab11a蛋白，并进行了atp酶活性测定、下拉和单分子运动测定。我们的研究结果表明，在生理离子条件下，Spire2和Rab11a都需要激活Myo5b的运动活性。Spire2的GTBM片段刺激Myo5b的atp酶活性，而Rab11a则增强这种激活。这种激活是通过破坏Myo5b的正反相互作用而发生的。此外，在单分子水平上，我们观察到Spire2的GTBM片段和Rab11a协同刺激Myo5b的运动活性。基于我们的研究结果，我们提出Myo5b、Spire2和Rab11a与囊泡膜结合后形成一个三联合物，被抑制的Myo5b被Spire2和Rab11a协同激活，从而触发囊泡的远距离运输。
Cellular vesicle long-distance transport along the cytoplasmic actin network has recently been uncovered in several cell systems. In metaphase mouse oocytes, the motor protein myosin-5b (Myo5b) and the actin nucleation factor Spire are recruited to the Rab11a-positive vesicle membrane, forming a ternary complex of Myo5b/Spire/Rab11a that drives the vesicle long-distance transport to the oocyte cortex. However, the mechanism underlying the intermolecular regulation of the Myo5b/Spire/Rab11a complex remains unknown. In this study, we expressed and purified Myo5b, Spire2, and Rab11a proteins, and performed ATPase activity measurements, pulldown and single-molecule motility assays. Our results demonstrate that both Spire2 and Rab11a are required to activate Myo5b motor activity under physiological ionic conditions. The GTBM fragment of Spire2 stimulates the ATPase activity of Myo5b, while Rab11a enhances this activation. This activation occurs by disrupting the head-tail interaction of Myo5b. Furthermore, at the single-molecule level, we observed that the GTBM fragment of Spire2 and Rab11a coordinate to stimulate the Myo5b motility activity. Based on our results, we propose that upon association with the vesicle membrane, Myo5b, Spire2 and Rab11a form a ternary complex, and the inhibited Myo5b is synergistically activated by Spire2 and Rab11a, thereby triggering the long-distance transport of vesicles.