Wada Yuuko, Baba Shoji A, Kamimura Shinji
Department of Biological Sciences, Faculty of Science and Engineering, Chuo University, Tokyo, Japan.
Department of Biology, Ochanomizu University, Tokyo, Japan.
Cytoskeleton (Hoboken). 2015 Apr;72(4):182-92. doi: 10.1002/cm.21218. Epub 2015 May 25.
Ciliobrevin has recently been found to be a membrane-permeable inhibitor that is specific to AAA+ molecular motors such as cytoplasmic dyneins. In this study, we investigated how ciliobrevin inhibited the motility of sperm from sea urchins: Hemicentrotus pulcherrimus, Pseudocentrotus depressus, and Anthocidaris crassispina. After application of 100 μM of ciliobrevin A to live spermatozoa, swimming speed decreased gradually and flagellar motion stopped almost completely within 5 to 10 min. This inhibition was reversible and the frequency of flagellar beating was reduced in a concentration-dependent manner. Ciliobrevin had similar inhibitory effects on the flagellar beating of demembranated and reactivated sperm and the sliding disintegration of trypsin-treated axonemes. We also analyzed the curvature and shear angle of the beating flagella and found that the proximal region of the sperm flagellum was less sensitive to ciliobrevin compared with more distal regions, where bending motions were blocked completely. Interestingly, the shear angle analysis of flagellar motility showed that ciliobrevin induced highly asymmetric bends in the proximal region of the flagellum. These results suggest that there is heterogeneity in the inhibitory thresholds of dynein motors, which depend on the regions along the flagellar shaft (proximal or distal) and on the sites of doublets in the flagellar cross-section (doublet numbers). We expect that it will be possible to map the functional differences in dynein subtypes along and/or around the cross-sections of flagellar axonemes by analyzing the inhibitory effects of ciliobrevin.
纤毛抑素最近被发现是一种可透过细胞膜的抑制剂，它对诸如胞质动力蛋白等AAA+分子马达具有特异性。在本研究中，我们探究了纤毛抑素如何抑制海胆精子的运动：即马粪海胆、凹裂星海胆和厚刺海胆的精子。将100μM的纤毛抑素A应用于活精子后，游动速度逐渐降低，鞭毛运动在5至10分钟内几乎完全停止。这种抑制是可逆的，并且鞭毛摆动频率以浓度依赖的方式降低。纤毛抑素对去膜并重新激活的精子的鞭毛摆动以及胰蛋白酶处理的轴丝的滑动解体具有类似的抑制作用。我们还分析了摆动鞭毛的曲率和剪切角，发现与鞭毛更远端区域相比，精子鞭毛的近端区域对纤毛抑素不太敏感，在远端区域弯曲运动被完全阻断。有趣的是，鞭毛运动的剪切角分析表明，纤毛抑素在鞭毛近端区域诱导了高度不对称的弯曲。这些结果表明，动力蛋白马达的抑制阈值存在异质性，这取决于沿鞭毛轴的区域（近端或远端）以及鞭毛横截面中双联体的位置（双联体数量）。我们期望通过分析纤毛抑素的抑制作用，能够绘制出沿鞭毛轴丝横截面和/或围绕其横截面的动力蛋白亚型的功能差异图。
