Garcés J A, Hoey J G, Gavin R H
Biology Doctoral Program of The City University of New York, Brooklyn College 11210, USA.
J Cell Sci. 1995 Mar;108 ( Pt 3):869-81. doi: 10.1242/jcs.108.3.869.
The basal body cage is a fibrillar chamber which surrounds each basal body in the ciliate cytoskeleton. The function of this chamber is unknown. In Tetrahymena, the cage contains actin filaments which connect the cage to triplet microtubules. In this study, we have examined the cage for the presence of myosin. Skeletal muscle myosin-II heavy and light chains were used to affinity-purify anti-MHC and anti-MLC antibodies, respectively, from an antiserum raised against Tetrahymena oral apparatus proteins. On western immunoblots of ATP-solubilized Tetrahymena proteins, the anti-MHC antibody detected a putative myosin heavy (180 kDa) chain, and the anti-MLC antibody detected a putative myosin light (18 kDa) chain. The anti-MHC antibody specifically labeled the AI zone of sarcomeres. In cosedimentation assays with an ATP-solubilized protein fraction, the 180 kDa polypeptide associated with skeletal muscle actin filaments in an ATP-dependent manner. The sedimented actin filaments appeared to be organized into bundles. Immunodepletion of the 180 kDa rendered the ATP-solubilized protein fraction ineffective in bundling actin filaments in a cosedimentation assay. ATP-solubilized Tetrahymena proteins, which included the 180 kDa polypeptide, exhibited F-actin-stimulated, Mg2+ ATPase activity and K+, EDTA ATPase activity which are characteristic of myosin ATPases. Immunodepletion of the 180 kDa polypeptide reduced the F-actin, Mg2+ ATPase activity of the ATP-solubilized protein fraction by more than 80%. Based on these various observations, we conclude that the 180 kDa polypeptide is a putative myosin heavy chain, probably a myosin-II and that the 18 kDa polypeptide is probably a myosin-II light chain. We have used the affinity-purified, anti-myosin antibodies with immunofluorescence microscopy and immunogold electron microscopy to map the location of the putative myosin heavy and light chains in Tetrahymena. Immunofluorescence microscopy showed that the anti-myosin antibodies localized to Tetrahymena somatic and oral region basal bodies. At the ultrastructural level, the anti-myosin antibodies localized to filaments in the basal body-cage complex. The labeling patterns with both anti-myosin antibodies were identical to the labeling pattern observed with an anti-actin antibody reported in a previous study. The co-localization of myosin and actin argue for a motility system within the basal body-cage complex.
基体笼是纤毛虫细胞骨架中围绕每个基体的纤维状腔室。该腔室的功能尚不清楚。在四膜虫中，该笼含有肌动蛋白丝，可将笼子与三联体微管相连。在本研究中，我们检测了该笼中是否存在肌球蛋白。分别用骨骼肌肌球蛋白-II重链和轻链从针对四膜虫口器蛋白产生的抗血清中亲和纯化抗MHC和抗MLC抗体。在ATP溶解的四膜虫蛋白的western免疫印迹上，抗MHC抗体检测到一条假定的肌球蛋白重链（180 kDa），抗MLC抗体检测到一条假定的肌球蛋白轻链（18 kDa）。抗MHC抗体特异性标记肌节的AI区。在与ATP溶解的蛋白组分的共沉降试验中，180 kDa多肽以ATP依赖的方式与骨骼肌肌动蛋白丝结合。沉降的肌动蛋白丝似乎被组织成束。在共沉降试验中，去除180 kDa的蛋白后，ATP溶解的蛋白组分在使肌动蛋白丝成束方面变得无效。ATP溶解的四膜虫蛋白（包括180 kDa多肽）表现出F-肌动蛋白刺激的、Mg2+ATP酶活性和K+、EDTA ATP酶活性，这些是肌球蛋白ATP酶的特征。去除180 kDa多肽后，ATP溶解的蛋白组分的F-肌动蛋白、Mg2+ATP酶活性降低了80%以上。基于这些不同的观察结果，我们得出结论，180 kDa多肽是一条假定的肌球蛋白重链，可能是肌球蛋白-II，18 kDa多肽可能是肌球蛋白-II轻链。我们使用亲和纯化的抗肌球蛋白抗体，通过免疫荧光显微镜和免疫金电子显微镜来定位四膜虫中假定的肌球蛋白重链和轻链的位置。免疫荧光显微镜显示，抗肌球蛋白抗体定位于四膜虫体细胞和口区基体。在超微结构水平上，抗肌球蛋白抗体定位于基体-笼复合体中的细丝。两种抗肌球蛋白抗体的标记模式与先前研究中报道的抗肌动蛋白抗体的标记模式相同。肌球蛋白和肌动蛋白的共定位表明基体-笼复合体中存在一个运动系统。
