Pitzen Valentin, Askarzada Sophie, Gräf Ralph, Meyer Irene
Department of Cell Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam-Golm, Germany.
Cells. 2018 Apr 23;7(4):32. doi: 10.3390/cells7040032.
centrosomes consist of a nucleus-associated cylindrical, three-layered core structure surrounded by a corona consisting of microtubule-nucleation complexes embedded in a scaffold of large coiled-coil proteins. One of them is the conserved CDK5RAP2 protein. Here we focus on the role of CDK5RAP2 for maintenance of centrosome integrity, its interaction partners and its dynamic behavior during interphase and mitosis. GFP-CDK5RAP2 is present at the centrosome during the entire cell cycle except from a short period during prophase, correlating with the normal dissociation of the corona at this stage. RNAi depletion of CDK5RAP2 results in complete disorganization of centrosomes and microtubules suggesting that CDK5RAP2 is required for organization of the corona and its association to the core structure. This is in line with the observation that overexpressed GFP-CDK5RAP2 elicited supernumerary cytosolic MTOCs. The phenotype of CDK5RAP2 depletion was very reminiscent of that observed upon depletion of CP148, another scaffolding protein of the corona. BioID interaction assays revealed an interaction of CDK5RAP2 not only with the corona markers CP148, γ-tubulin, and CP248, but also with the core components Cep192, CP75, and CP91. Furthermore, protein localization studies in both depletion strains revealed that CP148 and CDK5RAP2 cooperate in corona organization.
中心体由一个与细胞核相关的圆柱形三层核心结构组成，其周围是一个晕环，该晕环由嵌入大型卷曲螺旋蛋白支架中的微管成核复合物组成。其中之一是保守的CDK5RAP2蛋白。在这里，我们重点研究CDK5RAP2在维持中心体完整性中的作用、其相互作用伙伴以及在间期和有丝分裂期间的动态行为。除了前期的短时间外，GFP-CDK5RAP2在整个细胞周期中都存在于中心体中，这与该阶段晕环的正常解离相关。CDK5RAP2的RNAi缺失导致中心体和微管完全紊乱，这表明CDK5RAP2是晕环组织及其与核心结构关联所必需的。这与过表达的GFP-CDK5RAP2引发多余的胞质微管组织中心（MTOC）的观察结果一致。CDK5RAP2缺失的表型与另一种晕环支架蛋白CP148缺失时观察到的表型非常相似。生物素邻近标记（BioID）相互作用分析表明，CDK5RAP2不仅与晕环标记物CP148、γ-微管蛋白和CP248相互作用，还与核心成分Cep192、CP75和CP91相互作用。此外，对两种缺失菌株的蛋白质定位研究表明，CP148和CDK5RAP2在晕环组织中相互协作。
