Biochemistry and Molecular Biology and the Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA 16802.
MSTP Program, Milton S. Hershey College of Medicine, Hershey, PA 17033.
Mol Biol Cell. 2020 Sep 1;31(19):2125-2138. doi: 10.1091/mbc.E20-04-0237-T. Epub 2020 Jul 16.
Kinetochores connect centromeric chromatin to spindle microtubules during mitosis. Neurons are postmitotic, so it was surprising to identify transcripts of structural kinetochore (KT) proteins and regulatory chromosome passenger complex (CPC) and spindle assembly checkpoint (SAC) proteins in neurons after dendrite injury. To test whether these proteins function during dendrite regeneration, postmitotic RNA interference (RNAi) was performed and dendrites or axons were removed using laser microsurgery. Reduction of KT, CPC, and SAC proteins decreased dendrite regeneration without affecting axon regeneration. To understand whether neuronal functions of these proteins rely on microtubules, we analyzed microtubule behavior in uninjured neurons. The number of growing plus, but not minus, ends increased in dendrites with reduced KT, CPC, and SAC proteins, while axonal microtubules were unaffected. Increased dendritic microtubule dynamics was independent of dual leucine zipper kinase (DLK)-mediated stress but was rescued by concurrent reduction of γ-tubulin, the core microtubule nucleation protein. Reduction of γ-tubulin also rescued dendrite regeneration in backgrounds containing kinetochore RNAi transgenes. We conclude that kinetochore proteins function postmitotically in neurons to suppress dendritic microtubule dynamics by inhibiting nucleation.
在有丝分裂过程中，着丝粒将着丝粒染色质连接到纺锤体微管上。神经元是有丝分裂后的，因此在树突损伤后在神经元中鉴定到结构着丝粒（KT）蛋白和调节染色体乘客复合物（CPC）和纺锤体组装检查点（SAC）蛋白的转录本是令人惊讶的。为了测试这些蛋白质是否在树突再生过程中起作用，进行了有丝分裂后 RNA 干扰（RNAi），并使用激光显微手术去除树突或轴突。减少 KT、CPC 和 SAC 蛋白会减少树突再生，而不会影响轴突再生。为了了解这些蛋白质的神经元功能是否依赖于微管，我们分析了未受伤神经元中的微管行为。在 KT、CPC 和 SAC 蛋白减少的树突中，生长的微管端（而不是负端）的数量增加，而轴突微管不受影响。减少 KT、CPC 和 SAC 蛋白会增加树突微管动力学，这与双亮氨酸拉链激酶（DLK）介导的应激无关，但可以通过同时减少核心微管成核蛋白γ-微管蛋白得到挽救。γ-微管蛋白的减少也挽救了含有动粒 RNAi 转基因的背景中的树突再生。我们的结论是，动粒蛋白在神经元中有丝分裂后发挥作用，通过抑制成核来抑制树突微管动力学。
