Laboratory of Plant Developmental Genetics, School of Biological Sciences, Plant Genomics and Breeding Institute, Seoul National University, Seoul, 08826, Korea.
Plant J. 2018 Mar;93(6):977-991. doi: 10.1111/tpj.13823. Epub 2018 Feb 20.
Kinetochore, a protein super-complex on the centromere of chromosomes, mediates chromosome segregation during cell division by providing attachment sites for spindle microtubules. The NDC80 complex, composed of four proteins, NDC80, NUF2, SPC24 and SPC25, is localized at the outer kinetochore and connects spindle fibers to the kinetochore. Although it is conserved across species, functional studies of this complex are rare in Arabidopsis. Here, we characterize a recessive mutant, meristem unstructured-1 (mun-1), exhibiting an abnormal phenotype with unstructured shoot apical meristem caused by ectopic expression of the WUSCHEL gene in unexpected tissues. mun-1 is a weak allele because of the insertion of T-DNA in the promoter region of the SPC24 homolog. The mutant exhibits stunted growth, embryo arrest, DNA aneuploidy, and defects in chromosome segregation with a low cell division rate. Null mutants of MUN from TALEN and CRISPR/Cas9-mediated mutagenesis showed zygotic embryonic lethality similar to nuf2-1; however, the null mutations were fully transmissible via pollen and ovules. Interactions among the components of the NDC80 complex were confirmed in a yeast two-hybrid assay and in planta co-immunoprecipitation. MUN is co-localized at the centromere with HTR12/CENH3, which is a centromere-specific histone variant, but MUN is not required to recruit HTR12/CENH3 to the kinetochore. Our results support that MUN is a functional homolog of SPC24 in Arabidopsis, which is required for proper cell division. In addition, we report the ectopic generations of stem cell niches by the malfunction of kinetochore components.
着丝粒是位于染色体着丝粒上的一种蛋白质超级复合物，通过为纺锤体微管提供附着位点来介导细胞分裂过程中的染色体分离。由四个蛋白质（NDC80、NUF2、SPC24 和 SPC25）组成的 NDC80 复合物定位于外着丝粒，将纺锤体纤维与着丝粒连接起来。尽管该复合物在物种间是保守的，但在拟南芥中对其功能的研究却很少。在这里，我们对隐性突变体 meristem unstructured-1（mun-1）进行了表征，该突变体表现出异常表型，由于 WUSCHEL 基因在意外组织中的异位表达，导致茎尖分生组织无序。mun-1 是一个弱等位基因，因为 T-DNA 插入到 SPC24 同源物的启动子区域。该突变体表现出生长迟缓、胚胎停滞、DNA 非整倍体和染色体分离缺陷，细胞分裂率低。TALEN 和 CRISPR/Cas9 介导的诱变产生的 MUN 的 null 突变体表现出与 nuf2-1 相似的合子胚胎致死性；然而，这些 null 突变体可以通过花粉和胚珠完全传递。在酵母双杂交测定和体内共免疫沉淀中证实了 NDC80 复合物的成分之间的相互作用。MUN 与 HTR12/CENH3 共定位于着丝粒，后者是一种着丝粒特异性组蛋白变体，但 MUN 不需要将 HTR12/CENH3 募集到着丝粒。我们的结果支持 MUN 是拟南芥中 SPC24 的功能同源物，它是正确细胞分裂所必需的。此外，我们报告了由于着丝粒成分的功能障碍，干细胞龛的异位发生。
