Takáč Tomáš, Šamajová Olga, Pechan Tibor, Luptovčiak Ivan, Šamaj Jozef
From the ‡Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Šlechtitelů 27, 783 71 Olomouc, Czech Republic.
§Institute for Genomics, Biocomputing and Biotechnology, Mississippi Agricultural and Forestry Experiment Station, Mississippi State University, Starkville, Mississippi 39759.
Mol Cell Proteomics. 2017 Sep;16(9):1591-1609. doi: 10.1074/mcp.M117.068015. Epub 2017 Jul 13.
Microtubule organization and dynamics are critical for key developmental processes such as cell division, elongation, and morphogenesis. Microtubule severing is an essential regulator of microtubules and is exclusively executed by KATANIN 1 in In this study, we comparatively studied the proteome-wide effects in two mutants. Thus, shotgun proteomic analysis of roots and aerial parts of single nucleotide mutant and T-DNA insertion mutant was carried out. We have detected 42 proteins differentially abundant in both and KATANIN 1 dysfunction altered the abundance of proteins involved in development, metabolism, and stress responses. The differential regulation of tubulins and microtubule-destabilizing protein MDP25 implied a feedback microtubule control in mutants. Furthermore, deregulation of profilin 1, actin-depolymerizing factor 3, and actin 7 was observed. These findings were confirmed by immunoblotting analysis of actin and by microscopic observation of actin filaments using fluorescently labeled phalloidin. Results obtained by quantitative RT-PCR analysis revealed that changed protein abundances were not a consequence of altered expression levels of corresponding genes in the mutants. In conclusion, we show that abundances of several cytoskeletal proteins as well as organization of microtubules and the actin cytoskeleton are amended in accordance with defective microtubule severing.
微管组织和动力学对于细胞分裂、伸长和形态发生等关键发育过程至关重要。微管切断是微管的一种重要调节机制，且仅由katanin 1执行。在本研究中，我们比较研究了两个突变体中蛋白质组范围内的影响。因此，对单核苷酸突变体和T-DNA插入突变体的根和地上部分进行了鸟枪法蛋白质组分析。我们检测到在两个突变体中42种蛋白质的丰度存在差异。katanin 1功能障碍改变了参与发育、代谢和应激反应的蛋白质丰度。微管蛋白和微管去稳定蛋白MDP25的差异调节暗示了突变体中存在微管反馈控制。此外，还观察到了肌动蛋白结合蛋白1、肌动蛋白解聚因子3和肌动蛋白7的失调。通过肌动蛋白的免疫印迹分析以及使用荧光标记鬼笔环肽对肌动蛋白丝进行显微镜观察，证实了这些发现。定量RT-PCR分析结果表明突变体中蛋白质丰度的变化并非相应基因表达水平改变的结果。总之，我们表明，随着微管切断缺陷，几种细胞骨架蛋白的丰度以及微管和肌动蛋白细胞骨架的组织都发生了改变。
