University of Debrecen, Department of Botany, Debrecen, Hungary.
Ann Bot. 2012 Sep;110(4):797-808. doi: 10.1093/aob/mcs154. Epub 2012 Jul 20.
Microcystin-LR (MCY-LR) is a cyanobacterial toxin, a specific inhibitor of type 1 and 2A protein phosphatases (PP1 and PP2A) with significant impact on aquatic ecosystems. It has the potential to alter regulation of the plant cell cycle. The aim of this study was improved understanding of the mitotic alterations induced by cyanotoxin in Vicia faba, a model organism for plant cell biology studies.
Vicia faba seedlings were treated over the long and short term with MCY-LR purified in our laboratory. Short-term treatments were performed on root meristems synchronized with hydroxylurea. Sections of lateral root tips were labelled for chromatin, phosphorylated histone H3 and β-tubulin via histochemical and immunohistochemical methods. Mitotic activity and the occurrence of mitotic alterations were detected and analysed by fluorescence microscopy. The phosphorylation state of histone H3 was studied by Western blotting.
Long-term MCY-LR exposure of lateral root tip meristems increased the percentage of either early or late mitosis in a concentration-dependent manner. We observed hypercondensed chromosomes and altered sister chromatid segregation (lagging chromosomes) leading to the formation of micronuclei, accompanied by the formation of disrupted, multipolar and monopolar spindles, disrupted phragmoplasts and the hyperphosphorylation of histone H3 at Ser10. Short-term MCY-LR treatment of synchronized cells showed that PP1 and PP2A inhibition delayed the onset of anaphase at 1 µg mL(-1) MCY-LR, accelerated cell cycle at 10 µg mL(-1) MCY-LR and induced the formation of lagging chromosomes. In this case mitotic microtubule alterations were not detected, but histone H3 was hyperphosphorylated.
MCY-LR delayed metaphase-anaphase transition. Consequently, it induced aberrant chromatid segregation and micronucleus formation that could be associated with both H3 hyperphosphorylation and altered microtubule organization. However, these two phenomena seemed to be independent. The toxin may be a useful tool in the study of plant cell cycle regulation.
微囊藻毒素-LR（MCY-LR）是一种蓝藻毒素，可特异性抑制蛋白磷酸酶 1（PP1）和 2A（PP2A），对水生生态系统有重大影响。它可能改变植物细胞周期的调控。本研究旨在提高对 MCY-LR 诱导的蚕豆根尖细胞有丝分裂改变的认识，蚕豆是植物细胞生物学研究的模式生物。
我们用实验室纯化的 MCY-LR 对蚕豆幼苗进行了长期和短期处理。短期处理在羟基脲同步的根分生组织上进行。通过组织化学和免疫组织化学方法对侧根根尖的染色质、磷酸化组蛋白 H3 和 β-微管蛋白进行标记。通过荧光显微镜检测和分析有丝分裂活性和有丝分裂改变的发生。通过 Western 印迹研究组蛋白 H3 的磷酸化状态。
侧根分生组织根尖的 MCY-LR 长期暴露以浓度依赖的方式增加了早期或晚期有丝分裂的比例。我们观察到染色体高度浓缩和姐妹染色单体分离异常（滞后染色体），导致微核形成，同时形成断裂、多极和单极纺锤体、断裂的成膜体和组蛋白 H3 的 Ser10 高度磷酸化。同步细胞的 MCY-LR 短期处理表明，1μg/mL MCY-LR 抑制 PP1 和 PP2A 可延迟后期起始，10μg/mL MCY-LR 加速细胞周期并诱导滞后染色体形成。在这种情况下，没有检测到有丝分裂微管改变，但组蛋白 H3 高度磷酸化。
MCY-LR 延迟了中期-后期转变。因此，它诱导了异常的染色单体分离和微核形成，这可能与 H3 高度磷酸化和微管组织改变有关。然而，这两种现象似乎是独立的。该毒素可能是研究植物细胞周期调控的有用工具。
