Akiyoshi Bungo, Faktorová Drahomíra, Lukeš Julius
Centre for Cell Biology, Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, UK.
Institute of Parasitology, Biology Centre, Czech Academy of Sciences, České Budějovice, Czech Republic.
Open Biol. 2025 Aug;15(8):250096. doi: 10.1098/rsob.250096. Epub 2025 Aug 6.
Diplonemids are highly diverse and abundant marine plankton with significant ecological importance. However, little is known about their biology, even in the model diplonemid whose genome sequence is available. Examining the subcellular localization of proteins using fluorescence microscopy is a powerful approach to infer their putative function. Here, we report a plasmid-based method that enables YFP-tagging of a gene at the endogenous locus. By examining the localization of proteins whose homologs are involved in chromosome organization or segregation in other eukaryotes, we discovered several notable features in mitotically dividing cells. Cohesin is enriched on condensed interphase chromatin. During mitosis, chromosomes organize into two rings (termed mitotic rings herein) that surround the elongating nucleolus and align on a bipolar spindle. Homologs of chromosomal passenger complex components (INCENP, two Aurora kinases and KIN-A), a CLK1 kinase, meiotic chromosome axis protein SYCP2L1, spindle checkpoint protein Mad1 and microtubule regulator XMAP215 localize in between the two mitotic rings. In contrast, a Mad2 homolog localizes near basal bodies as in trypanosomes. By representing the first molecular characterization of mitotic mechanisms in and raising many questions, this study forms the foundation for dissecting mitotic mechanisms in diplonemids.
双滴虫是高度多样化且数量丰富的海洋浮游生物，具有重要的生态意义。然而，即便对于其基因组序列已可知的模式双滴虫，人们对其生物学特性仍知之甚少。利用荧光显微镜检查蛋白质的亚细胞定位是推断其假定功能的一种有效方法。在此，我们报告一种基于质粒的方法，该方法能够在内源基因座对基因进行黄色荧光蛋白（YFP）标记。通过检查其同源物在其他真核生物中参与染色体组织或分离的蛋白质的定位，我们在有丝分裂细胞中发现了几个显著特征。黏连蛋白富集于浓缩的间期染色质上。在有丝分裂期间，染色体组织成两个环（本文中称为有丝分裂环），围绕伸长的核仁并在双极纺锤体上排列。染色体乘客复合体组分（INCENP、两种极光激酶和KIN - A）、CLK1激酶、减数分裂染色体轴蛋白SYCP2L1、纺锤体检查点蛋白Mad1和微管调节蛋白XMAP215的同源物定位于两个有丝分裂环之间。相比之下，Mad2同源物如在锥虫中一样定位于基体附近。通过展示双滴虫有丝分裂机制的首次分子特征并提出许多问题，本研究为剖析双滴虫的有丝分裂机制奠定了基础。
