Biology Centre, Institute of Plant Molecular Biology, Czech Academy of Sciences, České Budějovice, Czech Republic.
Department of Chromosome Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany.
Chromosome Res. 2024 Feb 26;32(1):3. doi: 10.1007/s10577-024-09747-x.
Centromere is the chromosomal site of kinetochore assembly and microtubule attachment for chromosome segregation. Given its importance, markers that allow specific labeling of centromeric chromatin throughout the cell cycle and across all chromosome types are sought for facilitating various centromere studies. Antibodies against the N-terminal region of CENH3 are commonly used for this purpose, since CENH3 is the near-universal marker of functional centromeres. However, because the N-terminal region of CENH3 is highly variable among plant species, antibodies directed against this region usually function only in a small group of closely related species. As a more versatile alternative, we present here antibodies targeted to the conserved domains of two outer kinetochore proteins, KNL1 and NDC80. Sequence comparison of these domains across more than 350 plant species revealed a high degree of conservation, particularly within a six amino acid motif, FFGPVS in KNL1, suggesting that both antibodies would function in a wide range of plant species. This assumption was confirmed by immunolabeling experiments in angiosperm (monocot and dicot) and gymnosperm species, including those with mono-, holo-, and meta-polycentric chromosomes. In addition to centromere labeling on condensed chromosomes during cell division, both antibodies detected the corresponding regions in the interphase nuclei of most species tested. These results demonstrated that KNL1 and NDC80 are better suited for immunolabeling centromeres than CENH3, because antibodies against these proteins offer incomparably greater versatility across different plant species which is particularly convenient for studying the organization and function of the centromere in non-model species.
着丝粒是动粒组装和微管附着的染色体分离的染色体位点。鉴于其重要性，人们一直在寻找能够在整个细胞周期和所有染色体类型中特异性标记着丝粒染色质的标记物，以促进各种着丝粒研究。针对 CENH3 的 N 端区域的抗体通常用于此目的，因为 CENH3 是功能着丝粒的近乎普遍的标记物。然而，由于 CENH3 的 N 端区域在植物物种中高度可变，针对该区域的抗体通常仅在一小部分密切相关的物种中起作用。作为一种更通用的替代方法，我们在这里提出了针对两个外动粒蛋白 KNL1 和 NDC80 的保守结构域的抗体。对 350 多种植物物种的这些结构域进行序列比较显示出高度的保守性，特别是在 KNL1 中的六个氨基酸基序 FFGPVS 内，表明这两种抗体都将在广泛的植物物种中起作用。在被子植物（单子叶植物和双子叶植物）和裸子植物物种中的免疫标记实验证实了这一假设，包括那些具有单、全和多核着丝粒的物种。除了在细胞分裂过程中对浓缩染色体上的着丝粒进行标记外，这两种抗体还在大多数测试物种的间期核中检测到相应区域。这些结果表明，KNL1 和 NDC80 比 CENH3 更适合免疫标记着丝粒，因为针对这些蛋白质的抗体在不同植物物种中提供了无与伦比的更大通用性，这对于研究非模式物种的着丝粒的组织和功能特别方便。
