Abstract
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.
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Acknowledgements
This research was financially supported by grants from the Czech Science Foundation (20-25440S) and the Czech Academy of Sciences (RVO:60077344). AM is financially supported by the Max Planck Society and Deutsche Forschungsgemeinschaft (grant number MA 9363/3-1). YMS was financially supported by a PROBRAL—Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Deutscher Akademischer Austauschdienst grant (number 495995/2020-00 and 88881.144086/2017-01). YTK was financially supported by the Deutsche Forschungsgemeinschaft (grant number HO 1779/32-2). Computational resources and data storage facilities were provided by the ELIXIR-CZ Research Infrastructure Project (LM2023055). We thank J. Látalová and V. Tetourová for technical assistance.
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LO, PN and JM conceived the study and designed the experiments. LO, YMS and YTK performed the cytogenetics experiments and conventional fluorescence microscopy. VS carried out the super-resolution structured illumination microscopy. PN analyzed the sequence data. AM provided Luzula nivea sequencing data. LO and PN wrote the manuscript with input from YMS, YTK, AM, VS and JM. All authors read and approved the final manuscript.
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Oliveira, L., Neumann, P., Mata-Sucre, Y. et al. KNL1 and NDC80 represent new universal markers for the detection of functional centromeres in plants. Chromosome Res 32, 3 (2024). https://doi.org/10.1007/s10577-024-09747-x
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DOI: https://doi.org/10.1007/s10577-024-09747-x