Origin recognition complex (ORC) architecture has only been explored in depth in the opisthokont supergroup of eukaryotes, which includes yeast and mammals, with little work in protists. The Kinetoplastida is a well-studied order of eukaryotic microbes and contains several important human parasites, such as Trypanosoma brucei. Genome sequencing of T. brucei identified only a single ORC-related protein – TbORC1/CDC6. A number of TbORC1/CDC6-interacting factors have been identified, raising the possibility that an ORC is present in T. brucei. However, many of these interactors are highly diverged in sequence from canonical ORC subunits and none has been shown to have a role in replication. To compare the localization pattern of TbORC1/CDC6 with two of these factors (TbORC1b, and TbORC4), and with a subunit (TbMCM3) of the replicative MCM helicase that ORC is through to recruit, each protein was tagged with 12 copies of the c-myc epitope (12myc). Analysis of the ratio and morphology of the nucleus (N) and kinetoplast (K) delineates the cell cycle stage of cells within the population: 1N1K for G1 phase; 1N1eK (elongated – replicating – but not yet divided kinetoplast) for S phase; 1N2K cells for S-G2 phase; and 2N2K cells for postmitosis. Using Super-resolution structured illumination microscopy (SR-SIM) we characterized the distribution pattern of each factor using anti-myc antiserum and compared this with replicating DNA, which was detected with EdU labeling, and with chromatin (DAPI). TbORC1/CDC6 and TbORC4 presented a punctate distribution in non-replicating cells, and did not localize to a specific region within the nucleus. In early S phase cells there was limited co-localization between either TbORC1/CDC6 or TbORC4 and EdU, which was also seen in puncta. An overlap in the myc and EdU signals became more pronounced in 1N2K cells, where S phase is more complete, and at that cell cycle stage the protein and replication puncta become more abundant and appeared more diffuse. In 2N2K cells, after completion of replication, localization of TbORC1/CDC6 and TbORC4 returned to a similar pattern to that of 1N1K cells. SR-SIM imaging revealed that TbORC1B is only seen in the nucleus of S phase cells (1N1eK and 1N2K), where a comparable pattern to TbORC1/CDC6 and TbORC4 was seen, with signal throughout the nucleus in a similarly large number of puncta and with some overlap with EdU. TbMCM3 signal was more abundant and more homogenous in the nucleus that any of the putative ORC-factors, and displayed little obvious variation in the different cell cycle stages, indicating that each of TbORC1/CDC6, TbORC4 and TbORC1B display distinct sub-nuclear localisation to that of TbMCM3. SR-SIM imaging has helped reveal that ORC architecture and regulation appear to be diverged features of replication initiation in T. brucei.
Acknowledgements: This work was supported by the Wellcome Trust , the BBSRC [BB/K006495/1] and Fundação para a Ciência e Tecnologia (FCT, Portugal) [SFRH/BD/68784/2010]. The Wellcome Trust Centre for Molecular Parasitology is supported by core funding from the Wellcome Trust .
To cite this abstract:Leandro Lemgruber, Catarina Marques, Richard McCulloch; Nanoscopic localization of he components of the origin recognition complex during Trypanosome brucei cell cycle. The 16th European Microscopy Congress, Lyon, France. https://emc-proceedings.com/abstract/nanoscopic-localization-of-he-components-of-the-origin-recognition-complex-during-trypanosome-brucei-cell-cycle/. Accessed: December 5, 2022
EMC Abstracts - https://emc-proceedings.com/abstract/nanoscopic-localization-of-he-components-of-the-origin-recognition-complex-during-trypanosome-brucei-cell-cycle/