Differences in the Detection of BrdU/EdU Incorporation Assays Alter the Calculation for G1, S, and G2 Phases of the Cell Cycle in Trypanosomatids

Abstract

Trypanosomatids are the etiologic agents of various infectious diseases in humans. They diverged early during eukaryotic evolution and have attracted attention as peculiar models for evolutionary and comparative studies. Here, we show a meticulous study comparing the incorporation and detection of the thymidine analogs BrdU and EdU in Leishmania amazonensis, Trypanosoma brucei, and Trypanosoma cruzi to monitor their DNA replication. We used BrdU- and EdU-incorporated parasites with the respective standard detection approaches: indirect immunofluorescence to detect BrdU after standard denaturation (2 M HCl) and "click" chemistry to detect EdU. We found a discrepancy between these two thymidine analogs due to the poor detection of BrdU, which is reflected on the estimative of the duration of the cell cycle phases G1, S, and G2. To solve this discrepancy, we increase the exposure of incorporated BrdU using different concentrations of HCl. Using a new value for HCl concentration, we re-estimated the phases G1, S, G2 + M, and cytokinesis durations, confirming the values found by this approach using EdU. In conclusion, we suggest that the studies using BrdU with standard detection approach, not only in trypanosomatids but also in others cell types, should be reviewed to ensure an accurate estimation of DNA replication monitoring.

Transcription activity contributes to the firing of non-constitutive origins in Trypanosoma brucei

“…Thus, G1 phase duration was estimated to be 3.37 h or 0.397 ccu ( Figure 1D). 15 These new precise values for the duration of G1 and S phase in T. brucei TREU927 share close similarities to those obtained for T. brucei Lister strain 427, which were also estimated by EdU incorporation (15) or by counter-flow elutriation (27). This similarity suggests that different strains of the same species (T. brucei) probably do not have significant differences in cell cycle lengths.…”

“…The duration of S phase starts when the first cytokinesis-labeled cell appears, extends throughout the period of detection for all cytokinesislabeled cells and ends when some unlabeled cells appear again. Of note, this assay was carried out according to a protocol described in a previous study (15).…”

“…To quickly measure S phase length during transcription inhibition, avoiding possible transcription-dependent interference, we decided to use an approach that does not rely on the measurement of other phases of the cell cycle, such as those performed in Figure 1. To this end, we followed an established assay in trypanosomatids (15), which directly yields kinetic determinations of S phase through the measurement of cytokinesis (2N2K)-labeled nuclei. In this assay, we performed a 15-min EdU pulse to subsequently measure the cytokinesis of EdU-labeled nuclei until the EdU signal disappeared.…”

“…Trypanosoma brucei, one of the parasites known as African trypanosomes, has the best-studied DNA and RNA synthesis, although this knowledge is limited relative to that of model eukaryotes (14). The duration of the by using a most sensitive thymidine analog, 5-ethynyl-2'-deoxyuridine (EdU), to monitor DNA replication, although there are still no similar assays for T. brucei TREU927 (15). The number of DNA replication origins per chromosome and the replication rate are a matter of debate according to the technique used to obtain these data and the choice of either T. brucei Lister strain 427 or TREU927 (3,14,16,17).…”

Investigation of pyrimidine nucleoside analogues as chemical probes to assess compound effects on the proliferation of Trypanosoma cruzi intracellular parasites

“…Additionally, an image-based assay to assess parasite replication utilising EdU as a chemical probe was developed to determine compound activity against Leishmania donovani [20]. The incorporation of EdU and BrdU has been compared in T. cruzi epimastigotes and it was found that EdU was most effective for monitoring DNA replication because of its sensitivity and that DNA denaturation is not required [21].…”