Leishmania amazonensis Promastigotes Present Two Distinct Modes of Nucleus and Kinetoplast Segregation during Cell Cycle

Abstract

Here, we show the morphological events associated with organelle segregation and their timing in the cell cycle of a reference strain of Leishmania (L.) amazonensis promastigotes, the main causative agent of Tegumentary leishmaniasis in the Americas. We show evidences that during the cell cycle, L. amazonensis promastigotes present two distinct modes of nucleus and kinetoplast segregation, which occur in different temporal order in different proportions of cells. We used DAPI-staining and EdU-labeling to monitor the segregation of DNA-containing organelles and DNA replication in wild-type parasites. The emergence of a new flagellum was observed using a specific monoclonal antibody. The results show that L. amazonensis cell cycle division is peculiar, with 65% of the dividing cells duplicating the kinetoplast before the nucleus, and the remaining 35% doing the opposite or duplicating both organelles concomitantly. In both cases, the new flagellum appeared during S to G2 phase in 1N1K cells and thus before the segregation of both DNA-containing organelles; however, we could not determine the exact timing of flagellar synthesis. Most of these results were confirmed by the synchronization of parasites using hydroxyurea. Altogether, our data show that during the cell cycle of L. amazonensis promastigotes, similarly to L. donovani, the segregation of nucleus and kinetoplast do not follow a specific order, especially when compared to other trypanosomatids, reinforcing the idea that this characteristic seems to be species-specific and may represent differences in cellular biology among members of the Leishmania genus.

KHARON Is an Essential Cytoskeletal Protein Involved in the Trafficking of Flagellar Membrane Proteins and Cell Division in African Trypanosomes

“…Synchronization of L. mexicana with Hydroxyurea-Promastigotes of L. mexicana were synchronized with hydroxyurea as described by da Silva et al (60). Briefly, mid-log phase promastigotes were incubated in RPMI culture medium containing 5 mM hydroxyurea for 14 h at 26°C.…”

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

“…; da Silva et al. ). The duration of each cell cycle phase varies considerably in different protozoans and appears to be species‐specific, although it also depends on many other factors, such as parasite strain, life cycle stage, and molecular mediators (Domingo‐Sananes et al.…”

“…; Salic and Mitchison ; da Silva et al. ). EdU detection requires a cycloaddition reaction, which involves the covalent crosslinking of the EdU ethynyl group with a fluorescent azide catalyzed by Cu (I) ions in the presence of ascorbic acid.…”

N -Butyl-[1-(4-Methoxy)Phenyl-9 H -β-Carboline]-3-Carboxamide Prevents Cytokinesis in Leishmania amazonensis

“…New flagellum growth begins during the S phase and becomes visible outside the flagellar pocket near the end of this phase of the cell cycle; segregation of nuclear DNA occurs during the anaphase of mitosis, closely followed by kinetoplast division and cytokinesis, which is initiated at the anterior part of the parasite between the two flagella. However, differences in the order and timing of cell cycle events among Leishmania species have been described (22)(23)(24). Our findings indicate that, although the alkaloid ␤-CB did not prevent the duplication of flagella, kinetoplasts, or nuclei, cytokinesis was impaired, resulting in abnormal phenotypes of the parasite.…”