Morphological Events during the Cell Cycle of Leishmania major

Abstract

The morphological events involved in the Leishmania major promastigote cell cycle have been investigated in order to provide a detailed description of the chronological processes by which the parasite replicates its set of single-copy organelles and generates a daughter cell. Immunofluorescence labeling of ␤-tubulin was used to follow the dynamics of the subcellular cytoskeleton and to monitor the division of the nucleus via visualization of the mitotic spindle, while RAB11 was found to be a useful marker to track flagellar pocket division and to follow mitochondrial DNA (kinetoplast) segregation. Classification and quantification of these morphological events were used to determine the durations of phases of the cell cycle. Our results demonstrate that in L. major promastigotes, the extrusion of the daughter flagellum precedes the onset of mitosis, which in turn ends after kinetoplast segregation, and that significant remodelling of cell shape accompanies mitosis and cytokinesis. These findings contribute to a more complete foundation for future studies of cell cycle control in Leishmania.Leishmania spp. are protozoan parasites that are the causative agents of the leishmaniases, a spectrum of vector-borne diseases endemic in tropical and subtropical countries. The cutaneous form of leishmaniasis is the most common form, and it is estimated that it afflicts about 10 million people. In Africa and Asia, this disease is caused mainly by Leishmania tropica and L. major. The two major replicative developmental stages in the life cycle of Leishmania are the procyclic promastigote, which occurs in the sand fly insect vector, and the amastigote, which resides in the phagolysosome of mammalian macrophages. Leishmania procyclic promastigotes are highly polarized cells that possess a number of single-copy organelles with defined subcellular locations. These include the nucleus, the Golgi apparatus, the basal body, the mitochondrion (which incorporates the kinetoplast), and the flagellum, which protrudes from the cell body via the flagellar pocket. The generation of viable progeny relies upon precise control of the duplication and segregation of these organelles (11,17,29,32).The cell cycle of procyclic form Trypanosoma brucei has been characterized extensively and forms a basis for comparison with other trypanosomatids, including Leishmania. Notably, in T. brucei, replication of the nuclear and kinetoplast DNAs (S phase) starts almost synchronously, whereas the division and segregation periods for the nucleus (M and C phases, respectively) and kinetoplast (D and A phases, respectively) are separated in time. In procyclic form T. brucei as well as in L. tarentolae (28), kinetoplast division is completed before the onset of nuclear mitosis, while in L. mexicana and L. donovani these events appear to occur in the reverse order (11,17,32). The chromosomes of T. brucei do not visibly condense, the nuclear envelope remains intact during mitosis, and no structural equivalents of the mammalian spindle pole bodies (centrosomes) hav...

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.…”

Trypanosomatid Cell Division Kinases

“…For example, L. mexicana nuclear replication is reported to occur at about half the rate observed in T. brucei and T. cruzi, while T. brucei replicates its kDNA far more slowly than the other trypanosomatids [17]. Similar to T. brucei, the kinetoplast has been reported to divide before the nucleus in promastigote L. tarentolae [22] and L. major [18]. In T. cruzi epimastigotes, the kinetoplast S phase may commence after the start of nuclear replication, but the very low proportion (<2%) of cells with one nucleus and two kinetoplasts indicates that the segregation of nuclei and kinetoplasts occurs almost simultaneously [15].…”

“…Recent studies performed in T. cruzi and Leishmania [15][16][17][18] have highlighted differences in the cell division cycles of these trypanosomatids relative to T. brucei, and thus it is likely that such structural differences will be reflected at the molecular level for example, kinase function. Differences are observed in the timing of flagellar elongation, flagellar pocket duplication and diversity of flagellar length.…”

“…In T. brucei, flagellum elongation commences early in the cell cycle, and daughter pocket formation is complete by the onset of mitosis [19]. In contrast, in L. major the new flagellum exits the old flagellar pocket at the end of S phase, and formation of the daughter flagellar pocket commences just prior to mitosis [18]. In T. cruzi, however, elongation of the new flagellum only occurs in G 2 phase, after DNA synthesis is complete, and the daughter flagellar pocket is not formed until cytokinesis [15].…”

Nuclear DNA replication and repair in parasites of the genusLeishmania: Exploiting differences to develop innovative therapeutic approaches

“…In other species, such as L. donovani, nuclear division precedes kinetoplast segregation only in 80% of cells, while in the other 20%, the kinetoplast segregates first (Minocha et al, 2011a). In L. major, nuclear DNA replication precedes kinetoplast segregation, but nuclear division is accomplished after kinetoplast segregation (Ambit et al, 2011). A similar order of events has been described in Trypanosoma brucei and Trypanosoma cruzi, in which kinetoplast segregation precedes karyokinesis (Godoy et al, 2009).…”