Metacaspases (MCAs) are distant orthologues of caspases and have been proposed to play a role in programmed cell death in yeast and plants, but little is known about their function in parasitic protozoa. The MCA gene of Leishmania major (LmjMCA) is expressed in actively replicating amastigotes and procyclic promastigotes, but at a lower level in metacyclic promastigotes. LmjMCA has a punctate distribution throughout the cell in interphase cells, but becomes concentrated in the kinetoplast (mitochondrial DNA) at the time of the organelle's segregation. LmjMCA also translocates to the nucleus during mitosis, where it associates with the mitotic spindle. Overexpression of LmjMCA in promastigotes leads to a severe growth retardation and changes in ploidy, due to defects in kinetoplast segregation and nuclear division and an impairment of cytokinesis. LmjMCA null mutants could not be generated and following genetic manipulation to express LmjMCA from an episome, the only mutants that were viable were those expressing LmjMCA at physiological levels. Together these data suggest that in L. major active LmjMCA is essential for the correct segregation of the nucleus and kinetoplast, functions that could be independent of programmed cell death, and that the amount of LmjMCA is crucial. The absence of MCAs from mammals makes the enzyme a potential drug target against protozoan parasites.

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...