Leishmania major possesses, apparently uniquely, four families of ATG8-like genes, designated ATG8, ATG8A, ATG8B and ATG8C, and 25 genes in total. L. major ATG8 and examples from the ATG8A, ATG8B and ATG8C families are able to complement a Saccharomyces cerevisiae ATG8-deficient strain, indicating functional conservation. Whereas ATG8 has been shown to form putative autophagosomes during differentiation and starvation of L. major, ATG8A primarily form puncta in response to starvation-suggesting a role for ATG8A in starvation-induced autophagy. Recombinant ATG8A was processed at the scissile glycine by recombinant ATG4.2 but not ATG4.1 cysteine peptidases of L. major and, consistent with this, ATG4.2-deficient L. major mutants were unable to process ATG8A and were less able to withstand starvation than wild-type cells. GFP-ATG8-containing puncta were less abundant in ATG4.2 overexpression lines, in which unlipidated ATG8 predominated, which is consistent with ATG4.2 being an ATG8-deconjugating enzyme as well as an ATG8A-processing enzyme. In contrast, recombinant ATG8, ATG8B and ATG8C were all processed by ATG4.1, but not by ATG4.2. ATG8B and ATG8C both have a distinct subcellular location close to the flagellar pocket, but the occurrence of the GFP-labeled puncta suggest that they do not have a role in autophagy. L. major genes encoding possible ATG5, ATG10 and ATG12 homologues were found to complement their respective S. cerevisiae mutants, and ATG12 localized in part to ATG8-containing puncta, suggestive of a functional ATG5-ATG12 conjugation pathway in the parasite. L. major ATG12 is unusual as it requires C-terminal processing by an as yet unidentified peptidase.

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...

The transforming protozoan Theileria recruits Plk1, a host kinase that regulates mitosis, to its surface and engages spindle microtubules to secure its division and inheritance into both daughter cells.