Whole cell reconstructions of Leishmania mexicana through the cell cycle

The unicellular parasite Leishmania has a precisely defined cell architecture that is inherited by each subsequent generation, requiring a highly coordinated pattern of duplication and segregation of organelles and cytoskeletal structures. A framework of nuclear division and morphological changes is known from light microscopy, yet this has limited resolution and the intrinsic organisation of organelles within the cell body and their manner of duplication and inheritance is unknown. Using volume electron microscopy approaches, we have produced three-dimensional reconstructions of different promastigote cell cycle stages to give a spatial and quantitative overview of organelle positioning, division and inheritance. The first morphological indications seen in our dataset that a new cell cycle had begun were the assembly of a new flagellum, the duplication of the contractile vacuole and the increase in volume of the nucleus and kinetoplast. We showed that the progression of the cytokinesis furrow created a specific pattern of membrane indentations, while our analysis of sub-pellicular microtubule organisation indicated that there is likely a preferred site of new microtubule insertion. The daughter cells retained these indentations in their cell body for a period post-abscission. By comparing cultured and sand fly derived promastigotes, we found an increase in the number and overall volume of lipid droplets in the promastigotes from the sand fly, reflecting a change in their metabolism to ensure transmissibility to the mammalian host. Our insights into the cell cycle mechanics of Leishmania will support future molecular cell biology analyses of these parasites. The parasite Leishmania causes the insect-transmitted neglected tropical disease, leishmaniasis. Leishmania is a single-celled parasite with a distinctive and highly defined shape. It is critical that each generation of the parasite retains the same shape. To achieve this, the parasite has a highly coordinated cell cycle in which organelles and key structures are duplicated and segregated at specific points. Previous work has defined a framework of cellular changes at the light microscopy level but this has limited resolution. We used volume electron microscopy to reconstruct Leishmania cells at different points in the cell cycle, which provided a three-dimensional overview of organelle positioning, duplication, and segregation. Moreover, we found that cytokinesis created membrane indentations that persisted in the daughter cells for a limited time. Finally, we examined Leishmania parasites in their insect vector, the sand fly. We found that the parasites in the sand fly had a greater number of lipid droplets, indicative of changes to their metabolism. Our insights into the cell cycle mechanics of Leishmania will provide a framework for future analyses of these parasites.