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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 cell cycle is fundamental for Leishmania survivability and to maintain pathogenicity, ensuring that each generation retains the same cytoskeletal morphology and organelles. This requires a tightly coordinated process of organelle duplication and segregation at specific stages. Using volume electron microscopy, we reconstructed Leishmania mexicana promastigote cells at different cell cycle stages, to produce a 3D view of organelle positioning and division. Our findings show that the new flagellum does not rotate around the old flagellum and cytokinesis creates a membrane indentation that is retained in daughter cells until early in the next cell cycle. Transition fibres serve as docking sites for axonemal proteins, ensuring proper flagellum assembly and length maintenance. In Leishmania, flagella elongate over multiple cell cycles, and we identified CEP164C as a regulator of flagellum growth, instead of as part of a locking mechanism. We observed CEP164C localising to the old flagellum and being recruited to the new flagellum later in the cell cycle. Knockdown of CEP164C caused dysregulation, with the old flagellum elongating and the new one shortening. In T. brucei, CEP164C localizes exclusively to the old flagellum. We investigated whether CEP164C interacts with differentially expressed transition fibre proteins found at the old and new flagella. Our findings showed that knockdown of CEP164C results in the upregulation of four of these transition fibre proteins. We also studied gametogenesis in Plasmodium berghei which occurs in the Anopheles mosquito and is critical for sexual reproduction and transmission to mammalian hosts. Using volume electron microscopy, we examined key structures in P. berghei microgametocytes and microgametes. Our findings revealed axonemes coiling around the nucleus in opposite directions, forming a central axonemal band. We also discovered that the nucleus tightly coils around the axoneme in microgametes before exflagellation. This de