Patterns of organelle ontogeny through a cell cycle revealed by whole-cell reconstructions using 3D electron microscopy

Abstract

The major mammalian bloodstream form of the African sleeping sickness parasite Trypanosoma brucei multiplies rapidly, and it is important to understand how these cells divide. Organelle inheritance involves complex spatiotemporal re-arrangements to ensure correct distribution to daughter cells. Here, serial block face scanning electron microscopy (SBF-SEM) was used to reconstruct whole individual cells at different stages of the cell cycle to give an unprecedented temporal, spatial and quantitative view of organelle division, inheritance and abscission in a eukaryotic cell. Extensive mitochondrial branching occurred only along the ventral surface of the parasite, but the mitochondria returned to a tubular form during cytokinesis. Fission of the mitochondrion occurred within the cytoplasmic bridge during the final stage of cell division, correlating with cell abscission. The nuclei were located underneath each flagellum at mitosis and the mitotic spindle was located along the ventral surface, further demonstrating the asymmetric arrangement of cell cleavage in trypanosomes. Finally, measurements demonstrated that multiple Golgi bodies were accurately positioned along the flagellum attachment zone, suggesting a mechanism for determining the location of Golgi bodies along each flagellum during the cell cycle.

Bidirectional intraflagellar transport is restricted to two sets of microtubule doublets in the trypanosome flagellum

“…2A). The shape, size and distribution of these organelles were in agreement with published data based on electron tomography (Lacomble et al, 2009) or serial block face sectioning (Hughes et al, 2017). Flagella were clearly recognised in both cross and longitudinal sections, including multiple cases where the proximal portion was seen to emerge from the flagellar pocket (Video S1).…”

The single mitochondrion of the kinetoplastid parasite Crithidia fasciculata is a dynamic network

“…By imaging live cells, we were able to observe mitochondrial division and, indirectly, kDNA division. Division of the mitochondrion in C. fasciculata was closely coordinated with cytokinesis, as has been observed for T. brucei [30,50,58]. However, the mechanism of kDNA division is different between these two species.…”

“…Branching may be important for establishing mitochondrial network topology and may be mechanistically distinct from DLP-mediated fission. Fenestrated sheets have also been observed in bloodstream form (BSF) T. brucei and are thought to resolve into tubules [58]. In addition, branching of the mitochondrion increases just prior to cell division, which may facilitate symmetrical division of the network [50,58].…”

“…Fenestrated sheets have also been observed in bloodstream form (BSF) T. brucei and are thought to resolve into tubules [58]. In addition, branching of the mitochondrion increases just prior to cell division, which may facilitate symmetrical division of the network [50,58]. Whether these events are mediated by proteins that are conserved in the eukaryotic lineage, or whether they involve novel proteins specific to kinetoplastids, remains to be discovered.…”

“…brucei involves fusion of parts of the network [50,58]. The presence of fusion indicates that there must be an opposing process of fission (or an alternative, branching activity), one that is not necessarily restricted to final division of the network.…”