Differences in external morphology during the cell division cycle of the procyclic and bloodstream forms. Composite scanning electron micrograph images of stages of procyclic (A–H) and bloodstream (I–O) form cell division.
A and I. G1 cell with a single attached flagellum. The exit point of the flagellum from the flagellar pocket is arrowed and the anterior and posterior indicated.
B and J. A new flagellum has grown to extend from the flagellar pocket (arrow). The distal tip of the new flagellum is laterally connected to the old flagellum in the procyclic (B) and is laterally embedded in the groove structure in the flank of the cell in the bloodstream form (J), indicated by dashed circles.
C, K–L. The flagellar pocket associated with the new flagellum (arrowed) is positioned posterior to the flagellar pocket associated with the old flagellum (arrowhead). The new flagellum is located to the left of the old when viewed looking from posterior to anterior.
D and M. A division fold is evident between the two flagella (arrowed) which is located along the long axis and begins to define the daughter cell shape. There are two distinct posterior end profiles. The new‐flagellum daughter inherits the existing posterior end and a new posterior end is formed for the old‐flagellum daughter (circled). The new flagellum is still attached to the old by the flagella connector in the procyclic form (D), but has grown free of the cell body in the bloodstream form (M), indicated by dashed circles.
E–F, N. A division cleft has opened up between the daughters (arrow) and the new flagellum tip remains attached to the old flagellum by the flagella connector in the procyclic form (E–F).
G–H, O. Pre‐abscission stage. In the procyclic form (G–H) the two daughter cells are attached by the posterior end of the old‐flagellum daughter (circled) to the side of the new‐flagellum daughter by a cytoplasmic bridge connection. In the bloodstream form (O) a posterior‐to‐posterior (circled) configuration is typical.

Microtubule‐mediated flagella segregation and unidirectional division fold ingression.
A. Cartoon illustrating the ingression of the division fold and furrow. A procyclic form cell is shown, but the same structures arise in the bloodstream form during cytokinesis.
B and C. Two examples of thin section transmission electron micrographs (TEMs) of transverse sections through PCF cells during unidirectional division fold (large open arrow) formation. The old flagellum is indicated with an asterisk and the basal body (in B), or flagellum within the flagellar pocket (in C), at the proximal end of the new flagellum are indicated with an arrow.
D–F. Thin section TEMs of transverse sections through bloodstream cells at different stages of new flagellum and FAZ growth. FAZ filaments (arrows) and the microtubule quartet (MtQ, dashed circles) are indicated. (D) Section with a single flagellum closely associated with the FAZ. (E) Section with two flagella closely associated with their respective FAZ. There are no sub‐pellicular microtubules between the old FAZ MtQ and the new FAZ. (F) TEM of a transverse section through a bloodstream form cell with two segregated flagella closely associated with their respective FAZ. Indentation of the plasma membrane (large arrow) is unidirectional.
G–I. Cartoon models of insertion of the new FAZ filament and MtQ among the sub‐pellicular microtubules, approximately corresponding to the micrographs in D–F. (G) Cartoon structure of a normal G1 phase single (old) flagellum and FAZ. Note the opposing polarities of the sub‐pellicular microtubules and the MtQ. Three distinct putative molecular‐scale structural linkages (pellicular microtubule‐MtQ, MtQ‐FAZ and FAZ‐pellicular microtubule) may be predicted. (H) Cartoon structure following invasion of the growing FAZ and microtubule quarter associated with the new flagellum. Invasion occurs along the pellicular microtubule ‐MtQ seam. This introduces fourth distinct putative molecular‐scale structural linkage; FAZ‐MtQ. (I) Cartoon structure following invasion of sub‐pellicular microtubules to segregate the new and old flagella. Invasion occurs along the FAZ‐MtQ seam. This restores FAZ‐pellicular microtubule and pellicular microtubule‐MtQ linkages. Note that either microtubule sliding or minus end sub‐pellicular microtubule polymerization is required for sub‐pellicular microtubule invasion.

Differences in microtubule remodelling during cell division visualized by tubulin detyrosination dynamics.
A and B. Cartoon representations of three zones of procyclic (A) and bloodstream form (B) cells that undergo distinct growth and morphogenetic events. Zone 1 (the posterior) is inherited by the new‐flagellum daughter cell. Zone 2 (the centre) is the dynamic area during division. Zone 3 (the anterior) is inherited by the old‐flagellum daughter cell. The forming new posterior end is indicated with an arrowhead.
C–N. Maximum projections of YL1/2 (anti‐tyrosinated α‐tubulin) immunofluorescence, DAPI fluorescence and phase contrast micrographs of procyclic (C–H) and bloodstream (I–N) form cells during the cell division cycle. The basal bodies (arrowheads in C, D, I, J), new growing flagellum (arrows in D, J), mitotic spindle (arrows in E, K) and a region near the flagella during mitosis (arrowheads in E, K) are strongly labelled by YL1/2 in addition to regions of the sub‐pellicular microtubule array. The forming new (dashed circle) and old (solid circle) posterior ends of the procyclic form are indicated.
O and P. Detail of YL1/2 labelling of posterior (a), central (b) and anterior (c) portions of the sub‐pellicular array in G2/cytokinetic procyclic (O) and bloodstream (P) form cells.

Morphogenesis of the cell posterior visualized by the tip‐tracking microtubule polymerase XMAP215.
A. Detail of YFP::XMAP215 localization at the posterior of a detergent‐extracted G1 procyclic form cell.
B–M. Native fluorescence of YFP::XMAP215, DAPI fluorescence and phase contrast micrographs of procyclic (B–G) and bloodstream (H–M) form cells during the cell division cycle. The cell posterior (arrowheads) and mitotic spindle (arrows) are indicated and an inset shows the structure of the forming new posterior end during cytokinesis (F, L).

Cytoplasmic bridge and abscission.
A–D. Still images from DIC videomicroscopy of live dividing procyclic (A, B) and bloodstream (C, D) form cells, connected by a cytoplasmic bridge (arrow).
E. TEM of a whole mount cytoskeleton of a pre‐abscission procyclic form cell. Detail of the old and newly forming posterior are shown with arrowheads indicating examples of microtubule plus ends, and an arrow indicating a bundle of microtubules which extend from the newly forming posterior end into the portion of the sub‐pellicular array which will be inherited by the daughter cell with the new flagellum.
F–O. Examples of XMAP215 labelling of G1 procyclic (F, G) and bloodstream (N, O) cells with a more rounded (F, N) or pointed (G, O) posterior end.

Quadriflagellate doublet cells.
A. SEM of quadriflagellate doublet cells in the bloodstream form. Daughter cells are attached by their posterior ends and both possess two flagella.
B. L8C4 (anti‐PFR) immunofluorescence, DAPI fluorescence and phase contrast micrographs of a quadriflagellate bloodstream form cell with 4 kinetoplasts, 4 nuclei and 4 flagella.
C. Cartoon depicting two hypothetical routes to cell division of quadriflagellate doublet cells resulting in 4 1K1N cells. The 2K2N cells generated by route 1 would be morphologically indistinguishable from 2K2N cells from the normal cell cycle.

Differences in morphogenesis of the bloodstream and procyclic forms of T. brucei through the cell division cycle. Major morphogenetic events are shown alongside cartoon representations of the associated cell morphologies. Vertical spacing is purely illustrative and does not represent relative time spent during each of these stages.