Model of the kinetoplast duplication cycle. T. brucei procyclic forms were prepared for TEM. Images were captured of serial thin sections through kinetoplasts at different stages in their duplication cycle, and 3D reconstructions were generated from these images. (A) The duplication cycle can be divided into five distinct stages, I to V, based on the morphology of the kDNA disk and the replication status of the basal body (bb)/probasal body (pbb) and adjacent flagellar pocket (fp); mm, mitochondrial membrane. bb1 denotes the basal body subtending the mature flagellum, and bb2 is the basal body of the new flagellum. In stage I, the kDNA forms a unit-sized disk; in stage II, the kDNA disk has prominent antipodal sites (AS); stage III kinetoplasts are bilobed and flagellar pocket duplication is complete; stage IV kinetoplasts are connected by a nabelschnur (n); and stage V kinetoplasts (not shown) are morphologically the same as stage I kinetoplasts. (B) Model of a stage IIa kinetoplast superimposed onto a single image from the series (oriented such that the anterior end of the cell is facing right). In this cell, bb2 is in a position anterior to bb1. (C) Model of a stage IIb kinetoplast superimposed onto a single image from the series (oriented as in panel B). In this cell, bb2 is now in a position posterior to bb1. nf, new flagellum; of, old flagellum; r, ridge forming between the old and new flagellar pocket. Scale bars, 200 nm.

3D reconstructions of the nabelschnur in segregating kinetoplasts. T. brucei procyclic forms were prepared for TEM, stained with E-PTA, and used to generate 3D reconstructions from serial sections, as described for Fig. 2. (A) Bilobed kinetoplast (the arrowhead marks the midzone between the lobes); (B and C) kinetoplasts with a short and a long nabelschnur, respectively (arrows). A1 to A3, B1 to B3, and C1 to C3 show adjacent serial sections; A4, B4, and C4 show the full 3D reconstruction of the corresponding kinetoplasts. of, old flagellum; nf, new flagellum; pbb, probasal body. Scale bar, 100 nm.

Quantitation of frequency and length of maxicircle threads in wild-type (927) cells. (A) The first five rows show representative FISH images of cells containing threads of various lengths. Cells were treated with the nicking enzyme DNase I prior to FISH. The last row shows a 2K2N cell lacking a thread. Scale bar, 2 μm. (B) Quantitation of frequency and length of threads in various stages of the cell cycle. In all cells containing two kinetoplasts (K), the distance between them was measured in microns. Cells then were scored according to whether or not they contained a thread. Green dots, cells containing a thread. Black dots, cells lacking a thread. A thread was never observed in 2K2N cells. Horizontal bars indicate the means. Error bars show standard errors of the means. N, nucleus; K, kinetoplast; n, nabelschnur/maxicircle thread.

Morphology of the kinetoplast during the cell cycle in T. brucei procyclic forms. The top row of images shows an overlay of the phase-contrast image and DAPI fluorescence (pseudocolored cyan) to show the number and location of kinetoplasts (K) and nuclei (N) in the cell. The second row shows the DAPI signal at twice the magnification to provide a clear view of K shapes. Images are arranged in the order in which the respective K/N configurations occur in the cell cycle. The cell in panel A has a unit-sized kinetoplast (1K), cells in panels B and C have bilobed kinetoplasts (Kdiv), and cells in panels D to G have two kinetoplasts (2K). Ndiv, nucleus with a mitotic spindle. Scale bars, 2 μm. bb, basal body.

BrdU labeling of antipodal sites. T. brucei procyclic cells in asynchronous culture were incubated with BrdU for 15 min before processing for the immunofluorescence detection of incorporated BrdU (MAb PRB-1) and basal bodies (MAb BBA4). DNA was stained with DAPI. (A to G) Cells in successive stages of the cell cycle. BrdU incorporation was detected at the poles of kinetoplasts in a subset of 1K1N cells (B and C) and 1Kdiv1N cells (D and E). The arrow in panel C indicates the short new flagellum. Cells with BrdU-positive kinetoplasts also had numerous BrdU foci throughout the nucleus. Scale bar, 5 μm.

Visualization of replicating and nonreplicating kDNA networks by FISH. (A) A representative field showing cells prepared using the standard FISH protocol. Only minicircles and maxicircles that are nicked or gapped due to replication are accessible to FISH probes. In the merged images minicircles are shown in red and maxicircles in green. Scale bar, 5 μm. Areas outlined by the white boxes in the merged image are enlarged in panel B. (B) The numbers refer to the order in which they are discussed in the text. Arrow indicates concentration of maxicircles in the center of a dumbbell-shaped kDNA network. The arrowhead indicates a thin thread of maxicircles that connects two networks that have already separated. Scale bar, 1 μm. (C) A representative field showing cells that were nicked by DNase treatment prior to the FISH procedure. Here, all kDNA networks regardless of cell cycle stage are labeled by the minicircle and maxicircle FISH probes. Scale bar, 5 μm.

Frequency and length of threads increases during overexpression of the mitochondrial helicase PIF2. (A) Quantitation of length and frequency of maxicircle threads in wild-type (927) cells (n = 22). FISH was performed in the absence of DNase treatment, meaning that threads were observed less frequently, most likely because some gaps in the replicated maxicircles are filled prior to maxicircle division, rendering them inaccessible to our FISH probes. Analysis was performed as described for Fig. 6. Green dots, cells containing a thread. Black dots, cells lacking a thread. Horizontal bars indicate the means. Error bars show standard errors of the means. K, kinetoplast; N, nucleus; n, nabelschnur/maxicircle thread. The image shows a 2K1N cell with a very fine maxicircle thread (minicircles, red; maxicircles, green). Scale bar, 1 μm. (B) A similar experiment was performed on cells that had overexpressed TbPIF2 for 48 h. TbPIF2 is a mitochondrial helicase involved in maxicircle replication. The overexpression of this protein leads to an increase in the number of maxicircles. In these cells (n = 32), the frequency and length of the maxicircle thread increases and can now be observed in 2K2N cells. The nabelschnur also appears thicker. The image shows a 2K1N cell (minicircles, red; maxicircles, green). Scale bar, 1 μm.