Fig 1.
mitoGFP is a mitochondrial marker in C. fasciculata.
A) Mitochondrial staining by the potential-dependent dye MitoTracker Red (MitoTracker, top panel) and by mitochondrial-targeted GFP (mitoGFP, lower panel). mitoGFP expression is driven by an ectopic plasmid causing heterogeneous fluorescence among cells. Scale bar is 10 μm. B) Higher magnification images of two representative cells show colocalization between MitoTracker and mitoGFP signals. The phase contrast image shows the cell boundaries. Nuclear DNA (arrowhead) and mitochondrial DNA (kDNA, arrow) are stained with DAPI. In merged images MitoTracker is shown in magenta and mitoGFP in green. Scale bar is 5 μm.
Fig 2.
Mitochondrial shape during the cell cycle of C. fasciculata.
A) Typical mitochondrial morphology in 1N1K (G1 phase) cells as shown by mitoGFP. Arrowheads show a small mitochondrial loop that indicates the presence of the kDNA disk. Arrows point to areas containing thickened mitochondrial tubules. DAPI signal is shown in magenta in merged images. Scale bar is 5 μm. B) Relative numbers of cells in different phases of the cell cycle in asynchronous cultures and in hydroxyurea (HU)-synchronized cultures at various times after removal of HU. This procedure allows for enrichment of mitotic cell cycle phases and demonstrates cell cycle progression from 1N1K to 2N1K, 2N2K, and back to 1N1K. N, nucleus. K, kDNA. C) Synchronized cells (n = 119) in different phases of the cell cycle according to DAPI staining were measured for overall cell length and width using phase contrast images. For length, cells were measured along their longest axis from the base of the flagellum to the posterior end. Cell width was measured at the widest point of the cell. P-values indicated *<0.05, **<0.005, *** = 0.001, and ****<0.0001 by ANOVA. D) Examples of typical mitochondrial shapes found in 1N1K, 2N1K, and 2N2K cells. Note that the nuclei are not symmetrically positioned with respect to the cell body in 2N1K cells. The last three columns show cells undergoing cytokinesis. Scale bar is 5 μm.
Fig 3.
Confocal analysis of mitochondrial shape during the cell cycle of C. fasciculata.
A) Examples of maximum projections of fixed, synchronized C. fasciculata mitoGFP-expressing cells as imaged by confocal microscopy. Mitochondria are shown in green and DAPI in red. Cell cycle stages are shown above each row. The first row shows all “closed” networks. Rows 2–4 show networks that are “open” at the anterior end (containing tubule end points rather than loops). Each image is oriented such that the anterior of the cell is on the right. Scale bar is 5 μm. B) Quantitation of the percent of cells (n = 114) in each cell cycle stage that have closed or open mitochondrial networks at their anterior end. C) Mitochondrial area in z-stack maximum projections as a function of cell cycle stage (n = 115). **** indicates P-value <0.0001 by Mann-Whitney test. D) The numbers of mitochondrial nodes, or branch points, were counted manually in maximum projections (n = 115) *** indicates P-value of 0.0002 by unpaired t-test with Welch’s correction.
Fig 4.
Mitochondrial dynamics in C. fasciculata.
A) Frames from live-cell imaging of two cells by spinning-disk confocal microscopy. Cells are expressing mitoGFP. Mitochondrial remodeling reactions are indicated by arrows. Times are given as h:min:s. White numbers indicate frame sequence. Not all frames in the time-course are shown (see S1 and S2 Movies) Scale bar is 5 μm. B) Quantitation of mitochondrial remodeling events in spinning disk confocal experiments. Z-stacks of 17 cells were generated every 2 min for an average of 49 min. The resulting movies were then analyzed for different types of mitochondrial remodeling events. The frequency of each event per minute was calculated then multiplied by 30 to give number of events/30 min time period. The bar labeled total represents the sum of all the different types of events shown by the other bars. C) Live-cell imaging of mitochondrial remodeling with a laser-scanning confocal microscope and deconvolution. White numbers indicate different frames. Boxes indicate areas in which remodeling occurs between frames. These areas are shown enlarged (with corresponding numbers) in the right panels. Scale bars are 2 μm.
Fig 5.
Adherent C. fasciculata haptomonads also show rapid mitochondrial remodeling.
A) Max projections of a typical rosette of C. fasciculata imaged using a laser scanning confocal microscope. Mitochondria in live cells were visualized using mitoGFP. Mitochondrial remodeling reactions are indicated by arrows. Times are given as mm:ss. White numbers indicate the sequence of frames. Note that not every frame is shown (see S3 Movie). Scale bar is 10 μm. B) Time course of mitochondrial remodeling in a single adherent cell. Each frame is a max projection that has been color-coded by depth. White numbers indicate the sequence of frames. Note that not every frame is shown (see S4 Movie). Bracket indicates the formation of a net-like structure that then is rapidly remodeled to tubules during the course of our observation. Scale bar is 5 μm.
Fig 6.
Mitochondrial division occurs just prior to cytokinesis in C. fasciculata.
A) Time-lapse of mitochondrial/cell division in a swimming nectomonad immobilized in low melting point agarose. Image is a max projection of a Z-stack generated on a spinning disk confocal microscope and subjected to deconvolution. Times are given in mm:ss and numbers indicate the sequence of frames. The anterior of the cell is on the left side of each image. Complete mitochondrial division seems to occur between frames 3 and 4. A fenestrated sheet appears in the lower cell in frames 5 and 6 (arrowheads). Not all frames are shown (see S5 Movie). Scale bar is 2 μm. B) Mitochondrial and cell division of adherent haptomonads. Arrowheads indicate a dividing cell. Times are given in mm:ss and numbers indicate the sequence of frames. Not all frames are shown (see S6 Movie). Scale bar is 10 μm. C) Time lapse of a dividing haptomonad. The presumed position of the kinetoplast (identified by the small loop of mitochondrial matrix surrounding it, is indicated by white arrows. Numbers indicate the sequence of frames. Not all frames are shown (see S8 Movie). kDNA division occurs in frame 6–7. Scale bar is 2 μm.