Morphology of the trypanosome bilobe. (A) Endogenous TbMORN1 localization in detergent-extracted cells fixed with paraformaldehyde and labeled with anti-TbMORN1 antibodies. (B) An endogenous replacement cell line expressing YFP-TbMORN1 recapitulates the localization and morphology of the wild-type protein. The image shows an intact cell fixed with paraformaldehyde, with the nucleus stained with DAPI. (C) YFP-TbMORN1 localized in detergent-extracted cells by use of anti-GFP antibodies. The immunogold labeling traces a hook-shaped pattern (white arrowheads) reminiscent of the morphology seen by immunofluorescence microscopy. The basal and probasal bodies are indicated with black and white arrowheads, respectively. (D) The bilobe is not readily visible in unlabeled transmission EM images of detergent-extracted YFP::TbMORN1 cells. (E) TbMORN1 copurifies with the flagellum and the basal body. Preparations of isolated flagella were fixed with paraformaldehyde and labeled with anti-TbMORN1 antibodies (green; arrow) and the monoclonal antibody YL1/2 to label the basal body (red; arrowhead). Localization and morphology were unaffected by the purification process. (F) YFP-TbMORN1 recapitulates the behavior of the wild-type protein. Basal bodies were labeled with YL1/2 (red; arrowheads); anti-GFP antibodies labeled YFP-TbMORN1. All immunofluorescence images are maximum-intensity z projections. Bars, 2 μm (A, B, E, and F) and 500 nm (C and D).

TbMORN1 and TbLRRP1 colocalize on the bilobe. (A) Endogenous TbMORN1 and TbLRRP1 colocalize in single 0.1-μm z slices. The image shows detergent-extracted cells fixed with paraformaldehyde and labeled with anti-TbMORN1 and anti-TbLRRP1 antibodies. (B) YFP-TbMORN1 and TbLRRP1 colocalize in isolated flagella purified from YFP::TbMORN1 cells. These were fixed with paraformaldehyde, labeled with anti-GFP and anti-TbLRRP1, and imaged in single 0.1-μm z slices. (C) TbLRRP1 preferentially localizes by immuno-EM to the arm nearest the MTQ rather than the FAZf (see inset). Isolated flagellum preparations from YFP::TbMORN1 cells were fixed and labeled with anti-TbLRRP1 antibodies followed by 10-nm gold particles. (D) YFP-TbMORN1 and TbLRRP1 colocalize by immuno-EM. YFP-TbMORN1 was visualized using anti-GFP antibodies and 20-nm gold particles; TbLRRP1 was visualized using anti-TbLRRP1 antibodies and 10-nm gold particles. Bars, 1 μm (A and B) and 500 nm (C and D).

The posterior hooked part of TbMORN1 sits above the flagellar pocket collar. (A) The flagellar pocket collar is visible as an electron-dense region at the neck of the flagellar pocket in an EM (arrows). (B) Anti-TbBILBO1 antibodies recognize an approximately 67-kDa polypeptide in immunoblots of trypanosome whole-cell lysates. (C) Anti-TbBILBO1 antibodies label a ring-shaped structure close to the point of flagellum entry into the cell (arrow). Detergent-extracted cells were fixed with methanol and labeled with anti-TbBILBO1. (D) YFP-TbMORN1 and TbBILBO1 overlap only partially at oblique angles. Isolated flagella from YFP::TbMORN1 cells were labeled with anti-GFP and anti-TbBILBO1. (E and F) The posterior hooked part of YFP-TbMORN1 overlaps with TbBILBO1 in immunogold experiments. TbBILBO1 labeling is always more posterior to YFP-TbMORN1 labeling (arrows). Bars, 500 nm (A, E, and F) and 1 μm (C and D).

Two distinct populations of mEGFP-TbMORN1. (A) FRAP experiments were performed on live T. brucei cells immobilized on a low-melting-point agarose gel and stably expressing mEGFP-TbMORN1. Either the bilobe (upper or lower panels) or tendrillar projection (middle panels) was photobleached, and recovery of the bleached area (red boxes) was followed by time-lapse microscopy. The 0-min column represents first postbleach images. (B) Quantification of normalized fluorescence intensity (mean ± standard deviation) in the bleached area of the bilobe (left panel), a tendrillar projection (middle panel), or the bilobe in the presence of a tendrillar projection (right panel) (n = 5 independent experiments in all cases).

Visualizing the bilobe in isolated flagellum preparations. (A and B) YFP-TbMORN1 localized in isolated flagella, as shown with anti-GFP antibodies. The immunogold labeling pattern describes a hook shape (Bi) consistent with that in immunofluorescence microscopy images. The FAZ filament (FAZf) is indicated. (C to F) Gallery of cytoskeletal structures near the base of the flagellum. The MTQ, originating from between the basal body (black arrowhead) and the probasal body (white arrowhead), describes a helical path around the flagellar pocket (not visible) before following the line of the flagellum (F). The MTQ filaments are seen most readily in panels E and F (white arrows). The FAZf originates in the vicinity of the FPC and runs alongside the MTQ. Note that the arm of YFP-TbMORN1 seen in panels A and B is not readily visible in panels C to F. What can be seen is another arm (*) that runs alongside the FAZf but is labeled only faintly for YFP-MORN1 (A and B). Bars, 500 nm.

TbMORN1 and TbCentrin4 are on separate arms of the bilobe. (A) YFP-TbMORN1 and TbCentrin4 do not overlap in detergent-extracted cells (the third panel shows merged images with a differential interference contrast [DIC] overlay). (B) Image gallery showing minimal to no overlap between YFP-TbMORN1 (green) and TbCentrin4 (red) in purified flagella isolated from YFP::TbMORN1 cells. Where visible, TbCentrin4 labeling of the basal body and probasal body is indicated with an arrowhead. TbCentrin4 was labeled using anti-LdCentrin4 rabbit polyclonal antibodies (i to iv) or monoclonal antibodies to TbCentrin4 (v to viii). Images are maximum-intensity z projections. Each z stack was analyzed in single sections, and overlap was observed only if the two bilobe arms became twisted around one another. (C) TbCentrin4 is present largely on the bilobe arm adjacent to the FAZf. Anti-TbCentrin4 antibodies were used to label isolated flagella from YFP::TbMORN1 cells, followed by gold-conjugated secondary antibodies (10 nm). The MTQ, basal body (black arrowhead), and probasal body (white arrowhead) are indicated for orientation. (D and E) YFP-TbMORN1 and TbCentrin4 define separate arms of the bilobe. Isolated flagella from YFP::TbMORN1 cells were labeled with anti-GFP (20-nm gold particles) and anti-TbCentrin4 (10-nm gold particles) antibodies. There appears to be minimal overlap between the hook-shaped YFP-TbMORN1 labeling pattern and the TbCentrin4 distribution. Bars, 1 μm (A and B) and 500 nm (C to E).

Tendrillar extensions of TbMORN1. (A and B) YFP-TbMORN1 (arrowheads) is sometimes seen to exhibit tendrillar extensions (arrows) to its normal hook-shaped pattern. YFP::TbMORN1 cells were fixed with paraformaldehyde, permeabilized with detergent, and labeled with anti-GFP antibodies. (C and D) The YFP-TbMORN1 tendrils (arrows) contact the basal body and maturing probasal body. Flagella extracted from YFP::TbMORN1 cells were labeled with anti-GFP antibodies and the YL1/2 antibody. YL1/2 labels fibers emanating from the mature basal body. The probasal body and the basal body are both labeled (arrowheads), indicating that the probasal body has matured. (E) Gallery showing various morphological forms of the YFP-TbMORN1 tendrils. Variously, these are single thin tendrils (i), single thick tendrils (ii), coat-hanger shapes (iii and iv), and figure-eight structures (v). All images show isolated flagella from YFP::TbMORN1 cells, fixed with paraformaldehyde. (F) A tendril emanating from the probasal body is occasionally seen in EM images of isolated flagella. The MTQ, originating between the basal body (black arrowhead) and the probasal body (white arrowhead), is indicated. The FAZf is marked for orientation. (G) Both YFP-TbMORN1 and TbLRRP1 can be found on the tendril and as part of the MTQ. Isolated flagella from YFP::TbMORN1 cells were labeled with anti-GFP (20-nm gold particles) and anti-TbLRRP1 (10-nm gold particles). Only half of the MTQ (bar) appears to be labeled. (H) The YFP-TbMORN1 tendril (long arrow) does not appear to contain alpha-tubulin (short arrow). The probasal body (arrowhead) is indicated in the DIC overlay. Isolated flagella were fixed with paraformaldehyde and labeled with anti-GFP and anti-alpha-tubulin antibodies. All immunofluorescence images are maximum-intensity z projections. Bars, 1 μm (A to E and H) and 500 nm (F and G).

Morphological model of the trypanosome bilobe. (A) Drawing showing the bilobe's position relative to other key cytoskeletal components. The locations of the bilobe and nearby cytoskeletal elements relative to the anterior-posterior (A-P), top-bottom (T-B), and left-right (L-R) axes of the whole cell are indicated in the inset. (B) Schematics of the isolated bilobe. The positions of the MTQ, FAZ filament, and apparent central projection of the FPC (arrowheads) are shown in flagellar (“top”) (i), “bottom” (ii), left (iii), and right (iv) views. Comparison with previously published tomograms indicates that the arms of the bilobe and the hook/flagellar pocket collar are oriented at an obtuse angle to one another. Furthermore, the left end of the flagellar pocket collar may extend partway along the ventral face of the MORN1/LRRP1 hook.