Figure 5. S. rosetta microtubule doublets contain unique holes and MIPs.

(A–F). Tomographic slices (A–C) and isosurface renderings (D–F) of the subtomogram average of S. rosetta doublet microtubules shown in cross (A, D) and longitudinal (B, C, E, F) section at the level of RS1. The white and black lines in (A) and (D), respectively indicate the viewing positions of the longitudinal slices in (B, C, and E-H). Note: panels (B and E) portray the distal (D) flagellum to the left, and the proximal (P) flagellum to the right. MIPs (and their corresponding arrowheads) are colored as indicated in the legend below panels (E/F). Because the MIPs repeat with a periodicity of 48 nm or less, only a 48 nm long segment of the 96 nm axonemal unit is shown. (G, H) Classification analyses focused on the region with the newly identified rail-MIP (G) and A-tubule hole (H) indicating their presence only in subsets of the axonemal repeats. Class 1 (top rows) lack the rail-MIP or A-tubule hole (empty arrowheads), whereas class 2 (bottom rows) contain the rail-MIP (navy blue arrowhead) or A-tubule hole (olive arrowhead), respectively. Percentages of repeats out of 7584 averaged particles are indicated for each class. The isosurface renderings highlight the position of the rail-MIP (navy blue) between protofilaments A1 and A13, adjacent to MIP 6ab (jade) (G), and of the A-tubule hole (olive arrowhead) in protofilament A2 (H). The tables show the doublet-specific distribution of the classes. Note: the rail-MIP and A-tubule hole distributions only partially overlap (Figure 5—figure supplement 1). Figure 5—figure supplement 2 indicates the presence of the rail-MIP in the proximal flagellum. Figure 5—figure supplement 3 shows two additional holes in the S. rosetta inner junction. Scale bars: 10 nm (A, applies also to B, C); 20 nm (G, applies to all other images in the panel); 20 nm (H, applies to all other images in the panel).

Figure 5—figure supplement 1. Distribution of rail-MIP and A-tubule hole classes by tomogram and doublet microtubule.

Each tomogram (TOM #) is represented by a block in which the columns indicate microtubule doublet (DMT1-9), and the rows represent individual 96 nm subvolumes within those DMTs, listed from distal (top) to proximal (bottom). For each tomogram, the rail-MIP classification results are displayed on the left, and the A-tubule hole classes are displayed on the right. Purple indicates the presence of either the rail-MIP or the A-tubule hole, whereas green denotes their absence. Rail-MIPs seem to be strongly present in certain tomograms (left), and almost completely absent in others (right). Holes, on the other hand, exhibit a more scattered distribution. Both features show some DMT-specificity or -preference, but there is only partial overlap between the presence/absence of a rail-MIP and A-tubule hole in the same subvolume.

Figure 5—figure supplement 2. The rail-MIP is present in the proximal region of the S.rosetta flagellum.

(A–B) Longitudinal (A) and cross-sectional (B) tomographic slices through a cryo-FIB milled S. rosetta cell, targeting the proximal region of the flagellum close to the cell body. The cartoon in (A) denotes the cell’s orientation, with cell body to the left. Cyan arrowheads indicate vane filaments. Note: the flagellar membrane in this sample is slightly swollen, which is not unusual for cells that were actively swimming while they were plunge-frozen. DMT numbers are indicated, with DMT 9 in white to indicate its absence (the cryo-FIB-milling removed material from that side of the axoneme). (C, D) Two subtomogram averages of the 96 nm axonemal repeat were calculated from the tomogram shown in (A, B): in (C), repeats from DMTs 1, 2, 4, 5, and 8 were combined, showing an average that lacks the rail-MIP (white arrowheads), and in (D), repeats from DMTs 3, 6, and 7 were combined, showing the rail-MIP (navy blue arrowheads). Note: the doublet-specific averages shown here are noisy due to limited number of averaged particles. Scale bars: 200 nm (A, applies also to B); 20 nm (C, applies also to D).

Figure 5—figure supplement 3. Additional holes in the doublet inner junction of the S.rosetta flagellum.

(A–B) Tomographic slices through the subtomogram average of the S. rosetta 96 nm axonemal repeat shown in cross-section (A) and longitudinal section (B). The white line indicates the location of the section shown in (B).(C–D) Isosurface renderings of the averages shown in (A, B). The black line indicates the view shown in (D). In all panels, the pale violet arrowheads indicate the previously reported hole near the N-DRC, the olive arrowheads indicate the A-tubule gap (see Figure 5 and Figure 5—figure supplement 1), and the green and pink arrows denote additional inner junction holes that have not been previously reported in other species. Other labels: IDA, inner dynein arms; NDRC, nexin-dynein regulatory complex; ODA, outer dynein arms. Scale bar: 20 nm (A, applies also to B).