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. 2018 Oct 15;29(21):2566-2577.
doi: 10.1091/mbc.E18-06-0405. Epub 2018 Aug 22.

Tetrahymena RIB72A and RIB72B Are Microtubule Inner Proteins in the Ciliary Doublet Microtubules

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Free PMC article

Tetrahymena RIB72A and RIB72B Are Microtubule Inner Proteins in the Ciliary Doublet Microtubules

Daniel Stoddard et al. Mol Biol Cell. .
Free PMC article

Abstract

Doublet and triplet microtubules are essential and highly stable core structures of centrioles, basal bodies, cilia, and flagella. In contrast to dynamic cytoplasmic micro-tubules, their luminal surface is coated with regularly arranged microtubule inner proteins (MIPs). However, the protein composition and biological function(s) of MIPs remain poorly understood. Using genetic, biochemical, and imaging techniques, we identified Tetrahymena RIB72A and RIB72B proteins as ciliary MIPs. Fluorescence imaging of tagged RIB72A and RIB72B showed that both proteins colocalize to Tetrahymena cilia and basal bodies but assemble independently. Cryoelectron tomography of RIB72A and/or RIB72B knockout strains revealed major structural defects in the ciliary A-tubule involving MIP1, MIP4, and MIP6 structures. The defects of individual mutants were complementary in the double mutant. All mutants had reduced swimming speed and ciliary beat frequencies, and high-speed video imaging revealed abnormal highly curved cilia during power stroke. Our results show that RIB72A and RIB72B are crucial for the structural assembly of ciliary A-tubule MIPs and are important for proper ciliary motility.

Figures

FIGURE 1:
FIGURE 1:
Schematic models showing the general organization of MIPs in the doublet microtubules and predicted structural motifs of T. thermophila RIB72A and RIB72B. (A) Simplified cross-sectional view of a cilium with nine DMTs surrounding the central pair complex (CPC); the viewing direction is from the proximal cilium end. Outer and inner dynein arms (ODA, IDA), and the nexin-dynein regulatory complex (N-DRC) connect neighboring DMTs, whereas the radial spokes (RS) connect to the CPC. One DMT is boxed and shown larger in B. (B) Schematic of a DMT with known axonemal MIPs and other microtubule-associated structures in cross-sectional view. Note that MIP5 in the B-tubule is not shown, as it is too small to be resolved by conventional cryo-ET. (C) Domain structures of T. thermophila RIB72A and RIB72B as predicted by NCBI BLAST, showing three DM-10 domains of unknown function for both proteins, and for RIB72A a C-terminal EF-hand motif. At, A-tubule; Bt, B-tubule; IJ, inner junction; M, membrane.
FIGURE 2:
FIGURE 2:
Fluorescence images reveal that Tetrahymena RIB72A and RIB72B colocalize to cilia and basal bodies. Fluorescence light microscopy images of fixed Tetrahymena cells expressing RIB72A or RIB72B tagged by fluorescent proteins along with basal body and microtubule markers. (A–C) RIB72A-mCherry (B) coexpressed with the basal body marker GFP-CEN1 (A) shows colocalization at basal bodies (C) in a WT background. (D–F) RIB72A-mCherry (E) coexpressed with the microtubule marker GFP-ATU1 (D) showing colocalization along the ciliary length (F) in a WT background. (G–I) Coexpressed GFP-RIB72A (G) and RIB72B-mCherry (H) showing colocalization at basal bodies and cilia (I) in a WT background. Cilia displaying signal from a single tagged Rib72 protein moved during imaging as live cells were used to limit fixation artifacts. Scale bar: 10 μm.
FIGURE 3:
FIGURE 3:
Gene knockouts of RIB72A and RIB72B decrease swimming velocity of Tetrahymena cells. (A) Loss of RIB72A or RIB72B decreases swimming velocity as compared with WT (100%): RIB72A-KO 66%, RIB72BKO 44%, RIB72A/B-KO 46% (n = 20 cells per stain). Error bars represent standard deviations and asterisks indicate statistical significance at p < 0.01 (one-way analysis of variance [ANOVA]). n.s. stands for nonsignificant, p > 0.05. (B) The percentage of cells recovering motility (>25 μm/s) after deciliation was recorded.
FIGURE 4:
FIGURE 4:
Chronographs based on high-speed video imaging show that loss of either RIB72A or RIB72B or both proteins strongly affects the motility of cilia. (A) Outline of the image analysis workflow, based on Funfak et al. (2015) as applied here to Tetrahymena (see Supplemental Figure S2 for details). As shown in the top panel, the first frame of a video was used to mark the cell body (blue) and cilia (green) regions. Numbers correspond to the positions along the cell circumference starting at the cell’s anterior tip and going around the cell in a counterclockwise direction. The bottom panel shows a single frame of a movie of an extracted and unwrapped cilia region. Numbers 1–5 correspond to the positions marked around the cell circumference as shown in the top panel. The red line shows the position which was plotted over time to produce the chronograph seen in B. (B–E) Chronographs of the ciliary zones from wild-type (B) (Supplemental Video S1), single (C, D) (Supplemental Videos S2 and S3) and double-knockout cells (E) (Supplemental Video S4). The numbers on the x-axis correspond to the positions around the cell circumference as indicated in A; the y-axis represents time. Red arrows show examples of single beat duration measurements as distances between two consecutive diagonal gray-scale lines representing the same cilium in consecutive beat cycles. Note the presence of stalled or pivoting cilia indicated by red boxes in C and E. (F) Average beat frequencies in the wild-type (38.1 + 3.2 Hz, n cilia = 45, n cells = 9), RIB72A-KO (29.3 + 3.1 Hz, n cilia = 48, n cells = 8), RIB72B-KO (27.6 + 4.6 Hz, n cilia = 64, n cells = 9), and RIB72AB-KO cells (23.3 + 4.3 Hz, n cilia = 50, n cells = 8) cells. Error bars represent standard deviations, and asterisks indicate statistical significance at p < 0.001 (one-way ANOVA). Abbreviations: oa, oral apparatus. Scale bars are 10 microns.
FIGURE 5:
FIGURE 5:
Losses of RIB72A and RIB72B affect the MIP1 structure differently. (A–L) Averaged tomographic slices (A, B, D, E, G, H, J, K) and isosurface renderings (C, F, I, L) in cross-sectional (A, D, G; from proximal) and longitudinal views (B, C, E, F, H, I; proximal on the right) of Tetrahymena axonemal repeat units of RIB72A-KO (D-F), RIB72B-KO (G–I), and the double mutant (J–L) showed different defects in the MIP1a (blue) and/or MIP1b (green) structure compared with WT (A–C). The RIB72A-KO mutant is missing MIP1a (open blue arrow) but not MIP1b (solid green arrow); the RIB72B-KO mutant lacks MIP1b (open green arrow) but not MIP1a (solid blue arrow). The double KO shows both defects, that is, MIP1a and MIP1b are missing (open blue and green arrows). Red dotted line in A indicates slice position of longitudinal tomographic slices, for example, in B. Other labels: At, A-tubule; Bt, B-tubule; IDA, inner dynein arms; ODA, outer dynein arms; RS, radial spokes. Scale bars: 25 nm (A, valid also for D, G), 16 nm (B, valid also for E, H).
FIGURE 6:
FIGURE 6:
Loss of RIB72A/B affects multiple MIP4 subunits. (A–L) Tomographic slices (A, B, D, E, G, H, J, K) and isosurface renderings (C, F, I, L) in cross-sectional (A, D, G, J; from proximal) and longitudinal views (B, C, E, F, H, I, K, L; proximal on the left) of Tetrahymena axonemal repeat units. RIB72A-KO (D-F), RIB72B-KO (G–I), and the double mutant (J–L) have various defects in the MIP4 structures (4a, rose; 4b, light orange; 4c, dark orange; 4d, yellow; 4e tomato) as compared with WT (A–C); missing/reduced structures are indicated by open arrows outlined by the respective colors for MIP4 subunits. The RIB72A mutant has defects in the MIP4b (light orange arrow), MIP4d (yellow arrow), and a slight defect in MIP4e (tomato arrow). The RIB72B mutant has defects only in the MIP4e subunit and no others. The defects in the double KO were additive, that is, MIP4b, 4d, and 4e are missing. Red dotted line in A indicates slice position of longitudinal tomographic slices, for example, in B. Other labels: At, A-tubule; Bt, B-tubule; IDA, inner dynein arms; ODA, outer dynein arms; RS, radial spoke; red, MIP2a/b. Scale bars: 25 nm (A, valid also for D, G, J), 16 nm (B, valid also for E, H, K).
FIGURE 7:
FIGURE 7:
Loss of RIB72A/B affects the MIP6 structure and shows that MIP6 is composed of multiple subunits. (A–L) Tomographic slices (A, B, D, E, G, H, J, K) and isosurface renderings (C, F, I, L) in cross-sectional (A, D, G, J; from proximal) and longitudinal bottom view (B, C, E, F, H, I, K, L; proximal on the left) of Tetrahymena axonemal repeat units. RIB72A-KO (D–F), RIB72B-KO (G–I), and the double mutant (J–L) each have defects in the MIP6 structures (6a, teal; 6b, aqua; 6c, purple; 6d, mauve) as compared with WT (A–C); missing structures are indicated by open arrows outlined by the respective colors for MIP6 subunits. The RIB72A mutant lacked MIP6b (aqua arrow) and MIP6c (purple arrow), whereas the RIB72B mutant was missing only MIP6a (teal arrow) and MIP6d (mauve arrow). The defects in the double KO were additive, that is, all four MIP6 subunits were missing. Red dotted line in A indicates slice position of longitudinal tomographic slices, for example, in B. Other labels: At, A-tubule; Bt, B-tubule; I1, I1 dynein; IDA, inner dynein arms; MIP3; N-DRC, nexin dynein regulatory complex; ODA, outer dynein arms. Scale bars: 25 nm (A, valid also for D, G, J), 16 nm (B, valid also for E, H, K).
FIGURE 8:
FIGURE 8:
RIB72BKO axonemes rescued with RIB72B-GFP show no structural defects and an additional tag density. (A–L) Tomographic slices (A–F) and isosurface renderings (G–L) in longitudinal views (in A, D, G, J proximal is on the right; in all remaining panels proximal is on the left) of Tetrahymena axonemal repeat units. The axonemal average from RIB72B-KO rescued with RIB72B-GFP (D-F, J-L) showed the same structures as seen in WT (A-C, G-I), that is, the mutant defects were rescued. Specifically, the loss of MIPs 1b, 4e, 6a, and 6d in the RIB72B-KO mutant (as seen in Figures 5H, empty green arrow; 6H, empty tomato arrow; and 7, G–I, empty teal and mauve arrows, respectively) is rescued by expression of RIB72B-GFP (green arrow and structure in D and J, tomato arrow and structure in L and F, and teal and mauve arrows and structures in E and K, respectively) and resemble the WT structures (green arrow in A, tomato arrow in C, and teal arrow in B, respectively). In addition, the extra density of the GFP-tag was visible in the vicinity of MIP4e in the rescued strain (green circles and densities in F and L) that was not observed in WT (compared with C and I). Slice positions of the tomographic slices are the same as in Figures 5 (for MIP1), 6 (for MIP4), and 7 (for MIP6); MIP structures are colored as follows: MIP1a blue, MIP1b green, MIP4a rose, MIP4b light orange, MIP4c dark orange, MIP4d yellow, MIP4e tomato, MIP6a teal, MIP6b aqua, MIP6c purple, MIP6d mauve. Other labels: At, A-tubule; Bt, B-tubule; I1, I1 dynein; IDA, inner dynein arms; MIP3; N-DRC, nexin dynein regulatory complex; ODA, outer dynein arms; RS, radial spokes; GFP, green fluorescent protein. Scale bar: 16 nm (valid A–F).
FIGURE 9:
FIGURE 9:
Summary schematics of MIP locations in the DMTs of WT (A), RIB72A-KO (B), RIB72B-KO (C), and RIB72A/B-KO (D) cilia. The structural comparison between the averaged DMTs of WT and mutant axonemes revealed that loss of RIB72A causes the loss of MIPs 1a (blue) 4b (light orange), 4d (yellow), 4e (tomato) (partially missing), 6b (aqua), and 6c (purple). The absence of RIB72B results in the loss of MIPs 1b (green), 4e (tomato), 6a (teal), and 6d (mauve). The defects in the double knockouts are additive. Coloring of all MIPs is shown in the color legend. Other labels: At, A-tubule; Bt, B-tubule; IDA, inner dynein arms; IJ, inner junction; N-DRC, nexin dynein regulatory complex; ODA, outer dynein arms; RS, radial spokes; 1–13, PF numbers.

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