A microtubule-dynein tethering complex regulates the axonemal inner dynein f (I1)

Abstract

Motility of cilia/flagella is generated by a coordinated activity of thousands of dyneins. Inner dynein arms (IDAs) are particularly important for the formation of ciliary/flagellar waveforms, but the molecular mechanism of IDA regulation is poorly understood. Here we show using cryoelectron tomography and biochemical analyses of Chlamydomonas flagella that a conserved protein FAP44 forms a complex that tethers IDA f (I1 dynein) head domains to the A-tubule of the axonemal outer doublet microtubule. In wild-type flagella, IDA f showed little nucleotide-dependent movement except for a tilt in the f β head perpendicular to the microtubule-sliding direction. In the absence of the tether complex, however, addition of ATP and vanadate caused a large conformational change in the IDA f head domains, suggesting that the movement of IDA f is mechanically restricted by the tether complex. Motility defects in flagella missing the tether demonstrates the importance of the IDA f-tether interaction in the regulation of ciliary/flagellar beating.

FIGURE 1:

Characterization of the fap44 mutants. (A) Schematic illustrations of the genomic DNA sequences of FAP44, FAP43, and FAP244 genes. The boxes and the lines represent the exons and the introns/untranslated regions, respectively. The arrowheads indicate the position of the paromomycin-resistance gene cassettes inserted for the mutagenesis. (B) Immunoblots showing the absence of FAP44 protein in the fap44 axoneme. The fap43 mutant retained the FAP44 protein. (C) Swimming velocities of the wild-type and the mutant strains. The fap44 cells showed reduced motility. Asterisks indicate statistically significant differences (p < 0.01, Student’s t test). No swimming cells were observed in the fap44oda2 double mutant. Means ± SD for the swimming velocities were calculated from 20 cells. (D) Waveforms of the wild-type and the mutant strains. The fap44 flagella showed a slight reduction in the bend amplitude. (E) Sliding disintegration assay of the axoneme. Microtubule sliding velocities were measured by observing the sliding of protease-treated and ATP-activated axonemes. Means ± SEM for the sliding velocities were calculated from indicated number of sliding events. The fap44 axoneme showed reduced microtubule sliding activity. Asterisks indicate the corresponding probability values calculated by Student’s t test. Although there is no statistically significant difference between the sliding velocities of ida5 and fap44ida5 axonemes, few sliding events were observed in fap44ida5 (thus, n is only 5), suggesting a motility defect in the double mutant.

FIGURE 2:

fap44 axoneme lacks the tether. (A) Cross-sectional views of the three-dimensional structure of the axoneme. The upper left inset is the base-to-tip view of the 9+2 structure. The red box indicates the position of the enlarged DMTs on the right. Red: tether; yellow: fα head; orange: fβ head; green: stalks. The directions of the views in B and C are indicated. RS: radial spoke; A and B: A-tubule and B-tubule, respectively. (B, C) Longitudinal views of the IDA f. The tether in red is missing in the fap44 axoneme. Red broken line indicates the shift in the fβ head toward the ODA. IC-LC: the IC-LC complex of the IDA f; a and b: the IDA subspecies a and b, respectively. The DMT structures are oriented with the distal ends on the right.

FIGURE 3:

Nucleotide-dependent conformational change of IDA f in three dimensions. The IDA f structures in the apo and the ATP plus vanadate (ATP+Va) states. (A) Wild type and (B) fap44 mutant. Red: tether; yellow: fα head; orange: fβ head; green: stalks. (A) The head and the stalk of the fβ show a tilt vertical to the long axis. (B) There are three states (I, II, and III) in the conformation of the IDA f in the presence of ATP+Va. Both fα and fβ heads show a large displacement away from the ODA.

FIGURE 4:

Two-dimensional slice representations of the nucleotide-dependent conformational change in IDA f. (A) Wild type and (B) fap44 mutant. Cross-sections are in base-to-tip views. Schematics showing the DMT, heads, and stalks are displayed on the left of the corresponding cross-sections. Yellow: fα head; orange: fβ head; green: stalk. Longitudinal sections, with the distal ends on the right, are placed next to the cross-sections. The orientations of the longitudinal sections are indicated with blue lines. Protofilaments in green indicate the expected position of the microtubule binding domain of the stalk. (C) Head and stalk orientations of the ODA and IDA e are shown in cross- and longitudinal sections.

FIGURE 5:

Functional redundancy between FAP43 and FAP244. (A) Schematic illustrations showing the domain organizations of FAP44, FAP43, and FAP244. DAB: Duffy antigen-binding domain; WD: WD domain (polygons represent the number of WD repeats); CC: coiled-coil domain. “WD?” in FAP244 indicates low confidence in the assignment of the WD domain due to the degeneration of the motif sequences. Blue arrowheads indicate the positions of the BCCP tags. (B) Three-dimensional structure of the DMT. fap43 and fap244 retain the tether structure (red), while fap43fap244 mutant lacks the tether. Expression of BCCP-tagged FAP43 restored the tether in fap43fap244. (C) Immunoblots showing that the fap43fap244 double mutant lacks fap44.

FIGURE 6:

Immunolocalization of FAP44 and FAP43 within the axoneme. The nucleoflagellar apparatus were stained with anti-FAP44 or anti-FAP43 antibody. White arrowheads indicate the distal ends of the flagella. FAP44 staining in the fap43 flagella is diminished near the distal ends. Red arrowheads indicate the proximal regions of the wild-type flagella lack FAP43 staining. In contrast, the whole lengths of the fap244 flagella were stained with anti-FAP43 antibody.

FIGURE 7:

Three-dimensional localizations of the termini of FAP44 and FAP43. Top, Tip-to-base view; bottom, distal end on the right. Tag densities were visualized by comparing the wild-type and labeled DMT structures. The broken circle indicates the RS stump (Pigino et al., 2011). Colored densities correspond to positions of the labels indicated by arrowheads of the same color in Figure 5A.

FIGURE 8:

Biochemical characterization of the tether complex. (A) Chromatographic separation of the axonemal salt extracts from FAP44C strain using an UnoQ anion-exchange column. The dynein species of each peak was identified according to the previous study (Furuta et al., 2009). FAP44 and FAP43 were detected using immunoblotting. Comigration of IDA f with FAP44 and FAP43 was clearly observed. (B) Immunoprecipitation of the FAP44 protein. Fractions containing purified IDA f, FAP44, and FAP43 were collected and BCCP-tagged FAP44 proteins were immunoprecipitated (IP) using streptavidin-agarose. As a negative control (biotin block), the streptavidin-agarose was blocked with 1 mM biocytin (biotin-lysine). IDA f was detected using the IC140 antibody. IDA f and FAP43 were co-immunoprecipitated with FAP44. (C) Sarkosyl fractionation of axonemal proteins. Axonemal proteins were extracted with various concentration of sarkosyl. Supernatant (sup) and precipitate (ppt) after centrifugation were analyzed by immunoblotting. Although the phenotypes of fap43 and fap244 are virtually wild type, extraction patterns showed destabilization of the tether complex.

FIGURE 9:

Schematic models of the IDA f-tether complex. Yellow ring: fα head; orange ring: fβ head; green strings: stalks; green sphere: stalk heads (microtubule binding domains of IDA f ); red rings: WD domains of FAP44 and FAP43; red strings: coiled-coil domains of FAP44 and FAP43; gray tubes: protofilamens of the axonemal microtubules. The tether complex is thought to be a heterodimer of FAP44 and FAP43 bundled via their carboxy-terminal coiled-coil domains, which are anchored to the A-tubule or to the 96-nm ruler complex. The tether complex holds both fα and fβ heads, and the nucleotide-dependent movement of the IDA f is restricted to a tilt in the fα head and stalk, which brings the microtubule binding domain to the adjacent protofilament.