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. 2013 Apr 15;201(2):263-78.
doi: 10.1083/jcb.201211048. Epub 2013 Apr 8.

The MIA Complex Is a Conserved and Novel Dynein Regulator Essential for Normal Ciliary Motility

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Free PMC article

The MIA Complex Is a Conserved and Novel Dynein Regulator Essential for Normal Ciliary Motility

Ryosuke Yamamoto et al. J Cell Biol. .
Free PMC article

Abstract

Axonemal dyneins must be precisely regulated and coordinated to produce ordered ciliary/flagellar motility, but how this is achieved is not understood. We analyzed two Chlamydomonas reinhardtii mutants, mia1 and mia2, which display slow swimming and low flagellar beat frequency. We found that the MIA1 and MIA2 genes encode conserved coiled-coil proteins, FAP100 and FAP73, respectively, which form the modifier of inner arms (MIA) complex in flagella. Cryo-electron tomography of mia mutant axonemes revealed that the MIA complex was located immediately distal to the intermediate/light chain complex of I1 dynein and structurally appeared to connect with the nexin-dynein regulatory complex. In axonemes from mutants that lack both the outer dynein arms and the MIA complex, I1 dynein failed to assemble, suggesting physical interactions between these three axonemal complexes and a role for the MIA complex in the stable assembly of I1 dynein. The MIA complex appears to regulate I1 dynein and possibly outer arm dyneins, which are both essential for normal motility.

Figures

Figure 1.
Figure 1.
Schematic model of motility regulation in C. reinhardtii axonemes. (A and B) Models depicting mechanical/chemical signaling pathways (dotted red arrows) in C. reinhardtii axonemes. Transverse (A) and longitudinal (B) sections are shown. Based on genetic and pharmacological experiments, signals are transmitted from the CP, through the RS1 to I1 dynein, and through RS2 to the CSC and N-DRC. Signals are also thought to be transmitted from I1 dynein and N-DRC to ODAs through OIDLs (blue lines in B). The question addressed here is how signals are transmitted to I1 dynein (question marks in A and B).
Figure 2.
Figure 2.
The MIA1 and MIA2 genes in C. reinhardtii encode conserved coiled-coil proteins. (A) The genomic structure of the MIA1 and MIA2 genes, showing untranslated regions in blue, exons in red, and introns as black lines. The mutations in the mia1 alleles and mia2 are indicated. (B) Sequence analyses of the mia mutants (mia1-2/317, mia1-3/623, mia1-4/834, and mia2) reveal the consequences resulting from the indicated mutations. Coloring indicates DNA bases (blue, cytosine; red, thymine; green, adenine; black, guanine). Asterisks indicate mutations sites.
Figure 3.
Figure 3.
The Mia1 and Mia2 proteins form the MIA complex. (A) Anti-FAP100 and -FAP73 antibodies detect bands in wild-type axonemes. Both bands are missing or greatly reduced in mia1, whereas only the FAP73 band is missing in mia2. CBB staining of tubulin from each sample is shown as a loading control. (B) Axonemes from HA-tagged rescued strains (mia1R and mia2R) assemble FAP100 (top) and FAP73 (bottom). The expressed proteins have slower migration as a result of the expression of the 3×HA tags. (C) Immunoprecipitations from mia1R extracts using the anti-HA antibody pull down an ∼76-kD protein (FAP100-HA) and an ∼35-kD protein (FAP73; both indicated by red arrows). Plus and minus signs indicate addition of beads with or without the anti-HA antibody. Immunoblots using the specific antibodies also show that these proteins were precipitated only in the mia1R extracts. (D) The Mia1p (FAP100; top) and Mia2p (FAP73; bottom) domain structures were predicted using the SMART and COILS programs. Both proteins show a high probability of forming coiled-coil domains in the middle part of the protein. The color coding in the structure prediction by SMART analysis: green, regions having a high probability of forming coiled-coil structure; pink, regions of low complexity; gray, areas with no detectable domains. The window width in the structure prediction by COILS version 2.2: green, 14 residues; blue, 21 residues; red, 28 residues. wt, wild type.
Figure 4.
Figure 4.
FAP100 and FAP73 assemble in various motility mutants and the mia mutants contain ODAs and IDAs. (A) Both FAP100 and FAP73 assemble in mutant axonemes that are missing the ODAs (oda1), I1 dynein (ida1), the single-headed IDAs a, c, d, and e (ida5), the N-DRC (pf3), the PP2A phosphatase (pf4), the RSs (pf14 and pf17), the CP (pf18), and the beaklike structures (mbo1 and mbo2). The proteins are also present in double mutants (oda1 × ida1, oda1 × ida2, and ida1 × ida5). (B) Immunoblots of subunits of the ODAs (IC69), I1 dynein (IC140 and IC97), the single-headed IDAs (actin and p28), and the RSs (RSP1) indicate these axonemal structures are apparently normal in both mia1 and mia2. The dynein subunits were detected on a single gel and blot, and RSP1 was detected on a second gel and blot. Ponceau staining of tubulin is shown as a loading control for each gel. (C) Silver-stained 3–5% urea gel of isolated axonemes demonstrates that dynein HC composition appears unaltered in mia mutants relative to wild type. Three lanes are from the same gel. The predicted molecular mass of I1 dynein HCs is the expected value: dynein HCs migrate significantly more slowly than the 250-kD molecular mass marker, which is shown well below the region of the gel shown. wt, wild type.
Figure 5.
Figure 5.
The MIA complex functions in the I1 dynein phosphoregulatory pathway and interacts with multiple axonemal proteins. (A) Untreated flagella (fla), untreated axonemes (axo), and axonemes treated with the kinase inhibitor DRB were probed with the anti-IC138 antibody. In the mia untreated axonemes, IC138 migrates in multiple forms in a manner similar to pf17. In contrast, wild-type axonemes show a compact IC138 profile. DRB treatment results in a shift in migration of IC138, indicating that these multiple forms of IC138 are caused by phosphorylation. (B) Immunoblot analyses indicate that CK1, PP2A (B and C subunits [sub]), and PP1 are assembled normally in the mia mutants. (C) Isolated wild-type axonemes treated with or without 5 mM EDC were probed with the FAP100 and FAP73 antibodies. Upon EDC exposure, both Mia proteins form some cross-linked products (red arrowheads), indicating that the MIA complex is in direct contact with multiple axonemal proteins. We could not detect the cross-linked products between I1 dynein subunits and the Mia proteins as solid bands on these blots. Black arrows indicate the un–cross-linked FAP100 and FAP73. Ab, antibody; wt, wild type.
Figure 6.
Figure 6.
The MIA complex is localized to a unique position in each 96-nm repeat. (A) Nucleoflagellar apparatuses from oda1 and mia1R × oda1 were stained with the anti-HA antibody. The HA antibody detects FAP100-HA in both flagella in the mia1R × oda1 strain (fluorescence image on the bottom). Very weak or no signal was detected in oda1 flagella that do not express the FAP100-HA protein. Both basal bodies and nuclei have some nonspecific staining. (top) Differential interference contrast images show that all cells possess flagella. Arrowheads indicate cis- and trans-flagella, showing that both flagella are staining. (B) Longitudinal tomographic slices of the averaged 96-nm axonemal repeats from DMTs 1–9 of pWT, mia2, mia1, and pf9-3/ida1 reveal structural defects in the mutant axonemes. The density of the I1 dynein (red outlines) is completely missing in pf9-3/ida1 (white outlines) and appears reduced in the mia mutants. These defects are more prominent in mia1 than in mia2. The densities of other major axonemal structures, such as the ODAs, single-headed inner dynein arms (IDAs 2–6 and X), and the N-DRC are not significantly changed in the mia mutants. (C) 3D isosurface renderings of the averaged 96-nm axonemal repeats from pWT, mia2, mia1, and pf9-3/ida1 reveal the structural defects in more detail. Regions that are reduced in the mia averages are colored green; the most obvious defects are the distal density of the IC/LC of I1 dynein (orange) and the density between the I1 dynein and the N-DRC (yellow), which is reduced in mia2, missing in mia1, but fully assembled in pWT and pf9-3/ida1. The pWT and pf9-3/ida1 data were refined from data originally reported by Heuser et al. (2012b). Proximal is on the left in B and C. Bars: (A) 10 µm; (B) 25 nm.
Figure 7.
Figure 7.
The MIA complex affects I1 dynein stability in the axoneme. (A) I1 dynein is missing along the entire length of DMT1 in the mia mutants. Tomographic slices of axonemal average of individual outer DMTs from pWT (top row), mia2 (middle row), and mia1 (bottom row). The first two columns show the axonemal repeat of DMT1 in the proximal (left) and distal (middle) regions; the right column depicts the combined average of the axonemal repeats from DMTs 2–9 that do not show obvious structural differences between the proximal and distal regions (for individual DMT averages, see Fig. S3). In wild type, I1 dynein is missing from the proximal region of DMT1 (white outlines) but is present in the distal region (red outlines). In contrast, in both mia mutants, I1 dynein is missing along the entire length of DMT1 (white outlines). The pWT data were refined from data originally reported by Heuser et al. (2009) and Lin et al. (2012b). Other structures labeled are single-headed inner dynein arms (IDAs 2–6 and X), the N-DRC, and the inner SUB5-6 bridge. Bars, 25 nm. (B) Representative ion-exchange chromatography (Mono Q; GE Healthcare) elution profiles of axonemal dyneins show that I1 dynein levels (black arrows) appear relatively normal in mia1-2 extracts compared with that of wild-type extracts (left graph), consistent with the immunoblotting results using I1 dynein subunit antibodies (Fig. 4 B). In contrast, I1 dynein is drastically reduced in the mia1-2 × oda6 double mutant (red arrow) compared with the oda1 single mutant (right graph), suggesting that I1 dynein becomes unstable without both ODAs and the MIA complex. Typical Mono Q elution patterns are shown (A280 protein estimate in arbitrary units is indicated to the right of each scan [y axis]; elution time in minutes is shown on the x axis), and at least two experiments were performed for each strain. Elution of dynein complexes began at ∼12 min. (C) Immunoblot analysis of isolated axonemes using the anti-DHC1 (I1 dynein HC-α) and IC97 antibodies confirms the reduction of I1 dynein in mia × oda double mutants. I1 dynein appears normal to slightly reduced in the single mia mutants relative to wild type (wt).
Figure 8.
Figure 8.
Regulation of dynein-driven microtubule sliding is disrupted in the mia mutants. ATP- and protease-induced microtubule sliding measurements show the characteristic sliding velocities for wild-type (wt) and pf17 mutant axonemes. The reduced sliding velocity of pf17 is rescued to wild-type levels by the addition of a kinase inhibitor (KI). Microtubule sliding velocities of mia1 (mia1-1 and mia1-3) are similar or only slightly reduced relative to that of wild type but greatly reduced when coupled with the pf17 mutation (mia1-1 × pf17). Microtubule sliding velocities of mia2 axonemes are significantly reduced and are not altered by kinase inhibitor treatment (mia2 + kinase inhibitor). Error bars show standard deviations (n = 4–36).
Figure 9.
Figure 9.
Proposed model for the function of the MIA complex in regulation of I1 dynein. (A) In wild type, the MIA complex is localized in a position to mediate signals to I1 dynein motor complex (orange). Kinases and phosphatases (white in A and red in B), such as CK1, PKA, and PP2A, are predicted to be positioned at the base of the I1 dynein complex, in position to regulate phosphorylation of the IC138 substrate of I1 dynein. (B) In the absence of the MIA complex, these signaling proteins (red) are present but may be mispositioned relative to IC138 and I1 dynein, which are flexible, resulting in hyperphosphorylated IC138 in the mia mutants. KIP, kinase–phosphatase complex; p, phosphorylation.

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