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Cited by 140Genetic and Kidney Cyst DiseasesMicrotubule and mitosis dynamicsCystic Fibrosis Research Advances

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Inner lumen proteins stabilize doublet microtubules in cilia and flagella

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Owa, Mikito, Uchihashi, Takayuki, Yanagisawa, Haru-aki, Yamano, Takashi, Iguchi, Hiro, Fukuzawa, Hideya, Wakabayashi, Ken-ichi, Ando, Toshio, Kikkawa, Masahide·2019·Nature Communications
DOI 10.1038/s41467-019-09051-xPMC PMC6408466

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Abstract

Motile cilia are microtubule-based organelles that play important roles in most eukaryotes. Although axonemal microtubules are sufficiently stable to withstand their beating motion, it remains unknown how they are stabilized while serving as tracks for axonemal dyneins. To address this question, we have identified two uncharacterized proteins, FAP45 and FAP52, as microtubule inner proteins (MIPs) in Chlamydomonas . These proteins are conserved among eukaryotes with motile cilia. Using cryo-electron tomography (cryo-ET) and high-speed atomic force microscopy (HS-AFM), we show that lack of these proteins leads to a loss of inner protrusions in B-tubules and less stable microtubules. These protrusions are located near the inner junctions of doublet microtubules and lack of both FAP52 and a known inner junction protein FAP20 results in detachment of the B-tubule from the A-tubule, as well as flagellar shortening. These results demonstrate that FAP45 and FAP52 bind to the inside of microtubules and stabilize ciliary axonemes.

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Fig. 1 Characteristics of the FAP45 and FAP52 null mutants. a Top: a transverse view of the 9+2 structure of the Chlamydomonas axoneme. Scale bar = 50 nm. Bottom: a magnified DMT. Arrowheads indicate MIPs. Major structures are colored; Outer dynein arm (ODA, magenta); Inner dynein arm (IDA, green); Dynein regulatory complex (DRC, purple); Radial spoke (RS, blue). IJ inner junction, OJ outer junction. Scale bar = 25 nm. b Western blot analyses of wild type, fap45, fap52, and fap45fap52 double mutant axonemes stained with various antibodies. FAP45 and FAP52 proteins were not detected in fap45 and fap52, respectively. Proteins essential for flagellar motility (ODA-IC2: outer dynein arm-intermediate chain 2; IDA-IC140: inner dynein arm-intermediate chain 140; IDA-p28: inner dynein arm-light chain p28; RSP1: radial spoke protein 1; DRC2 and 4: dynein regulatory complex 2 and 4; FAP20: inner junction protein of DMT) were not reduced in the mutants. c, d Axonemes from wild type and mutant Chlamydomonas crosslinked using EDC (zero-length crosslinker) were immunoblotted with anti-FAP45 (c) and anti-FAP52 antibodies (d). Filled arrowheads indicate the crosslinked product of FAP45 and FAP52 in a 1:1 ratio. The open arrowhead indicates the crosslinked product of FAP45 and tubulin. e–g Motility phenotypes of the mutants were assayed using the CLONA system. Swimming velocity and beat frequency were slightly reduced in fap45, whereas no significant reduction was observed in fap52. fap45fap52 showed a more severe phenotype than did fap45
Fig. 1 Characteristics of the FAP45 and FAP52 null mutants. a Top: a transverse view of the 9+2 structure of the Chlamydomonas axoneme. Scale bar = 50 nm. Bottom: a magnified DMT. Arrowheads indicate MIPs. Major structures are colored; Outer dynein arm (ODA, magenta); Inner dynein arm (IDA, green); Dynein regulatory complex (DRC, purple); Radial spoke (RS, blue). IJ inner junction, OJ outer junction. Scale bar = 25 nm. b Western blot analyses of wild type, fap45, fap52, and fap45fap52 double mutant axonemes stained with various antibodies. FAP45 and FAP52 proteins were not detected in fap45 and fap52, respectively. Proteins essential for flagellar motility (ODA-IC2: outer dynein arm-intermediate chain 2; IDA-IC140: inner dynein arm-intermediate chain 140; IDA-p28: inner dynein arm-light chain p28; RSP1: radial spoke protein 1; DRC2 and 4: dynein regulatory complex 2 and 4; FAP20: inner junction protein of DMT) were not reduced in the mutants. c, d Axonemes from wild type and mutant Chlamydomonas crosslinked using EDC (zero-length crosslinker) were immunoblotted with anti-FAP45 (c) and anti-FAP52 antibodies (d). Filled arrowheads indicate the crosslinked product of FAP45 and FAP52 in a 1:1 ratio. The open arrowhead indicates the crosslinked product of FAP45 and tubulin. e–g Motility phenotypes of the mutants were assayed using the CLONA system. Swimming velocity and beat frequency were slightly reduced in fap45, whereas no significant reduction was observed in fap52. fap45fap52 showed a more severe phenotype than did fap45CC BY · PubMed Central
Fig. 2 Dynein b density is reduced in the fap45 and fap52 DMT. Isosurface renderings of averaged DMT repeats: wild type (a), fap45 (b), and fap52 (c). The density of dynein b is decreased in fap45 and fap52 (arrowheads). Other species of IDAs, ODAs, RS, and N-DRC were properly arranged on the DMT in fap45 and fap52. Scale bar = 25 nm
Fig. 2 Dynein b density is reduced in the fap45 and fap52 DMT. Isosurface renderings of averaged DMT repeats: wild type (a), fap45 (b), and fap52 (c). The density of dynein b is decreased in fap45 and fap52 (arrowheads). Other species of IDAs, ODAs, RS, and N-DRC were properly arranged on the DMT in fap45 and fap52. Scale bar = 25 nmCC BY · PubMed Central
Fig. 3 fap45 and fap52 mutant have structural defects in B-tubules. Isosurface renderings of averaged axonemal 96-nm repeats from wild type (a, d), fap45 (b, e), and fap52 (c, f). (a–c) are cross-sectional and oblique, and (d–f) are longitudinal views. The black dashed lines in (a–c) indicate the slicing plane of (d–f). Protofilament numbers are labeled in (a) and (d–f). Missing structures in fap45 and fap52 were determined by Student’s t-tests as previously described33. The t-value maps acquired by the t-tests were overlaid on the renderings of fap45 (e, light green) and fap52 (f, light yellow). In this figure, MIP3a and b were colored by yellow and orange, respectively. MIP3c (the missing structure in fap45) was colored by green, according to the t-value map shown in (e). Scale bar = 25 nm
Fig. 3 fap45 and fap52 mutant have structural defects in B-tubules. Isosurface renderings of averaged axonemal 96-nm repeats from wild type (a, d), fap45 (b, e), and fap52 (c, f). (a–c) are cross-sectional and oblique, and (d–f) are longitudinal views. The black dashed lines in (a–c) indicate the slicing plane of (d–f). Protofilament numbers are labeled in (a) and (d–f). Missing structures in fap45 and fap52 were determined by Student’s t-tests as previously described33. The t-value maps acquired by the t-tests were overlaid on the renderings of fap45 (e, light green) and fap52 (f, light yellow). In this figure, MIP3a and b were colored by yellow and orange, respectively. MIP3c (the missing structure in fap45) was colored by green, according to the t-value map shown in (e). Scale bar = 25 nmCC BY · PubMed Central
Fig. 4 B-tubules are partially depolymerized in fap45fap52. a, b A typical tomogram of a fap45fap52 axoneme in cross-sectional (a) and longitudinal view (b). A dashed line in (a) indicates the slicing plane of (b). Arrowheads in (b) show break points in the B-tubules. c Typical thin section TEM images of fap45fap52 axonemes. Broken B-tubules were observed in 33.8% of the DMTs (n = 552). The 9+2 structure was disorganized and the axonemes were frayed in regions containing broken B-tubules. Scale bar = 100 nm
Fig. 4 B-tubules are partially depolymerized in fap45fap52. a, b A typical tomogram of a fap45fap52 axoneme in cross-sectional (a) and longitudinal view (b). A dashed line in (a) indicates the slicing plane of (b). Arrowheads in (b) show break points in the B-tubules. c Typical thin section TEM images of fap45fap52 axonemes. Broken B-tubules were observed in 33.8% of the DMTs (n = 552). The 9+2 structure was disorganized and the axonemes were frayed in regions containing broken B-tubules. Scale bar = 100 nmCC BY · PubMed Central
Fig. 5 Direct observation of B-tubule depolymerization by HS-AFM. a Schematic of high speed-AFM observation of the DMT. The tip of the cantilever (tip diameter ~1–2 nm) intermittently tapped the DMT surface on a mica stage. At time 0 s, the tip of the cantilever was thrust into the B-tubule and made a small hole (circled in red in b and c), immediately followed by normal HS-AFM observation. b, c Representative time-lapse images of wild type (b) and fap45fap52 (c) DMTs acquired by HS-AFM. In the wild type, enlargement of the hole usually stopped within a few seconds, whereas most of the B-tubules in the field were broken in fap45fap52 by 40 s. Scale bar = 100 nm. d The average length of the depolymerized B-tubules was plotted against time (wild type: n = 11, total length = 7.25 μm; fap45: n = 8, total length = 4.92 μm; fap52: n = 7, total length = 3.83 μm; fap45fap52: n = 10, total length = 4.95 μm)
Fig. 5 Direct observation of B-tubule depolymerization by HS-AFM. a Schematic of high speed-AFM observation of the DMT. The tip of the cantilever (tip diameter ~1–2 nm) intermittently tapped the DMT surface on a mica stage. At time 0 s, the tip of the cantilever was thrust into the B-tubule and made a small hole (circled in red in b and c), immediately followed by normal HS-AFM observation. b, c Representative time-lapse images of wild type (b) and fap45fap52 (c) DMTs acquired by HS-AFM. In the wild type, enlargement of the hole usually stopped within a few seconds, whereas most of the B-tubules in the field were broken in fap45fap52 by 40 s. Scale bar = 100 nm. d The average length of the depolymerized B-tubules was plotted against time (wild type: n = 11, total length = 7.25 μm; fap45: n = 8, total length = 4.92 μm; fap52: n = 7, total length = 3.83 μm; fap45fap52: n = 10, total length = 4.95 μm)CC BY · PubMed Central
Fig. 6 MIP3a is important for B-tubule anchoring to the A-tubule at inner junctions. a Differential interference contrast (DIC) images of wild type, fap20, fap20fap45, fap20fap52, and fap20fap45fap52 cells. Scale bar = 10 μm. b Box and whisker plots of flagellar length in wild type, fap20, fap20fap45, and fap20fap52. fap20fap52 and fap20fap45fap52 flagella were significantly shorter than in wild type, fap20, and fap20fap45. The whiskers represent 95% confidence intervals. The box represents the interquartile range and the center line shows the median. c Cross-sectional EM images of fap20, fap20fap45, fap20fap52, and fap20fap45fap52 axonemes. Arrowheads indicate disconnections between the A-tubule and B-tubule. Scale bar = 100 nm. d Cross-sectional images of fap20, fap20fap45, fap20fap52, and fap20fap45fap52 DMTs. Scale bar = 50 nm
Fig. 6 MIP3a is important for B-tubule anchoring to the A-tubule at inner junctions. a Differential interference contrast (DIC) images of wild type, fap20, fap20fap45, fap20fap52, and fap20fap45fap52 cells. Scale bar = 10 μm. b Box and whisker plots of flagellar length in wild type, fap20, fap20fap45, and fap20fap52. fap20fap52 and fap20fap45fap52 flagella were significantly shorter than in wild type, fap20, and fap20fap45. The whiskers represent 95% confidence intervals. The box represents the interquartile range and the center line shows the median. c Cross-sectional EM images of fap20, fap20fap45, fap20fap52, and fap20fap45fap52 axonemes. Arrowheads indicate disconnections between the A-tubule and B-tubule. Scale bar = 100 nm. d Cross-sectional images of fap20, fap20fap45, fap20fap52, and fap20fap45fap52 DMTs. Scale bar = 50 nmCC BY · PubMed Central
Fig. 7 A schematic model of MIP3. a Isosurface rendering of the wild type density map. MIP3a-c is highlighted as shown in Fig. 2 (MIP3a: yellow; MIP3b: orange; MIP3c: green). The right map is the longitudinal view sliced by the line in the left map. A possible unit of MIP3c is circled in red. Scale bar = 25 nm. b A model of DMT stabilization. FAP45 (green) and FAP52 (yellow) are localized on the inner lumen of the B-tubule and stabilize the MT wall. In particular, FAP45 ties protofilaments B7–B9. FAP52 anchors B9 and B10 to A13 and binds to FAP45. Thus, these proteins strengthen the MT wall against DMT-bending. Loss of both proteins leads to destabilization of the B-tubule and the B-tubule is depolymerized by bending
Fig. 7 A schematic model of MIP3. a Isosurface rendering of the wild type density map. MIP3a-c is highlighted as shown in Fig. 2 (MIP3a: yellow; MIP3b: orange; MIP3c: green). The right map is the longitudinal view sliced by the line in the left map. A possible unit of MIP3c is circled in red. Scale bar = 25 nm. b A model of DMT stabilization. FAP45 (green) and FAP52 (yellow) are localized on the inner lumen of the B-tubule and stabilize the MT wall. In particular, FAP45 ties protofilaments B7–B9. FAP52 anchors B9 and B10 to A13 and binds to FAP45. Thus, these proteins strengthen the MT wall against DMT-bending. Loss of both proteins leads to destabilization of the B-tubule and the B-tubule is depolymerized by bendingCC BY · PubMed Central

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  1. Harris, E. in The Chlamydomonas Sourcebook Vol. 2 (Elsevier, San Diego, CA, 2009).

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Authors
Owa, M., Uchihashi, T., Yanagisawa, H.-A., Yamano, T., Iguchi, H., Fukuzawa, H., Wakabayashi, K.-I., Ando, T., & Kikkawa, M.
Affiliations
  • The University of Tokyo (JP)
  • Nagoya University (JP)
  • Kyoto University (JP)
  • Tokyo Institute of Technology (JP)
  • Kanazawa University (JP)
Corresponding author
Kikkawa, Masahide
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2019-03-08
Volume
10
Issue
1
Pages
1143-1143
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-019-09051-x
PMC
PMC6408466
OpenAlex
W2885457519
Cited by
140
References
61
Citations by year
2026: 6 · 2025: 17 · 2024: 12 · 2023: 32 · 2022: 20 · 2021: 13 · 2020: 22 · 2019: 17
FWCI
10.81
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Institutional Repositories DataBase (IRDB) (submittedVersion, cc-by)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Kyoto University Research Information Repository (Kyoto University) (submittedVersion, cc-by)
Funders
  • Japan Society for the Promotion of Science (17H06121, 25120714, #26119003, 18H01837, 16H04805, 15H01206, CREST, 16K07399)
  • Japan Science and Technology Agency (CREST, JPMJAL1105)
  • Core Research for Evolutional Science and Technology
  • Advanced Low Carbon Technology Research and Development Program (JPMJAL1105)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetic and Kidney Cyst Diseases
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Microtubule and mitosis dynamics
  • Health Sciences › Medicine › Pulmonary and Respiratory Medicine › Cystic Fibrosis Research Advances
Keywords
microtubule inner proteins, doublet microtubules, motile cilia, flagella, Chlamydomonas
MeSH terms
Cilia, Flagella, Gene Expression, Chlamydomonas reinhardtii, Microscopy, Atomic Force, Cryoelectron Microscopy, Algal Proteins, Axoneme, Electron Microscope Tomography, Axonemal Dyneins

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