Journal: Mol Biol Cell / Year: 2017 Title: Electrostatic interaction between polyglutamylated tubulin and the nexin-dynein regulatory complex regulates flagellar motility. Authors: Tomohiro Kubo / Toshiyuki Oda / Abstract: Tubulins undergo various posttranslational modifications. Among them, polyglutamylation is involved in the motility of eukaryotic flagella and the stability of the axonemal microtubules. However, it ...Tubulins undergo various posttranslational modifications. Among them, polyglutamylation is involved in the motility of eukaryotic flagella and the stability of the axonemal microtubules. However, it remains unclear where polyglutamylated tubulin localizes precisely within the axoneme and how tubulin polyglutamylation affects flagellar motility. In this study, we identified the three-dimensional localization of the polyglutamylated tubulin in flagella using antibody labeling and cryo-electron tomography. Polyglutamylated tubulins specifically located in close proximity to a microtubule-cross-bridging structure called the nexin-dynein regulatory complex (N-DRC). Because N-DRC is positively charged, we hypothesized that there is an electrostatic interaction between the polyglutamylated tubulin and the N-DRC, and therefore we mutated the amino acid sequences of DRC4 to modify the charge of the N-DRC. We found that both augmentation and reduction of the positive charge on DRC4 resulted in reduced flagellar motility Moreover, reduced motility in a mutant with a structurally defective N-DRC was partially restored by increasing the positive charge on DRC4. These results clearly indicate that beating motion of flagella is maintained by the electrostatic cross-bridge formed between the negatively charged polyglutamylated tubulins and the positively charged N-DRC.
History
Deposition
Jun 12, 2017
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Header (metadata) release
Jul 12, 2017
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Map release
Jul 12, 2017
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Update
Jun 20, 2018
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Current status
Jun 20, 2018
Processing site: PDBj / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
pH: 7.2 Details: 30 mM Hepes-NaOH pH 7.2, 5 mM MgCl2, 1 mM dithiothreitol, 1 mM EGTA, 50 mM NaCl
Grid
Model: Homemade / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 5.5 seconds before plunging.
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Electron microscopy
Microscope
JEOL 3100FEF
Specialist optics
Energy filter - Name: Omega / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Image recording
Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number grids imaged: 1 / Average exposure time: 1.0 sec. / Average electron dose: 1.7 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron optics
Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Sample stage
Specimen holder model: GATAN 914 HIGH TILT LIQUID NITROGEN CRYO TRANSFER TOMOGRAPHY HOLDER Cooling holder cryogen: NITROGEN
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Image processing
Final reconstruction
Resolution.type: BY AUTHOR / Resolution: 50.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: PEET / Number subtomograms used: 766
Extraction
Number tomograms: 8 / Number images used: 892 / Software - Name: PEET
CTF correction
Software - Name: IMOD
Final angle assignment
Type: NOT APPLICABLE / Software - Name: PEET
FSC plot (resolution estimation)
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