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A dynein-associated photoreceptor protein prevents ciliary acclimation to blue light

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Science Advances  26 Feb 2021:
Vol. 7, no. 9, eabf3621
DOI: 10.1126/sciadv.abf3621
  • Fig. 1 Characterization of DYBLUP from sperm flagella of the ascidian C. intestinalis.

    (A) Sucrose density gradient centrifugation of high salt/ATP extract from flagellar axonemes. Proteins separated in 6% SDS gels and corresponding Western blots using antibodies against orthologs of Chlamydomonas IC140 (IC116), IC138 (IC110), and IC97 (IC105) are shown. The bar represents the fractions subjected to Uno Q anion-exchange column chromatography (fig. S1B). (B) Electron micrographs of the negatively stained OAD and IAD f/I1 dynein. Both show two-headed structures but differ in the shape of their tails. Schematic drawings are shown on the right. Scale bars, 20 nm. (C) Subunit composition of Ciona f/I1 dynein. The 32-kDa protein DYBLUP is indicated in red. (D) Labeling of f/I1 dynein with anti-DYBLUP IgG. The images in the top and bottom rows correspond to two opposite orientations when the molecules are adsorbed to the carbon support film. The labeled motor domain is located on the convex side of the tail surface. Schematic drawings are shown on the right. Scale bar, 20 nm. (E) Images of IgG molecule. Scale bar, 20 nm. (F) Localization of DYBLUP on the 1β motor domain. The f/I1 dynein image extracted from the cryo-ET structure of an axoneme [EMD-5330; (18)] is oriented as in the top row of (D). Comparison with the IgG-labeled negative-stain images specifies the position of the label on the motor domain. Scale bar, 10 nm.

  • Fig. 2 Characterization of the DYBLUP ortholog MOT7 from flagella of the green alga C. reinhardtii.

    (A) Western blot analysis of MOT7 in the axonemes of the WT and several mutants. MOT7 is present in mutant axonemes with a defect in OAD (oda1 and oda6), two-headed IAD f/I1 (ida1), several single-headed IADs (ida4), most IADs (pf23), the modifier of inner arms (MIA) complex (mia2), the nexin-dynein regulatory complex (N-DRC) complex (pf3), radial spokes (pf14), and the central-pair complex (pf18). Loadings (tubulin bands, Coomassie Brilliant Blue) and the Western blot with anti-MOT7 are shown. (B) Western blot analysis of dynein subunits in axonemes of the WT and mot7 mutant. The mot7 mutant has no apparent axonemal defect in IC2 (OAD), p38 (IAD d), p28 (IADs a, c, d), FAP120, or IC138 (IAD f/I1) but lacks MOT7 (red arrowhead). (C) Immunofluorescence image showing MOT7 along the flagella. Images of staining with anti-HA antibody (left) and anti–acetylated α-tubulin antibody (right) are shown. The flagella of the mot7-rescue mutant expressing MOT7-BCCP-3HA are recognized with anti-HA antibody. The strong fluorescence in the cell body is autofluorescence. Scale bar, 20 μm. (D) Immunoblots of f/I1 dynein purified with a Mono Q column showing the presence of MOT7 in the WT strain and the oda6 and ida1 mutants. (E) Immunoprecipitation of axonemal extract with anti–HA-tag antibody. Four bands (red arrowheads) are specifically immunoprecipitated with antibody from the mot7-rescue mutant expressing MOT7-BCCP-3HA. Two of these (numbers 2 and 4) were identified as FAP44, a tether complex protein associated with the f/I1 motor domain (see table S2). (F) IC138 is hyperphosphorylated in the mot7 mutant. Western blots of axonemes from the WT strain and mot7 mutant using anti-IC138 antibody are shown. Phosphorylation was induced by addition of ATP and cAMP to the isolated axonemes. Phosphorylated IC138 bands are indicated by red dots. CIAP, calf intestine alkaline phosphatase.

  • Fig. 3 Cryo-ET showing the localization of MOT7 in the link structure between the tether complex and f/I1-1β motor domain.

    (A) Isosurface rendering longitudinal images of the axonemes from the WT C. reinhardtii strain (left), mot7 mutant (middle), and the mutant rescued with MOT7-BCCP-3HA (right). The 1α and 1β motor domains of f/I1dynein (58), the tether complex, and the enhanced biotin carboxyl carrier protein (BCCP) tag are indicated in orange, blue, red, and green, respectively. BCCP is associated with both the 1β domain and the tether complex. A part of the tether structure is absent in the mot7 mutant. (B) Tomographic slices of the averaged longitudinal view in axonemes from the WT strain (left), mot7 mutant (middle), and the mutant rescued with MOT7-BCCP-3HA (right). The density of BCCP is clearly seen between f/I1 dynein and the tether complex. The amount of tether complex is significantly decreased in the mot7 mutant, likely due to structural fluctuation. (C) Detailed comparison of isosurface rendering images. The WT strain (left), mot7 mutant (middle), and the mutant rescued with MOT7-BCCP-3HA (right) are shown. Two links between the f/I1 motor domain and the tether complex are colored in dark pink. The link from the tether to the 1β domain is lost in the mot7 mutant, but that from the tether to the 1α domain is retained.

  • Fig. 4 MOT7-deficient C. reinhardtii mutant shows acclimation to blue light.

    (A) Positive phototactic behavior induced by weak blue light is seen for both the WT strain and the mot7 mutant. (B) Negative phototaxis induced by strong blue light is also seen for both the WT strain and the mot7 mutant. However, the mot7 mutant soon acclimates and turns to the blue light (see also fig. S6A). This acclimation is significantly delayed in the mutant rescued with MOT7-3HA. (C) Irradiation with strong blue light in the center of a microscopic field under red light illumination over the full field. The area of blue light irradiation is shown with a blue line. mot7 mutants become accumulated to the blue light after a time, but the WT strain avoided the area. Scale bar, 50 μm. (D) Accumulation of Chlamydomonas at the opposite end (black bar) of a chamber to avoid strong blue light. WT cells stay and accumulate at the opposite end from the light, whereas mot7 mutant cells initially accumulate at that end but soon turn around toward the blue light, resulting in dispersion of the cell mass. Scale bar, 200 μm. (E) Swimming trajectories of WT cells and the mot7 mutant around the edge of the chamber, recorded for 20 s at 9 min after irradiation. (F) Circular histogram showing the increase in the number of mot7 mutants that swim toward the blue light. Swimming directions 9 min after irradiation are indicated in 18 sections with the bin of 20°; n = 421 WT and 397 mutant cells. Arrows show the direction of blue light. (G) Duration of swimming in a straight path toward the blue light 3 and 9 min after irradiation; n = 30 WT and 31 mutant cells. **P < 0.01 and ***P < 0.001 versus WT.

  • Fig. 5 Schematic model of MOT7-mediated avoidance of blue light acclimation.

    (A) In WT Chlamydomonas flagella, the motor activity of IAD f/I1 is regulated by the proper arrangement of motors and regulatory components, such as the tether complex, protein kinase CK1, and protein phosphatase PP1/2A. The molecular interactions among these components are essential for positive and negative phototaxis and suppression of light acclimation. MOT7 links the 1β motor domain and the tether component FAP44, regulating the motor activity for avoidance of acclimation to strong blue light. (B) The fap44 mutant lacks the entire tether structure but shows both positive and negative phototaxis, although these responses are weak compared with the WT strain. The lack of the tether complex decreases the binding of CK1 to the doublet microtubule, causing hyperphosphorylation of IC138. (C) In the mot7 mutant, the link between the 1β motor and the tether complex is missing, although that between the 1α motor and the tether complex is present. IC138 is constitutively hyperphosphorylated due to decreased binding of CK1 to the microtubule. The mutant shows both positive and negative phototaxis but becomes acclimated to strong light. The extent of three photoresponses is indicated on the right. The motility regulation of the two flagella during positive phototaxis is associated with anchoring of the 1α motor, but not 1β motor, to the tether complex and not related to dephosphorylation of IC138. Negative phototaxis does not require the tether-f/I1 connection or regulation of IAD f/I1 activity by IC138 dephosphorylation. Regarding the acclimation to strong blue light, MOT7-mediated binding of 1β motor to the tether complex is required for unbalanced flagellar beating, which prevents the cells from swimming toward the light source. The anchoring of 1α by the tether structure is thought to promote light acclimation.

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