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. 2015 Aug 1;26(15):2788-800.
doi: 10.1091/mbc.E15-01-0018. Epub 2015 Jun 10.

DRC3 Connects the N-DRC to Dynein G to Regulate Flagellar Waveform

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Free PMC article

DRC3 Connects the N-DRC to Dynein G to Regulate Flagellar Waveform

Junya Awata et al. Mol Biol Cell. .
Free PMC article

Abstract

The nexin-dynein regulatory complex (N-DRC), which is a major hub for the control of flagellar motility, contains at least 11 different subunits. A major challenge is to determine the location and function of each of these subunits within the N-DRC. We characterized a Chlamydomonas mutant defective in the N-DRC subunit DRC3. Of the known N-DRC subunits, the drc3 mutant is missing only DRC3. Like other N-DRC mutants, the drc3 mutant has a defect in flagellar motility. However, in contrast to other mutations affecting the N-DRC, drc3 does not suppress flagellar paralysis caused by loss of radial spokes. Cryo-electron tomography revealed that the drc3 mutant lacks a portion of the N-DRC linker domain, including the L1 protrusion, part of the distal lobe, and the connection between these two structures, thus localizing DRC3 to this part of the N-DRC. This and additional considerations enable us to assign DRC3 to the L1 protrusion. Because the L1 protrusion is the only non-dynein structure in contact with the dynein g motor domain in wild-type axonemes and this is the only N-DRC-dynein connection missing in the drc3 mutant, we conclude that DRC3 interacts with dynein g to regulate flagellar waveform.

Figures

FIGURE 1:
FIGURE 1:
The architecture of the N-DRC in the Chlamydomonas wild-type axoneme. (A) Three-dimensional isosurface rendering of the averaged 96-nm repeat shows the nexin linker (green) of the N-DRC and other axonemal structures (gray) in a longitudinal front view (proximal is on the left). (B) Magnified surface rendering of the N-DRC shows the nexin linker (green) and the base plate (blue) in a longitudinal bottom view (from the central pair toward the doublet microtubule). For better visualization of the N-DRC, other axonemal structures are not shown in B. Arrowheads indicate OID linkers. a–g, inner arms a–g; IDA, inner dynein arm; L1, L1 protrusion; L2, L2 protrusion; ODA, outer dynein arm; RS1 and RS2, radial spokes 1 and 2.
FIGURE 2:
FIGURE 2:
Characterization of Chlamydomonas drc3 mutant. (A) Diagram of Chlamydomonas DRC3 gene. The black rectangles are exons. Double-headed arrows indicate the positions of PCR products used to delimit the deleted region; plus and minus marks indicate whether the PCR products were amplified in the wild type (WT) or the mutant (drc3[B1179]). (B) Western blot of isolated flagella probed with anti-DRC3. The antibody recognized a band of the predicted size for DRC3 (60 kDa) in WT; the band was absent from the drc3 flagella but was restored in the rescued strain (DRC3-R). In the DRC3-SNAP strain, the band recognized by anti-DRC3 shifted upward because of the addition of the SNAP-tag (20 kDa). The outer dynein arm intermediate chain IC2 served as a loading control. Note that the levels of DRC3 and DRC3-SNAP in flagella of the rescued strains are similar to that of DRC3 in wild-type flagella. (C) Means ± SDs of swimming speed determined from 50 cells each of WT, drc3, DRC3-R, and DRC3-SNAP strains. The swimming speed of drc3 cells was slower than that of WT and was rescued by transforma­tion of the mutant with either the wild-type DRC3 gene or a gene expressing SNAP-tagged DRC3. Statistical significance was determined by the Tukey–Kramer method: NS, p ≥ 0.05; ***p < 0.001. (D) Swimming paths of WT, drc3, and DRC3-R cells. Positions of cells while swimming in the focal plane were plotted with Manual Tracking in ImageJ. Recording time (1.5 s) was identical in all samples; interval between dots is 33 ms. Even though the swimming speed of drc3 was only slightly less than that of WT, the tracks of drc3 cells are much shorter than those of WT cells because the former tended to swim out of the focal plane more rapidly.
FIGURE 3:
FIGURE 3:
Flagellar beat frequency of drc3 cells is higher than that of WT and other N-DRC mutant cells. (A) Beat frequency of WT, drc3, DRC3-R, and DRC3-SNAP cells was analyzed by the FFT method. Loss of DRC3 caused increased beat frequency compared with WT cells. Beat frequency was restored to nearly normal by transformation of the drc3 strain with the WT gene or the construct expressing SNAP-tagged DRC3. (B) FFT spectra of other N-DRC mutants. Except for pf2, beat frequency is comparable to WT. The vertical solid and broken lines are aligned to the peaks of the spectra of the WT and the drc3 mutant, respectively.
FIGURE 4:
FIGURE 4:
Loss of DRC3 affects flagellar waveform. (A) Repre­sentative flagellar waveforms of WT, drc3, and DRC3-R cells. Waveforms of at least six free-swimming cells were analyzed for each strain; cells were observed over 2–10 beat cycles. For each cell type, cis- and trans-flagella from the same digital image have the same color; tracings of different colors are not necessarily from the same beat cycle. Flagellar length is not consistent through the beat cycle because the flagellar tip was sometimes out of focus. (B) Schematic diagrams illustrating typical flagellar beat envelopes for the WT and the drc3 mutant. The drc3 cells have a smaller beat envelope.
FIGURE 5:
FIGURE 5:
Loss of DRC3 does not suppress the paralyzed-flagella phenotype of pf14. Stacked bar graphs showing percentage of cells of the indicated strains that were paralyzed (red), vibrating (yellow), spinning (green), or swimming (blue). Two independently isolated strains of the drc3 pf14 double mutant were analyzed. For each strain, 118 vegetative (left) and 113 gametic (right) cells were scored.
FIGURE 6:
FIGURE 6:
N-DRC proteins in the drc3 axoneme. (A) Silver-stained 2D gels of WT and drc3 axonemes. The drc3 axonemes lack DRC3 (right, white arrowhead) but contain normal amounts of DRC1, 2, and 4–7 and of FAP206, 230, and 252, previously identified as candidate N-DRC proteins (Lin et al., 2011; Bower et al., 2013). The drc3 axonemes also contain normal amounts of FAP61/IDA7, which are reduced in many other N-DRC mutants, and of “spot 11,” an unidentified protein also reduced in many other N-DRC mutants (Lin et al., 2011). (B) Western blots of isolated flagella from WT and drc3 cells probed with anti-DRC8 and anti-DRC11 antibodies. DRC8 and DRC11 are present at normal levels in the drc3 flagella. IC2 was used as a loading control.
FIGURE 7:
FIGURE 7:
Structure of the N-DRC in the drc3 axoneme. Three-dimensional isosurface renderings of cryo-ET averages of the 96-nm repeats from WT (A–C), drc3 (D–F), and DRC3-SNAP (G–I) axonemes. The N-DRC linker (red, green) and base plate (blue) are shown in cross-sectional (A, D, G) and longitudinal (B, E, H) views of the doublet microtubule. (C, F, I) Enlarged N-DRC images in a longitudinal bottom view (looking from the axoneme center outward); to show the entire N-DRC clearly, the inner dynein arms and radial spoke RS2 were removed from these three images. A portion of the distal lobe, the L1 protrusion, and the connection between these two structures (red) are missing in the drc3 axonemes but are specifically restored in the strain rescued with DRC3-SNAP. Outer dynein arms and inner arm dyneins a–g are labeled. Inner arm dynein g, to which the L1 protrusion connects, is indicated in pink.
FIGURE 8:
FIGURE 8:
DRC3 is in a subcomplex with DRC4, DRC7, and DRC11. Isolated axonemes of WT and the DRC3-HA strain were extracted with KI and the extract subjected to immunoprecipitation using the anti-HA antibody. (A) Silver-stained SDS–polyacrylamide gel of the resulting immunoprecipitate. An arrow indicates a band likely to contain HA-tagged DRC3. Arrowheads indicate bands with polypeptides specifically coprecipitated with HA-tagged DRC3. (B) The same samples as in A were analyzed by Western blotting using anti-DRC3, 4, 7, and 11 antibodies.

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