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. 2011 Aug 19;286(33):29175-91.
doi: 10.1074/jbc.M111.241760. Epub 2011 Jun 23.

Building Blocks of the Nexin-Dynein Regulatory Complex in Chlamydomonas Flagella

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Free PMC article

Building Blocks of the Nexin-Dynein Regulatory Complex in Chlamydomonas Flagella

Jianfeng Lin et al. J Biol Chem. .
Free PMC article

Abstract

The directional flow generated by motile cilia and flagella is critical for many processes, including human development and organ function. Normal beating requires the control and coordination of thousands of dynein motors, and the nexin-dynein regulatory complex (N-DRC) has been identified as an important regulatory node for orchestrating dynein activity. The nexin link appears to be critical for the transformation of dynein-driven, linear microtubule sliding to flagellar bending, yet the molecular composition and mechanism of the N-DRC remain largely unknown. Here, we used proteomics with special attention to protein phosphorylation to analyze the composition of the N-DRC and to determine which subunits may be important for signal transduction. Two-dimensional electrophoresis and MALDI-TOF mass spectrometry of WT and mutant flagellar axonemes from Chlamydomonas identified 12 N-DRC-associated proteins, including all seven previously observed N-DRC components. Sequence and PCR analyses identified the mutation responsible for the phenotype of the sup-pf-4 strain, and biochemical comparison with a radial spoke mutant revealed two components that may link the N-DRC and the radial spokes. Phosphoproteomics revealed eight proteins with phosphorylated isoforms for which the isoform patterns changed with the genotype as well as two components that may play pivotal roles in N-DRC function through their phosphorylation status. These data were assembled into a model of the N-DRC that explains aspects of its regulatory function.

Figures

FIGURE 1.
FIGURE 1.
Models showing the N-DRC in Chlamydomonas flagella. A, differential interference contrast image showing a Chlamydomonas cell with its two flagella and a zoomed in schematic of a flagellar axoneme in cross-sectional view, seen from the flagellar tip. Three structures that connect neighboring doublets are as follows: IDAs, ODAs, and the N-DRC complex. B and C, three-dimensional maps of the 96-nm axonemal repeat in WT Chlamydomonas flagella reconstructed by cryo-electron tomography (cryo-ET), in cross-sectional (B) and longitudinal views (C). The suggested locations of N-DRC subunits are summarized and colored. At, A-tubule; Bt, B-tubule; CPC, central pair complex; IDA, inner dynein arm; ODA, outer dynein arm; RS, radial spoke. Images are adapted from Heuser et al. (7).
FIGURE 2.
FIGURE 2.
Comparison of the axonemal polypeptides from WT Chlamydomonas, a rescued pf2 strain (PF2-HA) and four drc mutants (pf3, sup-pf-4, sup-pf-3, and pf2) using 2DE (with silver staining). Arrows, polypeptides with significantly altered abundance.
FIGURE 3.
FIGURE 3.
Details from 2DE analysis of WT, PF2-HA, and four drc mutants (pf3, sup-pf-4, sup-pf-3, and pf2) for the previously reported N-DRC components DRC1–7. A, close-up images of 2DE gel spots of the proteins of interest. The images for DRC2 (FAP250) are from Coomassie-stained gels (blue), and all other images are from silver-stained gels. Arrowheads, spots showing significant differences in abundance between WT and drc mutants. B, relative protein quantification of spots with abundance differences between axonemes from WT and drc mutants. Relative quantifications are based on the average of 6–16 independently replicated silver-stained gels (or 2–4 Coomassie-stained gels for DRC2) and are expressed as the mean ± S.D. Asterisks indicate significant changes in comparison with WT (over ±1.5-fold; p < 0.05). Spot numbers correspond to the numbers depicted in Fig. 2 and Tables 2 and 3.
FIGURE 4.
FIGURE 4.
Details from 2DE analysis of WT, PF2-HA, and four drc mutants (pf3, sup-pf-4, sup-pf-3, and pf2) for novel proteins that showed significant changes in abundance in the mutants. A, close-up images of silver-stained 2DE gel spots of the proteins of interest. Filled arrowheads indicate spots with significant differences between WT and drc mutants. Open arrowheads indicate spots in the panels for FAP230 and FAP252 that were not analyzed by MS but that most likely correspond to one of the shifted spots in the pf2 gels, based on the gel pattern. B, relative protein quantification of spots with abundance differences between axonemes from WT and drc mutants. Relative quantifications are based on the average of 6–16 independently replicated silver-stained gels and are expressed as the mean ± S.D. Asterisks indicate significant changes in comparison with WT (over ±1.5-fold; p < 0.05). Spot numbers correspond to the numbers depicted in Fig. 2 and Tables 2 and 3.
FIGURE 5.
FIGURE 5.
SDS-PAGE analysis of axonemal proteins from WT Chlamydomonas, PF2-HA, and four drc mutants (pf3, sup-pf-4, sup-pf-3, and pf2). A, DRC7 is marked with an arrow on the 5% SDS-polyacrylamide gel. B, relative quantification of DRC7 is based on the average of three independently replicated silver-stained gels and is expressed as the mean ± S.D. Asterisks indicate significant changes in comparison with WT (p < 0.01).
FIGURE 6.
FIGURE 6.
Twelve N-DRC candidates identified by comparative proteomic analysis of WT and drc mutants. No significant changes were observed in the spot patterns of the N-DRC components in mutants that lack either I1 dynein (pf9-3) or radial spokes (pf14), with the exception of FAP206, which is reduced in pf14. Note that many of the N-DRC candidates are present as multiple spots in the silver-stained 2DE gels.
FIGURE 7.
FIGURE 7.
Analysis of N-DRC transcripts in drc mutants by qRT-PCR and identification of a gene deletion by genomic PCR. A, changes in the transcript levels of N-DRC candidates after deflagellation were measured by qRT-PCR. B, relative abundance of each transcript in the drc mutants (sup-pf-4, sup-pf-3, pf2, and pf3). Arrowheads, qRT-PCR products that were not detected in a given drc mutant. C, identification of a gene deletion in sup-pf-4 by genomic PCR. PCR primers were designed for the amplification of genomic DNA fragments in the region around FAP155. The positions of PCR products 1–8 are shown in the left diagram. The region contains at least three genes, including FAP155. The results from agarose gel electrophoresis of genomic PCR products (PCR1, PCR2, PCR7, and PCR8) are shown in the right panel.
FIGURE 8.
FIGURE 8.
Close-up images of 2DE gel spots stained for comparative phosphorylation analysis. Unless otherwise indicated, images are from WT axonemes. For most proteins, six images are displayed; the upper and lower rows show the spot pattern in the absence (λ P−) or presence (λ P+) of λ-phosphatase, respectively. Phosphorylated isoforms (white arrowheads) were detected by comparison of phosphoprotein stain (ProQ Diamond, red spots) and total protein stain (SYPRO Ruby, green spots; silver stain, brown spots; or Coomassie stain, blue spots). Nonphosphorylated isoforms (magenta arrowheads) were only detected by total protein staining. For proteins FAP230, FAP252, and DRC2, a WT-to-mutant comparison is shown (see the label on the left of the last three image blocks).
FIGURE 9.
FIGURE 9.
Models for the N-DRC in Chlamydomonas WT and drc mutants. The probable locations of the N-DRC components are based on this study and previous structural results (7). Legends on the left of each diagram indicate the N-DRC candidates in each strain. The following labels and color codes for subunits were used: reduced proteins are indicated with downward arrows, and missing proteins are faded out on a white background; proteins with PTM changes are labeled with an asterisk, and truncated PF2 is labeled with two asterisks. The PTM annotations are based on our phosphoproteomic analysis of the N-DRC. Star-shaped blocks indicate N-DRC candidates with phosphorylated isoforms. The changing colors of FAP250 in sup-pf-3 and pf2 indicate the changes in phosphorylation status in these drc mutants. The interactions among candidate N-DRC proteins require further confirmation, and little is known about the stoichiometry of the candidates. At, A-tubule; Bt, B-tubule; RS2, radial spoke 2; IDA, inner dynein arm; ODA, outer dynein arm. For a detailed explanation, see the text.

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