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1
DiBella, Linda M., Miho Sakato, Ramila S. Patel-King, Gregory J. Pazour, and Stephen M. King. "The LC7 Light Chains of Chlamydomonas Flagellar Dyneins Interact with Components Required for Both Motor Assembly and Regulation." Molecular Biology of the Cell 15, no. 10 (October 2004): 4633–46. http://dx.doi.org/10.1091/mbc.e04-06-0461.
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Members of the LC7/Roadblock family of light chains (LCs) have been found in both cytoplasmic and axonemal dyneins. LC7a was originally identified within Chlamydomonas outer arm dynein and associates with this motor's cargo-binding region. We describe here a novel member of this protein family, termed LC7b that is also present in the Chlamydomonas flagellum. Levels of LC7b are reduced ∼20% in axonemes isolated from strains lacking inner arm I1 and are ∼80% lower in the absence of the outer arms. When both dyneins are missing, LC7b levels are diminished to <10%. In oda9 axonemal extracts that completely lack outer arms, LC7b copurifies with inner arm I1, whereas in ida1 extracts that are devoid of I1 inner arms it associates with outer arm dynein. We also have observed that some LC7a is present in both isolated axonemes and purified 18S dynein from oda1, suggesting that it is also a component of both the outer arm and inner arm I1. Intriguingly, in axonemal extracts from the LC7a null mutant, oda15, which assembles ∼30% of its outer arms, LC7b fails to copurify with either dynein, suggesting that it interacts with LC7a. Furthermore, both the outer arm γ heavy chain and DC2 from the outer arm docking complex completely dissociate after salt extraction from oda15 axonemes. EDC cross-linking of purified dynein revealed that LC7b interacts with LC3, an outer dynein arm thioredoxin; DC2, an outer arm docking complex component; and also with the phosphoprotein IC138 from inner arm I1. These data suggest that LC7a stabilizes both the outer arms and inner arm I1 and that both LC7a and LC7b are involved in multiple intradynein interactions within both dyneins.
2
Wang, Limei, Xuecheng Li, Guang Liu, and Junmin Pan. "FBB18 participates in preassembly of almost all axonemal dyneins ind of R2TP complex." PLOS Genetics 18, no. 8 (August 26, 2022): e1010374. http://dx.doi.org/10.1371/journal.pgen.1010374.
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Assembly of dynein arms requires cytoplasmic processes which are mediated by dynein preassembly factors (DNAAFs). CFAP298, which is conserved in organisms with motile cilia, is required for assembly of dynein arms but with obscure mechanisms. Here, we show that FBB18, a Chlamydomonas homologue of CFAP298, localizes to the cytoplasm and functions in folding/stabilization of almost all axonemal dyneins at the early steps of dynein preassembly. Mutation of FBB18 causes no or short cilia accompanied with partial loss of both outer and inner dynein arms. Comparative proteomics using 15N labeling suggests partial degradation of almost all axonemal dynein heavy chains (DHCs). A mutant mimicking a patient variant induces particular loss of DHCα. FBB18 associates with 9 DNAAFs and 14 out of 15 dynein HCs but not with IC1/IC2. FBB18 interacts with RuvBL1/2, components of the HSP90 co-chaperone R2TP complex but not the holo-R2TP complex. Further analysis suggests simultaneous formation of multiple DNAAF complexes involves dynein folding/stability and thus provides new insights into axonemal dynein preassembly.
3
Yamamoto, Ryosuke, Kangkang Song, Haru-aki Yanagisawa, Laura Fox, Toshiki Yagi, Maureen Wirschell, Masafumi Hirono, Ritsu Kamiya, Daniela Nicastro, and Winfield S. Sale. "The MIA complex is a conserved and novel dynein regulator essential for normal ciliary motility." Journal of Cell Biology 201, no. 2 (April 8, 2013): 263–78. http://dx.doi.org/10.1083/jcb.201211048.
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Axonemal dyneins must be precisely regulated and coordinated to produce ordered ciliary/flagellar motility, but how this is achieved is not understood. We analyzed two Chlamydomonas reinhardtii mutants, mia1 and mia2, which display slow swimming and low flagellar beat frequency. We found that the MIA1 and MIA2 genes encode conserved coiled-coil proteins, FAP100 and FAP73, respectively, which form the modifier of inner arms (MIA) complex in flagella. Cryo–electron tomography of mia mutant axonemes revealed that the MIA complex was located immediately distal to the intermediate/light chain complex of I1 dynein and structurally appeared to connect with the nexin–dynein regulatory complex. In axonemes from mutants that lack both the outer dynein arms and the MIA complex, I1 dynein failed to assemble, suggesting physical interactions between these three axonemal complexes and a role for the MIA complex in the stable assembly of I1 dynein. The MIA complex appears to regulate I1 dynein and possibly outer arm dyneins, which are both essential for normal motility.
4
Fox, L. A., and W. S. Sale. "Direction of force generated by the inner row of dynein arms on flagellar microtubules." Journal of Cell Biology 105, no. 4 (October 1, 1987): 1781–87. http://dx.doi.org/10.1083/jcb.105.4.1781.
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Our goal was to determine the direction of force generation of the inner dynein arms in flagellar axonemes. We developed an efficient means of extracting the outer row of dynein arms in demembranated sperm tail axonemes, leaving the inner row of dynein arms structurally and functionally intact. Sperm tail axonemes depleted of outer arms beat at half the beat frequency of sperm tails with intact arms over a wide range of ATP concentrations. The isolated, outer arm-depleted axonemes were induced to undergo microtubule sliding in the presence of ATP and trypsin. Electron microscopic analysis of the relative direction of microtubule sliding (see Sale, W. S. and P. Satir, 1977, Proc. Natl. Acad. Sci. USA, 74:2045-2049) revealed that the doublet microtubule with the row of inner dynein arms, doublet N, always moved by sliding toward the proximal end of the axoneme relative to doublet N + 1. Therefore, the inner arms generate force such that doublet N pushes doublet N + 1 tipward. This is the same direction of microtubule sliding induced by ATP and trypsin in axonemes having both inner and outer dynein arms. The implications of this result for the mechanism of ciliary bending and utility in functional definition of cytoplasmic dyneins are discussed.
5
Bui, Khanh Huy, Hitoshi Sakakibara, Tandis Movassagh, Kazuhiro Oiwa, and Takashi Ishikawa. "Asymmetry of inner dynein arms and inter-doublet links in Chlamydomonas flagella." Journal of Cell Biology 186, no. 3 (August 10, 2009): 437–46. http://dx.doi.org/10.1083/jcb.200903082.
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Although the widely shared “9 + 2” structure of axonemes is thought to be highly symmetrical, axonemes show asymmetrical bending during planar and conical motion. In this study, using electron cryotomography and single particle averaging, we demonstrate an asymmetrical molecular arrangement of proteins binding to the nine microtubule doublets in Chlamydomonas reinhardtii flagella. The eight inner arm dynein heavy chains regulate and determine flagellar waveform. Among these, one heavy chain (dynein c) is missing on one microtubule doublet (this doublet also lacks the outer dynein arm), and another dynein heavy chain (dynein b or g) is missing on the adjacent doublet. Some dynein heavy chains either show an abnormal conformation or were replaced by other proteins, possibly minor dyneins. In addition to nexin, there are two additional linkages between specific pairs of doublets. Interestingly, all these exceptional arrangements take place on doublets on opposite sides of the axoneme, suggesting that the transverse functional asymmetry of the axoneme causes an in-plane bending motion.
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Ishibashi, Kenta, Hitoshi Sakakibara, and Kazuhiro Oiwa. "Force-Generating Mechanism of Axonemal Dynein in Solo and Ensemble." International Journal of Molecular Sciences 21, no. 8 (April 18, 2020): 2843. http://dx.doi.org/10.3390/ijms21082843.
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In eukaryotic cilia and flagella, various types of axonemal dyneins orchestrate their distinct functions to generate oscillatory bending of axonemes. The force-generating mechanism of dyneins has recently been well elucidated, mainly in cytoplasmic dyneins, thanks to progress in single-molecule measurements, X-ray crystallography, and advanced electron microscopy. These techniques have shed light on several important questions concerning what conformational changes accompany ATP hydrolysis and whether multiple motor domains are coordinated in the movements of dynein. However, due to the lack of a proper expression system for axonemal dyneins, no atomic coordinates of the entire motor domain of axonemal dynein have been reported. Therefore, a substantial amount of knowledge on the molecular architecture of axonemal dynein has been derived from electron microscopic observations on dynein arms in axonemes or on isolated axonemal dynein molecules. This review describes our current knowledge and perspectives of the force-generating mechanism of axonemal dyneins in solo and in ensemble.
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Bui, Khanh Huy, Hitoshi Sakakibara, Tandis Movassagh, Kazuhiro Oiwa, and Takashi Ishikawa. "Molecular architecture of inner dynein arms in situ in Chlamydomonas reinhardtii flagella." Journal of Cell Biology 183, no. 5 (November 24, 2008): 923–32. http://dx.doi.org/10.1083/jcb.200808050.
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The inner dynein arm regulates axonemal bending motion in eukaryotes. We used cryo-electron tomography to reconstruct the three-dimensional structure of inner dynein arms from Chlamydomonas reinhardtii. All the eight different heavy chains were identified in one 96-nm periodic repeat, as expected from previous biochemical studies. Based on mutants, we identified the positions of the AAA rings and the N-terminal tails of all the eight heavy chains. The dynein f dimer is located close to the surface of the A-microtubule, whereas the other six heavy chain rings are roughly colinear at a larger distance to form three dyads. Each dyad consists of two heavy chains and has a corresponding radial spoke or a similar feature. In each of the six heavy chains (dynein a, b, c, d, e, and g), the N-terminal tail extends from the distal side of the ring. To interact with the B-microtubule through stalks, the inner-arm dyneins must have either different handedness or, more probably, the opposite orientation of the AAA rings compared with the outer-arm dyneins.
8
King, Stephen M. "Axonemal Dynein Arms." Cold Spring Harbor Perspectives in Biology 8, no. 11 (August 15, 2016): a028100. http://dx.doi.org/10.1101/cshperspect.a028100.
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Smith, E. F., and W. S. Sale. "Structural and functional reconstitution of inner dynein arms in Chlamydomonas flagellar axonemes." Journal of Cell Biology 117, no. 3 (May 1, 1992): 573–81. http://dx.doi.org/10.1083/jcb.117.3.573.
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The inner row of dynein arms contains three dynein subforms. Each is distinct in composition and location in flagellar axonemes. To begin investigating the specificity of inner dynein arm assembly, we assessed the capability of isolated inner arm dynein subforms to rebind to their appropriate positions on axonemal doublet microtubules by recombining them with either mutant or extracted axonemes missing some or all dyneins. Densitometry of Coomassie blue-stained polyacrylamide gels revealed that for each inner dynein arm subform, binding to axonemes was saturable and stoichiometric. Using structural markers of position and polarity, electron microscopy confirmed that subforms bound to the correct inner arm position. Inner arms did not bind to outer arm or inappropriate inner arm positions despite the availability of sites. These and previous observations implicate specialized tubulin isoforms or nontubulin proteins in designation of specific inner dynein arm binding sites. Further, microtubule sliding velocities were restored to dynein-depleted axonemes upon rebinding of the missing inner arm subtypes as evaluated by an ATP-induced microtubule sliding disintegration assay. Therefore, not only were the inner arm dynein subforms able to identify and bind to the correct location on doublet microtubules but they bound in a functionally active conformation.
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Piperno, G., and Z. Ramanis. "The proximal portion of Chlamydomonas flagella contains a distinct set of inner dynein arms." Journal of Cell Biology 112, no. 4 (February 15, 1991): 701–9. http://dx.doi.org/10.1083/jcb.112.4.701.
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A specific type of inner dynein arm is located primarily or exclusively in the proximal portion of Chlamydomonas flagella. This dynein is absent from flagella less than 6 microns long, is assembled during the second half of flagellar regeneration time and is resistant to extraction under conditions causing complete solubilization of two inner arm heavy chains and partial solubilization of three other heavy chains. This and other evidence described in this report suggest that the inner arm row is composed of five distinct types of dynein arms. Therefore, the units of three inner arms that repeat every 96 nm along the axoneme are composed of different dyneins in the proximal and distal portions of flagella.
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King, Stephen M., and Winfield S. Sale. "Fifty years of microtubule sliding in cilia." Molecular Biology of the Cell 29, no. 6 (March 15, 2018): 698–701. http://dx.doi.org/10.1091/mbc.e17-07-0483.
Full textAbstract:
Motility of cilia (also known as flagella in some eukaryotes) is based on axonemal doublet microtubule sliding that is driven by the dynein molecular motors. Dyneins are organized into intricately patterned inner and outer rows of arms, whose collective activity is to produce inter-microtubule movement. However, to generate a ciliary bend, not all dyneins can be active simultaneously. The switch point model accounts, in part, for how dynein motors are regulated during ciliary movement. On the basis of this model, supported by key direct experimental observations as well as more recent theoretical and structural studies, we are now poised to understand the mechanics of how ciliary dynein coordination controls axonemal bend formation and propagation.
12
Gardner, L. C., E. O'Toole, C. A. Perrone, T. Giddings, and M. E. Porter. "Components of a "dynein regulatory complex" are located at the junction between the radial spokes and the dynein arms in Chlamydomonas flagella." Journal of Cell Biology 127, no. 5 (December 1, 1994): 1311–25. http://dx.doi.org/10.1083/jcb.127.5.1311.
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Previous studies of flagellar mutants have identified six axonemal polypeptides as components of a "dynein regulatory complex" (DRC). The DRC is though to coordinate the activity of the multiple flagellar dyneins, but its location within the axoneme has been unknown (Huang et al., 1982; Piperno et al., 1992). We have used improved chromatographic procedures (Kagami and Kamiya, 1992) and computer averaging of EM images (Mastronarde et al., 1992) to analyze the relationship between the DRC and the dynein arms. Our results suggest that some of the DRC components are located at the base of the second radial spoke in close association with the inner dynein arms. (a) Averages of axoneme cross-sections indicate that inner arm structures are significantly reduced in three DRC mutants (pf3 < pf2 < sup-pf-3 < wt). (b) These defects are more pronounced in distal/medial regions of the axoneme than in proximal regions. (c) Analysis of flagellar extracts by fast protein liquid chromatography and SDS-PAGE indicates that a specific dynein I2 isoform is missing in pf3 and reduced in pf2 and sup-pf-3. Comparison with ida4 and pf3ida4 extracts reveals that this isoform differs from those missing in ida4. (d) When viewed in longitudinal section, all three DRC mutants lack a crescent-shaped density above the second radial spoke, and pf3 axonemes lack additional structures adjacent to the crescent. We propose that the crescent corresponds in part to the location of the DRC, and that this structure is also directly associated with a subset of the inner dynein arms. This position is appropriate for a complex that is thought to mediate signals between the radial spokes and the dynein arms.
13
Piperno, G., K. Mead, and S. Henderson. "Inner dynein arms but not outer dynein arms require the activity of kinesin homologue protein KHP1(FLA10) to reach the distal part of flagella in Chlamydomonas." Journal of Cell Biology 133, no. 2 (April 15, 1996): 371–79. http://dx.doi.org/10.1083/jcb.133.2.371.
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Inner dynein arms, but not outer dynein arms, require the activity of KHP1(FLA10) to reach the distal part of axonemes before binding to outer doublet microtubules. We have analyzed the rescue of inner or outer dynein arms in quadriflagellate dikaryons by immunofluorescence microscopy of p28(IDA4), an inner dynein arm light chain, or IC69(ODA6), an outer dynein arm intermediate chain. In dikaryons two strains with different genetic backgrounds share the cytoplasm. As a consequence, wild-type axonemal precursors are transported to and assembled in mutant axonemes to complement the defects. The rescue of inner dynein arms containing p28 in ida4-wild-type dikaryons progressively occurred from the distal part of the axonemes and with time was extended towards the proximal part. In contrast, the rescue of outer dynein arms in oda2-wild-type dikaryons progressively occurred along the entire length of the axoneme. Rescue of inner dynein arms containing p28 in ida4fla10-fla10 dikaryons was similar to the rescue observed in ida4-wild-type dikaryons at 21 degrees C, whereas it was inhibited at 32 degrees C, a nonpermissive temperature for KHP1(FLA10). In contrast, rescue of outer dynein arms in oda2fla10-fla10 dikaryons was similar to the rescue observed in oda2-wild-type dikaryons at both 21 degrees and 32 degrees C and was not inhibited at 32 degrees C. Positioning of substructures in the internal part of the axonemal shaft requires the activity of kinesin homologue protein 1.
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Ishijima, S., M. Kubo-Irie, H. Mohri, and Y. Hamaguchi. "Calcium-dependent bidirectional power stroke of the dynein arms in sea urchin sperm axonemes." Journal of Cell Science 109, no. 12 (December 1, 1996): 2833–42. http://dx.doi.org/10.1242/jcs.109.12.2833.
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Active sliding between doublet microtubules of sea urchin sperm axonemes that were demembranated with Triton X-100 in the presence or absence of calcium was induced with ATP and elastase at various concentrations of Ca2+ to examine the effects of Ca2+ on the direction of the power stroke of the dynein arms. Dark-field light microscopy of microtubule sliding revealed that the sliding from the axonemes demembranated with Triton and millimolar calcium and disintegrated with ATP and elastase showed various patterns of sliding disintegration, including loops of doublet microtubules formed near the head or the basal body. These loops were often thicker than the remaining axonemal bundle. In contrast, only thinner loops were found from the axonemes demembranated with Triton in the absence of calcium and disintegrated with ATP and elastase at high Ca2+ concentrations. Electron microscopic examination of the direction of microtubule sliding showed that the doublet microtubules in the axonemes demembranated in the presence of millimolar calcium moved toward the base of the axonemes by the dynein arms on the adjacent doublet microtubule as well as by their own dynein arms. Doublet microtubules in the axonemes demembranated in the absence of calcium moved toward the base of the axonemes only by their own dynein arms. Similar observations have been obtained from the axonemes from which the outer dynein arms were selectively extracted. From these observations, we can conclude that the dynein arms generate force in both directions and this feature of the dynein arms arises from at least the inner dynein arms.
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Howard, D. R., G. Habermacher, D. B. Glass, E. F. Smith, and W. S. Sale. "Regulation of Chlamydomonas flagellar dynein by an axonemal protein kinase." Journal of Cell Biology 127, no. 6 (December 15, 1994): 1683–92. http://dx.doi.org/10.1083/jcb.127.6.1683.
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Genetic, biochemical, and structural data support a model in which axonemal radial spokes regulate dynein-driven microtubule sliding in Chlamydomonas flagella. However, the molecular mechanism by which dynein activity is regulated is unknown. We describe results from three different in vitro approaches to test the hypothesis that an axonemal protein kinase inhibits dynein in spoke-deficient axonemes from Chlamydomonas flagella. First, the velocity of dynein-driven microtubule sliding in spoke-deficient mutants (pf14, pf17) was increased to wild-type level after treatment with the kinase inhibitors HA-1004 or H-7 or by the specific peptide inhibitors of cAMP-dependent protein kinase (cAPK) PKI(6-22)amide or N alpha-acetyl-PKI(6-22)amide. In particular, the peptide inhibitors of cAPK were very potent, stimulating half-maximal velocity at 12-15 nM. In contrast, kinase inhibitors did not affect microtubule sliding in axonemes from wild-type cells. PKI treatment of axonemes from a double mutant missing both the radial spokes and the outer row of dynein arms (pf14pf28) also increased microtubule sliding to control (pf28) velocity. Second, addition of the type-II regulatory subunit of cAPK (RII) to spoke-deficient axonemes increased microtubule sliding to wild-type velocity. Addition of 10 microM cAMP to spokeless axonemes, reconstituted with RII, reversed the effect of RII. Third, our previous studies revealed that inner dynein arms from the Chlamydomonas mutants pf28 or pf14pf28 could be extracted in high salt buffer and subsequently reconstituted onto extracted axonemes restoring original microtubule sliding activity. Inner arm dyneins isolated from PKI-treated axonemes (mutant strain pf14pf28) generated fast microtubule sliding velocities when reconstituted onto both PKI-treated or control axonemes. In contrast, dynein from control axonemes generated slow microtubule sliding velocities on either PKI-treated or control axonemes. Together, the data indicate that an endogenous axonemal cAPK-type protein kinase inhibits dynein-driven microtubule sliding in spoke-deficient axonemes. The kinase is likely to reside in close association with its substrate(s), and the substrate targets are not exclusively localized to the central pair, radial spokes, dynein regulatory complex, or outer dynein arms. The results are consistent with a model in which the radial spokes regulate dynein activity through suppression of a cAMP-mediated mechanism.
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Warner, F. D., J. G. Perreault, and J. H. McIlvain. "Rebinding of Tetrahymena 13 S and 21 S dynein ATPases to extracted doublet microtubules. The inner row and outer row dynein arms." Journal of Cell Science 77, no. 1 (August 1, 1985): 263–87. http://dx.doi.org/10.1242/jcs.77.1.263.
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Ciliary axonemes from Tetrahymena contain a second salt-extractable ATPase distinguishable from outer arm 21 S dynein by sedimentation velocity (congruent to 13 S), electrophoretic mobility and substrate specificity. As characterized by turbidimetric assay, gel electrophoresis in the presence of sodium dodecyl sulphate, ATPase activity and electron microscopy, the 13 S dynein ATPase rebinds to extracted doublet microtubules. Compared to structural-side (ATP-insensitive) 21 S dynein binding, which is moderately specific for the 24 nm outer row arm position, rebinding of 13 S dynein is highly specific but for the inner row arm position. However, 13 S dynein rebinds to the A subfibre with a spacing that coincides with the triplet spacing of the radial spokes (24–32-40 nm periods; 96 nm repeat). All of the major protein components present in the 13 S or 21 S fractions rebind to extracted doublets under conditions that both restore and activate dynein ATPase activity. Unlike active-side (ATP-sensitive) rebound 21 S dynein, rebound 13 S dynein is completely insensitive to dissociation by ATP-vanadate and does not independently decorate the B subfibre. The saturation profile for rebinding of 13 S dynein exhibits a lack of cooperativity between binding events (h = 1.0) similar to structural-side rebinding of 21 S dynein. At low 21 S/doublet stoichiometry there is no measureable competition between the 13 S and 21 S dyneins for binding sites on the A subfibre lattice, although at saturating concentrations of 21 S dynein, rebinding of 13 S dynein is blocked completely.
17
Fabczak, Hanna, and Anna Osinka. "Role of the Novel Hsp90 Co-Chaperones in Dynein Arms’ Preassembly." International Journal of Molecular Sciences 20, no. 24 (December 7, 2019): 6174. http://dx.doi.org/10.3390/ijms20246174.
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The outer and inner dynein arms (ODAs and IDAs) are composed of multiple subunits including dynein heavy chains possessing a motor domain. These complex structures are preassembled in the cytoplasm before being transported to the cilia. The molecular mechanism(s) controlling dynein arms’ preassembly is poorly understood. Recent evidence suggests that canonical R2TP complex, an Hsp-90 co-chaperone, in cooperation with dynein axonemal assembly factors (DNAAFs), plays a crucial role in the preassembly of ODAs and IDAs. Here, we have summarized recent data concerning the identification of novel chaperone complexes and their role in dynein arms’ preassembly and their association with primary cilia dyskinesia (PCD), a human genetic disorder.
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Tanner, Christopher A., Panteleimon Rompolas, Ramila S. Patel-King, Oksana Gorbatyuk, Ken-ichi Wakabayashi, Gregory J. Pazour, and Stephen M. King. "Three Members of the LC8/DYNLL Family Are Required for Outer Arm Dynein Motor Function." Molecular Biology of the Cell 19, no. 9 (September 2008): 3724–34. http://dx.doi.org/10.1091/mbc.e08-04-0362.
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The highly conserved LC8/DYNLL family proteins were originally identified in axonemal dyneins and subsequently found to function in multiple enzyme systems. Genomic analysis uncovered a third member (LC10) of this protein class in Chlamydomonas. The LC10 protein is extracted from flagellar axonemes with 0.6 M NaCl and cofractionates with the outer dynein arm in sucrose density gradients. Furthermore, LC10 is specifically missing only from axonemes of those strains that fail to assemble outer dynein arms. Previously, the oda12-1 insertional allele was shown to lack the Tctex2-related dynein light chain LC2. The LC10 gene is located ∼2 kb from that of LC2 and is also completely missing from this mutant but not from oda12-2, which lacks only the 3′ end of the LC2 gene. Although oda12-1 cells assemble outer arms that lack only LC2 and LC10, this strain exhibits a flagellar beat frequency that is consistently less than that observed for strains that fail to assemble the entire outer arm and docking complex (e.g., oda1). These results support a key regulatory role for the intermediate chain/light chain complex that is an integral and highly conserved feature of all oligomeric dynein motors.
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Takada, S., and R. Kamiya. "Functional reconstitution of Chlamydomonas outer dynein arms from alpha-beta and gamma subunits: requirement of a third factor." Journal of Cell Biology 126, no. 3 (August 1, 1994): 737–45. http://dx.doi.org/10.1083/jcb.126.3.737.
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The outer dynein arm of Chlamydomonas flagella, when isolated under Mg(2+)-free conditions, tends to dissociate into an 11 to 12S particle (12S dynein) containing the gamma heavy chain and a 21S particle (called 18S dynein) containing the alpha and beta heavy chains. We show here that functional outer arms can be reconstituted by the addition of 12S and 18S dyneins to the axonemes of the outer armless mutants oda1-oda6. A third factor that sediments at integral 7S is required for efficient reconstitution of the outer arms on the axonemes of oda1 and oda3. However, this factor is not necessary for reconstitution on the axonemes of oda2, oda4, oda5, and oda6. SDS-PAGE analysis indicates that the axonemes of the former two mutants lack a integral of 70-kD polypeptide that is present in those of the other mutants as well as in the 7S fraction from the wild-type extract. Furthermore, electron micrographs of axonemal cross sections revealed that the latter four mutants, but not oda1 or oda3, have small pointed structures on the outer doublets, at a position in cross section where outer arms normally occur. We suggest that the 7S factor constitutes the pointed structure on the outer doublets and facilitates attachment of the outer arm. The discovery of this structure raises a new question as to how the attachment site for the outer arm dynein is determined within the axoneme.
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Piperno, G., K. Mead, M. LeDizet, and A. Moscatelli. "Mutations in the "dynein regulatory complex" alter the ATP-insensitive binding sites for inner arm dyneins in Chlamydomonas axonemes." Journal of Cell Biology 125, no. 5 (June 1, 1994): 1109–17. http://dx.doi.org/10.1083/jcb.125.5.1109.
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To understand mechanisms of regulation of dynein activity along and around the axoneme we further characterized the "dynein regulatory complex" (drc). The lack of some axonemal proteins, which together are referred to as drc, causes the suppression of flagellar paralysis of radial spoke and central pair mutants. The drc is also an adapter involved in the ATP-insensitive binding of I2 and I3 inner dynein arms to doublet microtubules. Evidence supporting these conclusions was obtained through analyses of five drc mutants: pf2, pf3, suppf3, suppf4, and suppf5. Axonemes from drc mutants lack part of I2 and I3 inner dynein arms as well as subsets of seven drc components (apparent molecular weight from 29,000 to 192,000). In the absence of ATP-Mg, dynein-depleted axonemes from the same mutants bind I2 and I3 inner arms at both ATP-sensitive and -insensitive sites. At ATP-insensitive sites, they bind I2 and I3 inner arms to an extent that depends on the drc defect. This evidence suggested to us that the drc forms one binding site for the I2 and I3 inner arms on the A part of doublet microtubules.
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Yamamoto, Ryosuke, Haru-aki Yanagisawa, Toshiki Yagi, and Ritsu Kamiya. "Novel 44-Kilodalton Subunit of Axonemal Dynein Conserved from Chlamydomonas to Mammals." Eukaryotic Cell 7, no. 1 (November 2, 2007): 154–61. http://dx.doi.org/10.1128/ec.00341-07.
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ABSTRACT Cilia and flagella have multiple dyneins in their inner and outer arms. Chlamydomonas inner-arm dynein contains at least seven major subspecies (dynein a to dynein g), of which all but dynein f (also called dynein I1) are the single-headed type that are composed of a single heavy chain, actin, and either centrin or a 28-kDa protein (p28). Dynein d was found to associate with two additional proteins of 38 kDa (p38) and 44 kDa (p44). Following the characterization of the p38 protein (R. Yamamoto, H. A. Yanagisawa, T. Yagi, and R. Kamiya, FEBS Lett. 580:6357-6360, 2006), we have identified p44 as a novel component of dynein d by using an immunoprecipitation approach. p44 is present along the length of the axonemes and is diminished, but not absent, in the ida4 and ida5 mutants, both lacking this dynein. In the ida5 axoneme, p44 and p38 appear to form a complex, suggesting that they constitute the docking site of dynein d on the outer doublet. p44 has potential homologues in other ciliated organisms. For example, the mouse homologue of p44, NYD-SP14, was found to be strongly expressed in tissues with motile cilia and flagella. These results suggest that inner-arm dynein d and its subunit organization are widely conserved.
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Bower, Raqual, Kristyn VanderWaal, Eileen O'Toole, Laura Fox, Catherine Perrone, Joshua Mueller, Maureen Wirschell, R. Kamiya, Winfield S. Sale, and Mary E. Porter. "IC138 Defines a Subdomain at the Base of the I1 Dynein That Regulates Microtubule Sliding and Flagellar Motility." Molecular Biology of the Cell 20, no. 13 (July 2009): 3055–63. http://dx.doi.org/10.1091/mbc.e09-04-0277.
Full textAbstract:
To understand the mechanisms that regulate the assembly and activity of flagellar dyneins, we focused on the I1 inner arm dynein (dynein f) and a null allele, bop5-2, defective in the gene encoding the IC138 phosphoprotein subunit. I1 dynein assembles in bop5-2 axonemes but lacks at least four subunits: IC138, IC97, LC7b, and flagellar-associated protein (FAP) 120—defining a new I1 subcomplex. Electron microscopy and image averaging revealed a defect at the base of the I1 dynein, in between radial spoke 1 and the outer dynein arms. Microtubule sliding velocities also are reduced. Transformation with wild-type IC138 restores assembly of the IC138 subcomplex and rescues microtubule sliding. These observations suggest that the IC138 subcomplex is required to coordinate I1 motor activity. To further test this hypothesis, we analyzed microtubule sliding in radial spoke and double mutant strains. The results reveal an essential role for the IC138 subcomplex in the regulation of I1 activity by the radial spoke/phosphorylation pathway.
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Porter, M. E., S. Myster, C. Perrone, and E. O'Toole. "Molecular and Structural Studies on Dynein Associated Mutations in Chlamydomonas Flagella." Microscopy and Microanalysis 3, S2 (August 1997): 219–20. http://dx.doi.org/10.1017/s1431927600007984.
Full textAbstract:
The dynein ATPases are a large family of motor enzymes that provide the driving force for flagellar motility and contribute to microtubule-based transport inside cells. The challenge for the field is to appreciate the functional significance of the multiple dynein motors, to determine how the cell assembles a motor complex and targets each motor to its appropriate location, and to understand how a cell regulates the activity of each motor to accomplish its specific task(s). In our laboratory, we have capitalized on the highly ordered structural organization of the flagellar axoneme and on the ease of genetic analysis in Chlamydomonas to ask how a single cell controls the assembly and activity of its multiple flagellar dyneins. In particular, we have focused our efforts on the inner dynein arms, which are both necessary and sufficient for normal flagellar motility. The significance of this work derives from the conservation of axoneme structure across species, as well as the numerous functional homologies between the flagellar and cytoplasmic dyneins.
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Piperno, G. "Isolation of a sixth dynein subunit adenosine triphosphatase of Chlamydomonas axonemes." Journal of Cell Biology 106, no. 1 (January 1, 1988): 133–40. http://dx.doi.org/10.1083/jcb.106.1.133.
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This study of the axoneme led to the identification of a previously unknown adenosine triphosphatase (ATPase), which is likely a major component of inner dynein arms. The ATPase was isolated from a soluble fraction of axonemes obtained from pf 28, a Chlamydomonas mutant lacking the outer dynein arms. The activity hydrolyzed up to 2.3 mumol of ATP.min-1.mg-1 of protein (at pH 7.2, in the presence of both Ca++ and Mg++), had a sedimentation coefficient of 11S in sucrose gradient, and cosedimented with four polypeptides of apparent molecular weight 325,000, 315,000 140,000, and 42,000. Several arguments indicate that the new ATPase is a component of the inner dynein arms. Three or four polypeptides cosedimenting with the activity belong to a group of axonemal components that are deficient in the axonemes of pf 23 and pf 30, two mutants that display different levels of inner dynein arm deficiency. The 42,000 component is axonemal actin, a subunit of two other inner dynein ATPases. The two polypeptides of molecular weight greater than 300,000 have electrophoretic mobility similar to that of high molecular weight components of outer and inner dynein arms. In spite of some similarities each ATPase isolated from inner or outer arms is composed of a different set of polypeptides. Different ATPases may be required for the modulation of localized sliding of adjacent outer double microtubules in the axoneme.
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Hyams, J. S. "Binding of Tetrahymena dynein to axonemes of Marsilea vestita lacking the outer dynein arm." Journal of Cell Science 73, no. 1 (February 1, 1985): 299–310. http://dx.doi.org/10.1242/jcs.73.1.299.
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Axonemes from the heterosporous water fern Marsilea vestita were fixed in the presence of tannic acid and examined by thin-section electron microscopy. Transverse sections revealed the normal 9+2 configuration except for the absence of the outer of the two dynein arms. Both arms were normally preserved in parallel preparations of Chlamydomonas axonemes. Isolated dynein from the ciliated protozoon Tetrahymena bound to Marsilea axonemes at the site normally occupied by the outer arm. Dynein binding was partially reversed by ATP as judged by both electron microscopy and polyacrylamide gel electrophoresis. This system should provide a valuable insight into the biochemistry and function of the inner dynein arm and the relationship of the two arms to motility in more conventionally equipped axonemes.
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Warner, F. D., and J. H. McIlvain. "Kinetic properties of microtubule-activated 13 S and 21 S dynein ATPases251. Evidence for allosteric behaviour associated with the inner row and outer row dynein arms." Journal of Cell Science 83, no. 1 (July 1, 1986): 251–67. http://dx.doi.org/10.1242/jcs.83.1.251.
Full textAbstract:
The 13 S and 21 S dynein ATPases from Tetrahymena cilia rebind to extracted doublet microtubules as inner row and outer row arms. Rebinding is accompanied by four- to ninefold activation of the ATPase activity. The soluble (microtubule-free) forms of the two dyneins exhibit simple saturation kinetics (h = 1.0) with Vmax much less than mumol Pi mg-1 min-1 and Km = 20–40 microM-ATP. Mixing a fixed quantity of free dynein with increasing concentrations of extracted doublets results in systematic increases in all three kinetic parameters for each dynein. At infinite concentrations of doublets and ATP, each enzyme undergoes a significant shift to sigmoid saturation kinetics (h = 2–3), Vmax increases to a turnover rate of about 90 mol ATP per mol Es-1 and the Michaelis constant increases to much greater than 100 microM-ATP. These data suggest that both enzymes are allosteric and can be interpreted in terms of positive cooperativity relative to a minimum of two or three interacting sites. It is less clear whether this cooperativity is related to subunit interactions within the 21 S or 13 S particles, or to subunit interactions between adjacent particles (arms) on the microtubule lattice.
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Liu, Guang, Limei Wang, and Junmin Pan. "Chlamydomonas WDR92 in association with R2TP-like complex and multiple DNAAFs to regulate ciliary dynein preassembly." Journal of Molecular Cell Biology 11, no. 9 (November 14, 2018): 770–80. http://dx.doi.org/10.1093/jmcb/mjy067.
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Abstract The motility of cilia or eukaryotic flagella is powered by the axonemal dyneins, which are preassembled in the cytoplasm by proteins termed dynein arm assembly factors (DNAAFs) before being transported to and assembled on the ciliary axoneme. Here, we characterize the function of WDR92 in Chlamydomonas. Loss of WDR92, a cytoplasmic protein, in a mutant wdr92 generated by DNA insertional mutagenesis resulted in aflagellate cells or cells with stumpy or short flagella, disappearance of axonemal dynein arms, and diminishment of dynein arm heavy chains in the cytoplasm, suggesting that WDR92 is a DNAAF. Immunoprecipitation of WDR92 followed by mass spectrometry identified inner dynein arm heavy chains and multiple DNAAFs including RuvBL1, RPAP3, MOT48, ODA7, and DYX1C. The PIH1 domain-containing protein MOT48 formed a R2TP-like complex with RuvBL1/2 and RPAP3, while PF13, another PIH1 domain-containing protein with function in dynein preassembly, did not. Interestingly, the third PIH1 domain-containing protein TWI1 was not related to flagellar motility. WDR92 physically interacted with the R2TP-like complex and the other identified DNNAFs. Our data suggest that WDR92 functions in association with the HSP90 co-chaperone R2TP-like complex as well as linking other DNAAFs in dynein preassembly.
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SAKAKIBARA, HITOSHI, and RITSU KAMIYA. "Functional Recombination of Outer Dynein Arms with Outer Arm-Missing Flagellar Axonemes of a Chlamydomonas Mutant." Journal of Cell Science 92, no. 1 (January 1, 1989): 77–83. http://dx.doi.org/10.1242/jcs.92.1.77.
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A flagellar mutant of Chlamydomonas, oda, lacks the entire outer dynein arm but can swim at a speed of one third to half of that of the wild type. We found that the addition of a high-salt extract of wild-type axonemes to demembranated oda cell models restored up to 83% of the outer arms normally present on the outer-doublet microtubules of wild-type axonemes. Furthermore, when reactivated in the presence of ATP after being mixed with the extract, the oda cell models gained a higher level of motility, close to that of the wild type. The increase in flagellar beat frequency parallelled the increase in the number of restored outer dynein arms. These observations indicate that the axoneme of the oda mutant retains the binding sites for the outer dynein arms, and that the outer arms solubilized with high salt are functionally active. This in vitro recombination system with the oda mutant should be useful as an assay system for various preparations of outer-arm dynein. Evidence is presented that the two axonemes on an oda cell model beat at the same frequency, whereas those on a wild-type model beat at different frequencies. The two oda axonemes beat at the same frequency even after the higher level of motility has been restored by addition of crude dynein extract. We propose that a heterogeneity in the outer dynein arms is responsible for the frequency imbalance between the two flagella of wild-type Chlamydomonas.
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Lin, Jianfeng, Thuc Vy Le, Katherine Augspurger, Douglas Tritschler, Raqual Bower, Gang Fu, Catherine Perrone, et al. "FAP57/WDR65 targets assembly of a subset of inner arm dyneins and connects to regulatory hubs in cilia." Molecular Biology of the Cell 30, no. 21 (October 1, 2019): 2659–80. http://dx.doi.org/10.1091/mbc.e19-07-0367.
Full textAbstract:
Ciliary motility depends on both the precise spatial organization of multiple dynein motors within the 96 nm axonemal repeat and the highly coordinated interactions between different dyneins and regulatory complexes located at the base of the radial spokes. Mutations in genes encoding cytoplasmic assembly factors, intraflagellar transport factors, docking proteins, dynein subunits, and associated regulatory proteins can all lead to defects in dynein assembly and ciliary motility. Significant progress has been made in the identification of dynein subunits and extrinsic factors required for preassembly of dynein complexes in the cytoplasm, but less is known about the docking factors that specify the unique binding sites for the different dynein isoforms on the surface of the doublet microtubules. We have used insertional mutagenesis to identify a new locus, IDA8/BOP2, required for targeting the assembly of a subset of inner dynein arms (IDAs) to a specific location in the 96 nm repeat. IDA8 encodes flagellar-associated polypeptide (FAP)57/WDR65, a highly conserved WD repeat, coiled coil domain protein. Using high resolution proteomic and structural approaches, we find that FAP57 forms a discrete complex. Cryo-electron tomography coupled with epitope tagging and gold labeling reveal that FAP57 forms an extended structure that interconnects multiple IDAs and regulatory complexes.
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Ide, Takahiro, Wang Kyaw Twan, Hao Lu, Yayoi Ikawa, Lin-Xenia Lim, Nicole Henninger, Hiromi Nishimura, et al. "CFAP53 regulates mammalian cilia-type motility patterns through differential localization and recruitment of axonemal dynein components." PLOS Genetics 16, no. 12 (December 21, 2020): e1009232. http://dx.doi.org/10.1371/journal.pgen.1009232.
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Motile cilia can beat with distinct patterns, but how motility variations are regulated remain obscure. Here, we have studied the role of the coiled-coil protein CFAP53 in the motility of different cilia-types in the mouse. While node (9+0) cilia of Cfap53 mutants were immotile, tracheal and ependymal (9+2) cilia retained motility, albeit with an altered beat pattern. In node cilia, CFAP53 mainly localized at the base (centriolar satellites), whereas it was also present along the entire axoneme in tracheal cilia. CFAP53 associated tightly with microtubules and interacted with axonemal dyneins and TTC25, a dynein docking complex component. TTC25 and outer dynein arms (ODAs) were lost from node cilia, but were largely maintained in tracheal cilia of Cfap53-/- mice. Thus, CFAP53 at the base of node cilia facilitates axonemal transport of TTC25 and dyneins, while axonemal CFAP53 in 9+2 cilia stabilizes dynein binding to microtubules. Our study establishes how differential localization and function of CFAP53 contributes to the unique motion patterns of two important mammalian cilia-types.
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Shingyoji, Chikako, Hideo Higuchi, Misako Yoshimura, Eisaku Katayama, and Toshio Yanagida. "Dynein arms are oscillating force generators." Nature 393, no. 6686 (June 1998): 711–14. http://dx.doi.org/10.1038/31520.
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Yokota, E., and I. Mabuchi. "Isolation and characterization of a novel dynein that contains C and A heavy chains from sea urchin sperm flagellar axonemes." Journal of Cell Science 107, no. 2 (February 1, 1994): 345–51. http://dx.doi.org/10.1242/jcs.107.2.345.
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A novel dynein (C/A dynein), which is composed of C and A heavy chains, two intermediate chains and several light chains, was isolated from sea urchin sperm flagella. The C/A dynein was released by the treatment with 0.7 M NaCl plus 5 mM ATP from the axonemes depleted of outer arm 21 S dynein. Sedimentation coefficient of this dynein was estimated by sucrose density gradient centrifugation to be 22–23 S. The C/A dynein particle appeared to be composed of three distinct domains; two globular head domains and one rod domain as seen by negative staining electron microscopy. The mobility of ‘A’ heavy chain of C/A dynein on SDS-gel electrophoresis was similar to that of A heavy chains (A alpha and A beta) of 21 S dynein. However, UV-cleavage patterns of C and A heavy chains of C/A dynein were different from those of A heavy chains of 21 S dynein. Furthermore, an antiserum raised against A heavy chain of C/A dynein did not crossreact with A heavy chains of 21 S dynein. Under the conditions in which the C/A dynein was released, some of inner arms were removed concomitantly from axonemes as observed by electron microscopy. These results suggested that C/A dynein is a component of the inner arms.
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Horst, C. J., and G. B. Witman. "ptx1, a nonphototactic mutant of Chlamydomonas, lacks control of flagellar dominance." Journal of Cell Biology 120, no. 3 (February 1, 1993): 733–41. http://dx.doi.org/10.1083/jcb.120.3.733.
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A new mutant strain of Chlamydomonas, ptx1, has been identified which is defective in phototaxis. This strain swims with a rate and straightness of path comparable with that of wild-type cells, and retains the photoshock response. Thus, the mutation does not cause any gross defects in swimming ability or photoreception, and appears to be specific for phototaxis. Calcium is required for phototaxis in wild-type cells, and causes a concentration-dependent shift in flagellar dominance in reactivated, demembranated cell models. ptx1-reactivated models are defective in this calcium-dependent shift in flagellar dominance. This indicates that the mutation affects one or more components of the calcium-dependent axonemal regulatory system, and that this system mediates phototaxis. The reduction or absence of two 75-kD axonemal proteins correlates with the nonphototactic phenotype. Axonemal fractionation studies, and analysis of axonemes from mutant strains with known structural defects, failed to reveal the structural localization of the 75-kD proteins within the axoneme. The proteins are not components of the outer dynein arms, two of the three types of inner dynein arms, the radial spokes, or the central pair complex. Because changes in flagellar motility ultimately require the regulation of dynein activity, cell models from mutant strains defective in specific dynein arms were reactivated at various calcium concentrations. Mutants lacking the outer arms, or the I1 or I2 inner dynein arms, retain the wild-type calcium-dependent shift in flagellar dominance. Therefore, none of these arms are the sole mediators of phototaxis.
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King, Stephen M. "A solid-state control system for dynein-based ciliary/flagellar motility." Journal of Cell Biology 201, no. 2 (April 8, 2013): 173–75. http://dx.doi.org/10.1083/jcb.201302077.
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Ciliary and flagellar beating requires the coordinated action of multiple dyneins with different enzymatic and motor properties. In this issue, Yamamoto et al. (2013. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201211048) identify the MIA (modifier of inner arms) complex within the Chlamydomonas reinhardtii axoneme that physically links to a known regulatory structure and provides a signaling conduit from the radial spokes to an inner arm dynein essential for waveform determination.
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LeDizet, M., and G. Piperno. "ida4-1, ida4-2, and ida4-3 are intron splicing mutations affecting the locus encoding p28, a light chain of Chlamydomonas axonemal inner dynein arms." Molecular Biology of the Cell 6, no. 6 (June 1995): 713–23. http://dx.doi.org/10.1091/mbc.6.6.713.
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We recently determined the nucleotide sequence of the gene encoding p28, a light chain of inner dynein arms of Chlamydomonas axonemes. Here, we show that p28 is the protein encoded by the IDA4 locus. p28, and the dynein heavy chains normally associated with it, are completely absent from the flagella and cell bodies of three allelic strains of ida4, named ida4-1, ida4-2, and ida4-3. We determined the nucleotide sequence of the three alleles of the p28 gene and found in each case a single nucleotide change, affecting the splice sites of the first, second, and fourth introns, respectively. Reverse transcriptase-polymerase chain reaction amplification of RNAs prepared from ida4 cells confirmed that these mutations prevent the correct splicing of the affected introns, thereby blocking the synthesis of full-length p28. These are the first intron splicing mutations described in Chlamydomonas and the first inner dynein arm mutations characterized at the molecular level. The absence in ida4 axonemes of the dynein heavy chains normally found in association with p28 suggests that p28 is necessary for stable assembly of a subset of inner dynein arms or for the binding of these arms to the microtubule doublets.
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Stephens, R. E., and G. Prior. "Dynein from serotonin-activated cilia and flagella: extraction characteristics and distinct sites for cAMP-dependent protein phosphorylation." Journal of Cell Science 103, no. 4 (December 1, 1992): 999–1012. http://dx.doi.org/10.1242/jcs.103.4.999.
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Serotonin, an activator of adenylate cyclase, stimulates motility in molluscan gill cilia and sperm flagella. To determine and compare potential targets of cAMP action, dynein was prepared from the lateral gill.cilia and sperm flagella of the mussel Mytilus edulis and the clam Spisula solidissima. In the flagella of both species, high-salt extraction removes about half of the ATPase activity, half of the alpha and beta heavy chains, and the outer arms. The dynein from both species sediments at 18–20 S, contains two or three intermediate chains, and three light chains. High-salt plus detergent removes most of the remaining dynein ATPase, alpha and beta heavy chains, and inner arms, also yielding a stable 18–20 S particle. In gill cilia of both species, high-salt extraction removes only 12–18% of the ATPase, up to 1/3 of the alpha heavy chains, an equivalent amount of beta heavy chain, and a subset of the outer arms. The dynein sediments at 18–20 S and, in Spisula, the heavy, intermediate, and light chains precisely co-sediment. High-salt plus detergent removes another 1/3 of the alpha heavy chains, an equivalent amount of beta heavy chain, and the remaining outer arms. The ATPase sediments mainly as a 13–14 S form showing considerable dissociation of co-sedimenting intermediate and light chains. The inner arms and at least half of the ciliary dynein ATPase activity remain unextractable, corresponding in mass mainly to an apparent beta heavy chain that is vanadate-cleavable. Cyclic AMP-dependent, calcium-independent phosphorylation takes place on specific dynein light chains in cilia but on only the dynein alpha heavy chain in flagella. Pre-activation of the flagella prevents subsequent addition of labeled phosphate. Phosphorylation has no effect on the steady-state ATPase properties. The single phosphate added to the flagellar alpha chain is located within the LUV1 vanadate photocleavage fragment. Considering the probable locus of the light chains and the site of the alpha heavy chain phosphorylation, both beyond the active site and toward the base of the molecule, these distinct phosphorylations may regulate dynein action by modulating arm flexibility or interaction.
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Kubo, Tomohiro, Yuqing Hou, Deborah A. Cochran, George B. Witman, and Toshiyuki Oda. "A microtubule-dynein tethering complex regulates the axonemal inner dynein f (I1)." Molecular Biology of the Cell 29, no. 9 (May 2018): 1060–74. http://dx.doi.org/10.1091/mbc.e17-11-0689.
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Motility of cilia/flagella is generated by a coordinated activity of thousands of dyneins. Inner dynein arms (IDAs) are particularly important for the formation of ciliary/flagellar waveforms, but the molecular mechanism of IDA regulation is poorly understood. Here we show using cryoelectron tomography and biochemical analyses of Chlamydomonas flagella that a conserved protein FAP44 forms a complex that tethers IDA f (I1 dynein) head domains to the A-tubule of the axonemal outer doublet microtubule. In wild-type flagella, IDA f showed little nucleotide-dependent movement except for a tilt in the f β head perpendicular to the microtubule-sliding direction. In the absence of the tether complex, however, addition of ATP and vanadate caused a large conformational change in the IDA f head domains, suggesting that the movement of IDA f is mechanically restricted by the tether complex. Motility defects in flagella missing the tether demonstrates the importance of the IDA f-tether interaction in the regulation of ciliary/flagellar beating.
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Pazour, Gregory J., Anthony Koutoulis, Sharon E. Benashski, Bethany L. Dickert, Hong Sheng, Ramila S. Patel-King, Stephen M. King, and George B. Witman. "LC2, the Chlamydomonas Homologue of the tComplex-encoded Protein Tctex2, Is Essential for Outer Dynein Arm Assembly." Molecular Biology of the Cell 10, no. 10 (October 1999): 3507–20. http://dx.doi.org/10.1091/mbc.10.10.3507.
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Tctex2 is thought to be one of the distorter genes of the mouset haplotype. This complex greatly biases the segregation of the chromosome that carries it such that in heterozygous +/t males, the t haplotype is transmitted to >95% of the offspring, a phenomenon known as transmission ratio distortion. The LC2 outer dynein arm light chain ofChlamydomonas reinhardtii is a homologue of the mouse protein Tctex2. We have identified Chlamydomonasinsertional mutants with deletions in the gene encoding LC2 and demonstrate that the LC2 gene is the same as the ODA12 gene, the product of which had not been identified previously. Complete deletion of the LC2/ODA12 gene causes loss of all outer arms and a slow jerky swimming phenotype. Transformation of the deletion mutant with the cloned LC2/ODA12 gene restores the outer arms and rescues the motility phenotype. Therefore, LC2 is required for outer arm assembly. The fact that LC2 is an essential subunit of flagellar outer dynein arms allows us to propose a detailed mechanism whereby transmission ratio distortion is explained by the differential binding of mutant (t haplotype encoded) and wild-type dyneins to the axonemal microtubules oft-bearing or wild-type sperm, with resulting differences in their motility.
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Piperno, G., K. Mead, and W. Shestak. "The inner dynein arms I2 interact with a "dynein regulatory complex" in Chlamydomonas flagella." Journal of Cell Biology 118, no. 6 (September 15, 1992): 1455–63. http://dx.doi.org/10.1083/jcb.118.6.1455.
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We provide indirect evidence that six axonemal proteins here referred to as "dynein regulatory complex" (drc) are located in close proximity with the inner dynein arms I2 and I3. Subsets of drc subunits are missing from five second-site suppressors, pf2, pf3, suppf3, suppf4, and suppf5, that restore flagellar motility but not radial spoke structure of radial spoke mutants. The absence of drc components is correlated with a deficiency of all four heavy chains of inner arms I2 and I3 from axonemes of suppressors pf2, pf3, suppf3, and suppf5. Similarly, inner arm subunits actin, p28, and caltractin/centrin, or subsets of them, are deficient in pf2, pf3, and suppf5. Recombinant strains carrying one of the mutations pf2, pf3, or suppf5 and the inner arm mutation ida4 are more defective for I2 inner arm heavy chains than the parent strains. This evidence indicates that at least one subunit of the drc affects the assembly of and interacts with the inner arms I2.
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SULLIVAN, JEANELL, SUSAN A. LUDMANN, TOSHIKAZU HAMASAKI, and DAVID G. PENNOCK. "Analyses of 22S Dynein Binding to Tetrahymena Axonemes Lacking Outer Dynein Arms." Journal of Eukaryotic Microbiology 43, no. 1 (January 1996): 5–11. http://dx.doi.org/10.1111/j.1550-7408.1996.tb02466.x.
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Patel-King, Ramila S., Miho Sakato-Antoku, Maya Yankova, and Stephen M. King. "WDR92 is required for axonemal dynein heavy chain stability in cytoplasm." Molecular Biology of the Cell 30, no. 15 (July 15, 2019): 1834–45. http://dx.doi.org/10.1091/mbc.e19-03-0139.
Full textAbstract:
WDR92 associates with a prefoldin-like cochaperone complex and known dynein assembly factors. WDR92 has been very highly conserved and has a phylogenetic signature consistent with it playing a role in motile ciliary assembly or activity. Knockdown of WDR92 expression in planaria resulted in ciliary loss, reduced beat frequency and dyskinetic motion of the remaining ventral cilia. We have now identified a Chlamydomonas wdr92 mutant that encodes a protein missing the last four WD repeats. The wdr92-1 mutant builds only ∼0.7-μm cilia lacking both inner and outer dynein arms, but with intact doublet microtubules and central pair. When cytoplasmic extracts prepared by freeze/thaw from a control strain were fractionated by gel filtration, outer arm dynein components were present in several distinct high molecular weight complexes. In contrast, wdr92-1 extracts almost completely lacked all three outer arm heavy chains, while the IFT dynein heavy chain was present in normal amounts. A wdr92-1 tpg1-2 double mutant builds ∼7-μm immotile flaccid cilia that completely lack dynein arms. These data indicate that WDR92 is a key assembly factor specifically required for the stability of axonemal dynein heavy chains in cytoplasm and suggest that cytoplasmic/IFT dynein heavy chains use a distinct folding pathway.
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Short, Ben. "PIH proteins give dynein arms a hand." Journal of Cell Biology 190, no. 1 (July 5, 2010): 2. http://dx.doi.org/10.1083/jcb.1901iti3.
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Mitchell, D. R., and J. L. Rosenbaum. "A motile Chlamydomonas flagellar mutant that lacks outer dynein arms." Journal of Cell Biology 100, no. 4 (April 1, 1985): 1228–34. http://dx.doi.org/10.1083/jcb.100.4.1228.
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A new Chlamydomonas flagellar mutant, pf-28, which swims more slowly than wild-type cells, was selected. Thin-section electron microscopy revealed the complete absence of outer-row dynein arms in this mutant, whereas inner-row arms and other axonemal structures appeared normal. SDS PAGE analysis also indicated that polypeptides previously identified as outer-arm dynein components are completely absent in pf-28. The two ATPases retained by this mutant sediment at 17.7S and 12.7S on sucrose gradients that contain 0.6 M KCl. Overall swimming patterns of pf-28 differ little from wild-type except that forward swimming speed is reduced to 35% of the wild-type value, and cells show little or no backward movement during photophobic avoidance. Mutant cells will respond to phototactic stimuli, and their flagella will beat in either the forward or reverse mode. This is the first report of a mutant that lacks dynein arms that can swim.
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Dong, Fenglan, Kyosuke Shinohara, Yanick Botilde, Ryo Nabeshima, Yasuko Asai, Akemi Fukumoto, Toshiaki Hasegawa, et al. "Pih1d3 is required for cytoplasmic preassembly of axonemal dynein in mouse sperm." Journal of Cell Biology 204, no. 2 (January 13, 2014): 203–13. http://dx.doi.org/10.1083/jcb.201304076.
Full textAbstract:
Axonemal dynein complexes are preassembled in the cytoplasm before their transport to cilia, but the mechanism of this process remains unclear. We now show that mice lacking Pih1d3, a PIH1 domain–containing protein, develop normally but manifest male sterility. Pih1d3−/− sperm were immotile and fragile, with the axoneme of the flagellum lacking outer dynein arms (ODAs) and inner dynein arms (IDAs) and showing a disturbed 9+2 microtubule organization. Pih1d3 was expressed specifically in spermatogenic cells, with the mRNA being most abundant in pachytene spermatocytes. Pih1d3 localized to the cytoplasm of spermatogenic cells but was not detected in spermatids or mature sperm. The levels of ODA and IDA proteins were reduced in the mutant testis and sperm, and Pih1d3 was found to interact with an intermediate chain of ODA as well as with Hsp70 and Hsp90. Our results suggest that Pih1d3 contributes to cytoplasmic preassembly of dynein complexes in spermatogenic cells by stabilizing and promoting complex formation by ODA and IDA proteins.
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Sale, W. S., U. W. Goodenough, and J. E. Heuser. "The substructure of isolated and in situ outer dynein arms of sea urchin sperm flagella." Journal of Cell Biology 101, no. 4 (October 1, 1985): 1400–1412. http://dx.doi.org/10.1083/jcb.101.4.1400.
Full textAbstract:
Outer-arm dynein from the sperm of the sea urchin S. purpuratus was adsorbed to mica flakes and visualized by the quick-freeze, deep-etch technique. Replicas reveal particles comprised of two globular heads joined by two irregularly shaped stems which make contact along their length. One head is pear-shaped (18.5 X 12.5 nm) and the other is spherical (14.5-nm diam). The stems are decorated by a complex of bead-like subunits. The same two-headed protein is found in the 21S dynein-1 fraction of sucrose gradients. The beta-heavy chain/intermediate chain 1 (beta/IC-1) dynein subfraction, produced by low-salt dialysis and zonal centrifugation of the high-salt-extracted dynein-1, contains only single-headed molecules with single stems. These heads are predominantly pear-shaped (18.5 X 12.5 nm). Since 21S dynein-1 contains two heavy chains (alpha and beta), and the beta/IC-1 subfraction is comprised of only the beta-heavy chain (Tang et al., 1982, J. Biol. Chem. 257: 508-515), we conclude that each head is formed by a heavy chain, that the pear-shaped head contains the beta-heavy chain, and that the spherical head contains the alpha-heavy chain. The in situ outer dynein arms of demembranated sperm were also studied by the quick-freeze, deep-etch method. When frozen in reactivation buffer devoid of ATP, each arm consists of a large globular head that attaches to the A-microtubule by distally skewed subunits and attaches to the B-microtubule by a slender stalk. In ATP, this head shifts its orientation such that it can be seen to be constructed from two globular domains. We offer possible correlates between the in situ and the in vitro images, and we compare the structure of sea-urchin dynein with dynein previously described from Chlamydomonas and Tetrahymena.
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Kamiya, R., E. Kurimoto, and E. Muto. "Two types of Chlamydomonas flagellar mutants missing different components of inner-arm dynein." Journal of Cell Biology 112, no. 3 (February 1, 1991): 441–47. http://dx.doi.org/10.1083/jcb.112.3.441.
Full textAbstract:
Two types of Chlamydomonas reinhardtii flagellar mutants (idaA and idaB) lacking partial components of the inner-arm dynein were isolated by screening mutations that produce paralyzed phenotypes when present in a mutant missing outer-arm dynein. Of the currently identified three inner-arm subspecies I1, I2, and I3, each containing two heterologous heavy chains (Piperno, G., Z. Ramanis, E. F. Smith, and W. S. Sale. 1990. J. Cell Biol. 110:379-389), idaA and idaB lacked I1 and I2, respectively. The 13 idA isolates comprised three genetically different groups (ida1, ida2, ida3) and the two idaB isolates comprised a single group (ida4). In averaged cross-section electron micrographs, inner dynein arms in wild-type axonemes appeared to have two projections pointing to discrete directions. In ida1-3 and ida4 axonemes, on the other hand, either one of them was missing or greatly diminished. Both projections were weak in the double mutant ida1-3 x ida4. These observations suggest that the inner dynein arms in Chlamydomonas axonemes are aligned not in a single straight row, but in a staggered row or two discrete rows. Both ida1-3 and ida4 swam at reduced speed. Thus, the inner-arm subspecies missing in these mutants are not necessary for flagellar motility. However, the double mutants ida1-3 x ida4 were nonmotile, suggesting that axonemes with significant defects in inner arms cannot function. The inner-arm dynein should be important for the generation of axonemal beating.
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Aprea, Isabella, Johanna Raidt, Inga Marlena Höben, Niki Tomas Loges, Tabea Nöthe-Menchen, Petra Pennekamp, Heike Olbrich, et al. "Defects in the cytoplasmic assembly of axonemal dynein arms cause morphological abnormalities and dysmotility in sperm cells leading to male infertility." PLOS Genetics 17, no. 2 (February 26, 2021): e1009306. http://dx.doi.org/10.1371/journal.pgen.1009306.
Full textAbstract:
Axonemal protein complexes, such as outer (ODA) and inner (IDA) dynein arms, are responsible for the generation and regulation of flagellar and ciliary beating. Studies in various ciliated model organisms have shown that axonemal dynein arms are first assembled in the cell cytoplasm and then delivered into axonemes during ciliogenesis. In humans, mutations in genes encoding for factors involved in this process cause structural and functional defects of motile cilia in various organs such as the airways and result in the hereditary disorder primary ciliary dyskinesia (PCD). Despite extensive knowledge about the cytoplasmic assembly of axonemal dynein arms in respiratory cilia, this process is still poorly understood in sperm flagella. To better define its clinical relevance on sperm structure and function, and thus male fertility, further investigations are required. Here we report the fertility status in different axonemal dynein preassembly mutant males (DNAAF2/ KTU, DNAAF4/ DYX1C1, DNAAF6/ PIH1D3, DNAAF7/ZMYND10, CFAP300/C11orf70 and LRRC6). Besides andrological examinations, we functionally and structurally analyzed sperm flagella of affected individuals by high-speed video- and transmission electron microscopy as well as systematically compared the composition of dynein arms in sperm flagella and respiratory cilia by immunofluorescence microscopy. Furthermore, we analyzed the flagellar length in dynein preassembly mutant sperm. We found that the process of axonemal dynein preassembly is also critical in sperm, by identifying defects of ODAs and IDAs in dysmotile sperm of these individuals. Interestingly, these mutant sperm consistently show a complete loss of ODAs, while some respiratory cilia from the same individual can retain ODAs in the proximal ciliary compartment. This agrees with reports of solely one distinct ODA type in sperm, compared to two different ODA types in proximal and distal respiratory ciliary axonemes. Consistent with observations in model organisms, we also determined a significant reduction of sperm flagellar length in these individuals. These findings are relevant to subsequent studies on the function and composition of sperm flagella in PCD patients and non-syndromic infertile males. Our study contributes to a better understanding of the fertility status in PCD-affected males and should help guide genetic and andrological counselling for affected males and their families.
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Oda, Toshiyuki, Nobutaka Hirokawa, and Masahide Kikkawa. "Three-dimensional structures of the flagellar dynein–microtubule complex by cryoelectron microscopy." Journal of Cell Biology 177, no. 2 (April 16, 2007): 243–52. http://dx.doi.org/10.1083/jcb.200609038.
Full textAbstract:
The outer dynein arms (ODAs) of the flagellar axoneme generate forces needed for flagellar beating. Elucidation of the mechanisms underlying the chemomechanical energy conversion by the dynein arms and their orchestrated movement in cilia/flagella is of great importance, but the nucleotide-dependent three-dimensional (3D) movement of dynein has not yet been observed. In this study, we establish a new method for reconstructing the 3D structure of the in vitro reconstituted ODA–microtubule complex and visualize nucleotide-dependent conformational changes using cryoelectron microscopy and image analysis. As the complex went from the rigor state to the relaxed state, the head domain of the β heavy chain shifted by 3.7 nm toward the B tubule and inclined 44° inwards. These observations suggest that there is a mechanism that converts head movement into the axonemal sliding motion.
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King, S. J., W. B. Inwood, E. T. O'Toole, J. Power, and S. K. Dutcher. "The bop2-1 mutation reveals radial asymmetry in the inner dynein arm region of Chlamydomonas reinhardtii." Journal of Cell Biology 126, no. 5 (September 1, 1994): 1255–66. http://dx.doi.org/10.1083/jcb.126.5.1255.
Full textAbstract:
Strains of Chlamydomonas reinhardtii with a mutant allele at the BOP2 locus swim slowly and have an abnormal flagellar waveform similar to previously identified strains with defects in the inner arm region. Double mutant strains with the bop2-1 allele and any of 17 different mutations that affect the dynein arm region swim more slowly than either parent, which suggests that the bop2-1 mutation does not affect solely the outer dynein arms, the I1 or ida4 inner dynein arms, or the dynein regulatory complex. Flagellar axonemes isolated from bop2-1 cells are missing a phosphorylated polypeptide of 152 kD. Electron microscopic analysis shows that bop2-1 axonemes are missing density in the inner dynein arm region. Surprisingly, two populations of images were observed in longitudinal sections of axonemes from the bop2-1 strain. In the 10 longitudinal axonemes examined, a portion of the dynein regulatory complex and a newly identified structure, the projection, are affected. In five of these 10 longitudinal axonemes examined, two lobes of the ida4 inner arm are also missing. By examining the cross-sectional images of wild-type and bop2-1 axonemes at each outer doublet position around the axoneme, we have determined that the bop2-1 mutation affects the assembly of inner arm region components in a doublet specific manner. Doublets 5, 6, and 8 have the most severe deficiency, doublet 9 has an intermediate phenotype, and doublets 2, 3, 4, and 7 have the least severe phenotype. The bop2-1 mutation provides the first evidence of radial asymmetry in the inner dynein arm region.
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Ishikawa, Takashi, Hitoshi Sakakibara, and Kazuhiro Oiwa. "The Architecture of Outer Dynein Arms in Situ." Journal of Molecular Biology 368, no. 5 (May 2007): 1249–58. http://dx.doi.org/10.1016/j.jmb.2007.02.072.
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