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1.
Oda5p, a novel axonemal protein required for assembly of the outer dynein arm and an associated adenylate kinase
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Wirschell M Pazour G Yoda A Hirono M Kamiya R Witman GB 《Molecular biology of the cell》2004,15(6):2729-2741
Of the uncloned ODA genes required for outer dynein arm assembly in Chlamydomonas, ODA5 and ODA10 are of particular interest because they do not encode known subunits of the outer arm or the outer dynein arm-docking complex (ODA-DC), and because genetic studies suggest their products interact. Beginning with a tagged oda5 allele, we isolated genomic and cDNA clones of the wild-type gene. ODA5 predicts a novel, 66-kDa coiled-coil protein. Immunoblotting indicates Oda5p is an axonemal component that assembles onto the axoneme independently of the outer arm and ODA-DC and is uniquely missing in oda5 and oda10 axonemes. Oda5p is released from the axoneme by extraction with 0.6 M KCl, but the soluble Oda5p does not cosediment with the outer dynein arm/ODA-DC in sucrose gradients. Quantitative mass spectrometry by using isotope coded affinity tagging revealed that a previously unidentified adenylate kinase is reduced 35-50% in oda5 flagella. Direct enzymatic assays demonstrated a comparable reduction in adenylate kinase activity in oda5 flagella, and also in oda10 flagella, but not in flagella of other oda mutants. We propose that Oda5p is part of a novel axonemal complex that is required for outer arm assembly and anchors adenylate kinase in proximity to the arm. 相似文献
2.
We previously found that a mutation at the ODA7 locus in Chlamydomonas prevents axonemal outer row dynein assembly by blocking association of heavy chains and intermediate chains in the cytoplasm. We have now cloned the ODA7 locus by walking in the Chlamydomonas genome from nearby molecular markers, confirmed the identity of the gene by rescuing the mutant phenotype with genomic clones, and identified the ODA7 gene product as a 58-kDa leucine-rich repeat protein unrelated to outer row dynein LC1. Oda7p is missing from oda7 mutant flagella but is present in flagella of other outer row or inner row dynein assembly mutants. However, Oda7 levels are greatly reduced in flagella that lack both outer row dynein and inner row I1 dynein. Biochemical fractionation and rebinding studies support a model in which Oda7 participates in a previously uncharacterized structural link between inner and outer row dyneins. 相似文献
3.
Experiments were carried out to see if isolated inner arm dyneins could functionally combine with axonemes lacking them. High-salt extract from the axoneme of Chlamydomonas oda1 mutant lacking outer-arm dynein was added to the demembranated cell models of ida1oda1 lacking inner arm dynein f (dynein I1) and outer arm dynein. After incubation, the originally paralyzed ida1oda1 axonemes recovered the ability to beat in the presence of ATP. A similar good motility recovery after incubation with crude oda1 extract was observed in ida9oda2 lacking outer arm and inner arm dynein c, and partial recovery in ida4oda1 lacking outer arm and inner arm species a, c, and d. These observations indicate that dynein f and dynein c can functionally bind with mutant axonemes lacking them. A method for combining isolated inner arm dyneins with axonemes in a functionally active manner should provide a powerful experimental tool with which to study the mechanism of beating. 相似文献
4.
Functional reconstitution of Chlamydomonas outer dynein arms from alpha- beta and gamma subunits: requirement of a third factor 总被引:3,自引:3,他引:0
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《The Journal of cell biology》1994,126(3):737-745
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. 相似文献
5.
ODA16 aids axonemal outer row dynein assembly through an interaction with the intraflagellar transport machinery 总被引:2,自引:0,他引:2
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Formation of flagellar outer dynein arms in Chlamydomonas reinhardtii requires the ODA16 protein at a previously uncharacterized assembly step. Here, we show that dynein extracted from wild-type axonemes can rebind to oda16 axonemes in vitro, and dynein in oda16 cytoplasmic extracts can bind to docking sites on pf28 (oda) axonemes, which is consistent with a role for ODA16 in dynein transport, rather than subunit preassembly or binding site formation. ODA16 localization resembles that seen for intraflagellar transport (IFT) proteins, and flagellar abundance of ODA16 depends on IFT. Yeast two-hybrid analysis with mammalian homologues identified an IFT complex B subunit, IFT46, as a directly interacting partner of ODA16. Interaction between Chlamydomonas ODA16 and IFT46 was confirmed through in vitro pull-down assays and coimmunoprecipitation from flagellar extracts. ODA16 appears to function as a cargo-specific adaptor between IFT particles and outer row dynein needed for efficient dynein transport into the flagellar compartment. 相似文献
6.
Flagellar dynein activity is regulated by phosphorylation. One critical phosphoprotein substrate in Chlamydomonas is the 138-kDa intermediate chain (IC138) of the inner arm dyneins (Habermacher, G., and Sale, W. S. (1997) J. Cell Biol. 136, 167-176). In this study, several approaches were used to determine that casein kinase I (CKI) is physically anchored in the flagellar axoneme and regulates IC138 phosphorylation and dynein activity. First, using a videomicroscopic motility assay, selective CKI inhibitors rescued dynein-driven microtubule sliding in axonemes isolated from paralyzed flagellar mutants lacking radial spokes. Rescue of dynein activity failed in axonemes isolated from these mutant cells lacking IC138. Second, CKI was unequivocally identified in salt extracts from isolated axonemes, whereas casein kinase II was excluded from the flagellar compartment. Third, Western blots indicate that within flagella, CKI is anchored exclusively to the axoneme. Analysis of multiple Chlamydomonas motility mutants suggests that the axonemal CKI is located on the outer doublet microtubules. Finally, CKI inhibitors that rescued dynein activity blocked phosphorylation of IC138. We propose that CKI is anchored on the outer doublet microtubules in position to regulate flagellar dynein. 相似文献
7.
The outer dynein arm-docking complex: composition and characterization of a subunit (oda1) necessary for outer arm assembly
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Takada S Wilkerson CG Wakabayashi K Kamiya R Witman GB 《Molecular biology of the cell》2002,13(3):1015-1029
To learn more about how dyneins are targeted to specific sites in the flagellum, we have investigated a factor necessary for binding of outer arm dynein to the axonemal microtubules of Chlamydomonas. This factor, termed the outer dynein arm-docking complex (ODA-DC), previously was shown to be missing from axonemes of the outer dynein armless mutants oda1 and oda3. We have now partially purified the ODA-DC, determined that it contains equimolar amounts of M(r) approximately 105,000 and approximately 70,000 proteins plus a third protein of M(r) approximately 25,000, and found that it is associated with the isolated outer arm in a 1:1 molar ratio. We have cloned a full-length cDNA encoding the M(r) approximately 70,000 protein; the sequence predicts a 62.5-kDa protein with potential homologs in higher ciliated organisms, including humans. Sequencing of corresponding cDNA from strain oda1 revealed it has a mutation resulting in a stop codon just downstream of the initiator ATG; thus, it is unable to make the full-length M(r) approximately 70,000 protein. These results demonstrate that the ODA1 gene encodes the M(r) approximately 70,000 protein, and that the protein is essential for assembly of the ODA-DC and the outer dynein arm onto the doublet microtubule. 相似文献
8.
Tanner CA Rompolas P Patel-King RS Gorbatyuk O Wakabayashi K Pazour GJ King SM 《Molecular biology of the cell》2008,19(9):3724-3734
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 approximately 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. 相似文献
9.
The outer dynein arms of Chlamydomonas flagella are attached to a precise site on the outer doublet microtubules and repeat at a regular interval of 24 nm. This binding is mediated by the outer dynein arm docking complex (ODA-DC), which is composed of three protein subunits. In this study, antibodies against the 83- and 62-kD subunits (DC83 and DC62) of the ODA-DC were used to analyze its state of association with outer arm components within the cytoplasm, and its localization in the axonemes of oda mutants. Immunoprecipitation indicates that DC83 and DC62 are preassembled within the cytoplasm, but that they are not associated with outer arm dynein. Both proteins are lost or greatly diminished in oda1 and oda3, mutants in the structural genes of DC62 and DC83, respectively, demonstrating that their association is necessary for their stable presence in the cytoplasm. Immunoelectron microscopy indicates that DC83 repeats at 24-nm intervals along the length of the doublet microtubules of oda6, which lacks outer arms; thus, outer arm periodicity may be determined by the ODA-DC. Flagellar regeneration and temporary dikaryon experiments indicate that the ODA-DC can be rapidly transported into the flagellum and assembled on the doublet microtubules independently of the outer arms and independently of flagellar growth. Unexpectedly, the intensity of ODA-DC labeling decreased toward the distal ends of axonemes of oda6 but not wild-type cells, suggesting that the outer arms reciprocally contribute to the assembly/stability of the ODA-DC. 相似文献
10.
DiBella LM Gorbatyuk O Sakato M Wakabayashi K Patel-King RS Pazour GJ Witman GB King SM 《Molecular biology of the cell》2005,16(12):5661-5674
Tctex1 and Tctex2 were originally described as potential distorters/sterility factors in the non-Mendelian transmission of t-haplotypes in mice. These proteins have since been identified as subunits of cytoplasmic and/or axonemal dyneins. Within the Chlamydomonas flagellum, Tctex1 is a subunit of inner arm I1. We have now identified a second Tctex1-related protein (here termed LC9) in Chlamydomonas. LC9 copurifies with outer arm dynein in sucrose density gradients and is missing only in those strains completely lacking this motor. Zero-length cross-linking of purified outer arm dynein indicates that LC9 interacts directly with both the IC1 and IC2 intermediate chains. Immunoblot analysis revealed that LC2, LC6, and LC9 are missing in an IC2 mutant strain (oda6-r88) that can assemble outer arms but exhibits significantly reduced flagellar beat frequency. This defect is unlikely to be due to lack of LC6, because an LC6 null mutant (oda13) exhibits only a minor swimming abnormality. Using an LC2 null mutant (oda12-1), we find that although some outer arm dynein components assemble in the absence of LC2, they are nonfunctional. In contrast, dyneins from oda6-r88, which also lack LC2, retain some activity. Furthermore, we observed a synthetic assembly defect in an oda6-r88 oda12-1 double mutant. These data suggest that LC2, LC6, and LC9 have different roles in outer arm assembly and are required for wild-type motor function in the Chlamydomonas flagellum. 相似文献
11.
Identification of oda6 as a Chlamydomonas dynein mutant by rescue with the wild-type gene 总被引:12,自引:4,他引:8
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We find that two Chlamydomonas outer arm dynein assembly loci, oda6 and oda9, are located on the left arm of linkage group XII, in the vicinity of the previously mapped locus for a 70,000 Mr dynein intermediate chain protein. Restriction fragment length polymorphism mapping indicates that this dynein gene is very closely linked to the oda6 locus. A cDNA clone encoding the 70,000 Mr protein was isolated, sequenced, and used to select genomic clones spanning the corresponding locus from both wild-type and oda6 libraries. When wild-type clones were introduced into cells containing an oda6 allele, the mutant phenotype was rescued, while no rescue was observed after transformation with oda6 clones. Genetic analysis further revealed that newly introduced gene copies were responsible for the rescued phenotype and thus confirms that ODA6 encodes the 70,000 Mr dynein intermediate chain protein. The inability of oda6 mutants to assemble any major outer arm dynein subunits shows that this protein is essential for assembly of stable outer dynein arms. This is the first use of transformation with a wild-type gene to identify the product of a Chlamydomonas mutant. 相似文献
12.
Recent indirect observations have suggested that various axonemal proteins in cilia and flagella of live cells undergo turnover independently of shortening or elongation of the axoneme. To gain direct evidence, here we examined using a FRAP (fluorescence recovery after photobleaching) technique whether actin, a subunit of inner arm dynein, is being turned over in Chlamydomonas flagella. Fluorescently labeled rabbit actin was introduced by electroporation into the cells of ida5oda1, a double mutant between oda1 lacking outer arm dynein and ida5 lacking several species of inner arm dyneins due to the absence of a conventional-type actin. In actin-loaded cells, flagella became motile and fluorescent due to incorporation of inner-arm dyneins containing the labeled actin. Cells were sandwiched between an agar layer and a coverslip so as to restrict flagellar movement. After a small portion of a flagellum was photobleached, the fluorescence intensity in the bleached area was monitored with a sensitive video camera. The fluorescence intensity in the photobleached region was found to recover 10-40% of the original level over several tens of minutes without changing its position. The time course and extent of the recovery varied greatly from one cell to another, suggesting that the turnover depends on cellular conditions. Western blot analysis indicated that 70-80% of flagellar actin was associated with the axoneme. Hence this experiment provides direct evidence that an axonemal component undergoes dynamic exchange in stationary flagella. 相似文献
13.
The LC7 light chains of Chlamydomonas flagellar dyneins interact with components required for both motor assembly and regulation
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DiBella LM Sakato M Patel-King RS Pazour GJ King SM 《Molecular biology of the cell》2004,15(10):4633-4646
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 approximately 20% in axonemes isolated from strains lacking inner arm I1 and are approximately 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 approximately 30% of its outer arms, LC7b fails to copurify with either dynein, suggesting that it interacts with LC7a. Furthermore, both the outer arm gamma 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. 相似文献
14.
The cytoplasmic dynein regulatory factor Lis1, which induces a persistent tight binding to microtubules and allows for transport of cargoes under high-load conditions, is also present in motile cilia/flagella. We observed that Lis1 levels in flagella of Chlamydomonas strains that exhibit defective motility due to mutation of various axonemal substructures were greatly enhanced compared with wild type; this increase was absolutely dependent on the presence within the flagellum of the outer arm dynein α heavy chain/light chain 5 thioredoxin unit. To assess whether cells might interpret defective motility as a "high-load environment," we reduced the flagellar beat frequency of wild-type cells through enhanced viscous load and by reductive stress; both treatments resulted in increased levels of flagellar Lis1, which altered the intrinsic beat frequency of the trans flagellum. Differential extraction of Lis1 from wild-type and mutant axonemes suggests that the affinity of outer arm dynein for Lis1 is directly modulated. In cytoplasm, Lis1 localized to two punctate structures, one of which was located near the base of the flagella. These data reveal that the cell actively monitors motility and dynamically modulates flagellar levels of the dynein regulatory factor Lis1 in response to imposed alterations in beat parameters. 相似文献
15.
Kamiya R 《Methods (San Diego, Calif.)》2000,22(4):383-387
Flagellar mutants of Chlamydomonas have greatly contributed to our understanding of the function of axonemes and axonemal dyneins. An important step in studying mutants is to correlate the molecular and structural defects in the axoneme with motility. This is not always easy, however, partly because it is often necessary to quantify axonemal motility by measuring the cell's swimming velocity, the flagellar beat frequency, or flagellar waveform in a number of cells or axonemes. To skip this time-consuming step, a quick method for measuring the average flagellar beat frequency in a population of cells is developed based on fast Fourier transform (FFT) analysis of the vibration of cell bodies. This method yields the average beat frequency within 10-60 s and has been used as a powerful tool for identifying mutants lacking various dynein species. It is also particularly useful for studies analyzing detergent-extracted cell models under various reactivation conditions. 相似文献
16.
To elucidate the subunit composition of axonemal inner-arm dynein, we examined a 38 kDa protein (p38) co-purified with a Chlamydomonas inner arm subspecies, dynein d. We found it is a novel protein conserved among a variety of organisms with motile cilia and flagella. Immunoprecipitation using specific antibody verified its association with a heavy chain, actin and a previously identified light chain (p28). Unexpectedly, mutant axonemes lacking dynein d and other dyneins retained reduced amounts of p38. This finding suggests that p38 is involved in the docking of dynein d to specific loci. 相似文献
17.
Yagi T Minoura I Fujiwara A Saito R Yasunaga T Hirono M Kamiya R 《The Journal of biological chemistry》2005,280(50):41412-41420
Ciliary and flagellar axonemes contain multiple inner arm dyneins of which the functional difference is largely unknown. In this study, a Chlamydomonas mutant, ida9, lacking inner arm dynein c was isolated and shown to carry a mutation in the DHC9 dynein heavy chain gene. The cDNA sequence of DHC9 was determined, and its information was used to show that >80% of it is lost in the mutant. Electron microscopy and image analysis showed that the ida9 axoneme lacked electron density near the base of the S2 radial spoke, indicating that dynein c localizes to this site. The mutant ida9 swam only slightly slower than the wild type in normal media. However, swimming velocity was greatly reduced when medium viscosity was modestly increased. Thus, dynein c in wild type axonemes must produce a significant force when flagella are beating in viscous media. Because motility analyses in vitro have shown that dynein c is the fastest among all the inner arm dyneins, we can regard this dynein as a fast yet powerful motor. 相似文献
18.
《The Journal of cell biology》1996,133(2):371-379
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. 相似文献
19.
G J Attwell C S Bricker A Schwandt M A Gorovsky D G Pennock 《The Journal of protozoology》1992,39(2):261-266
We have characterized a novel, temperature-sensitive mutation affecting motility in Tetrahymena thermophila. Mutants grew and divided normally at the restrictive temperature (38 degrees C), but became nonmotile. Scanning electron microscopic analysis indicated that nonmotile mutants contained the normal number of cilia and that the cilia were of normal length. Transmission electron microscopic analysis indicated that axonemes isolated from nonmotile mutants lacked outer dynein arms, so the mutation was named oad 1 (outer arm deficient). Motile mutants shifted to 38 degrees C under conditions that prevent cell growth and division (starvation) remained motile suggesting that once assembled into axonemes at the permissive temperature (28 degrees C) the outer arm dyneins remain functional at 38 degrees C. Starved, deciliated mutants regenerated a full complement of functional cilia at 38 degrees C, indicating that the mechanism that incorporates the outer arm dynein into developing axonemes is not affected by the oad 1 mutation. Starved, nonmotile mutants regained motility when shifted back to 28 degrees C, but not when incubated with cycloheximide. We interpret these results to rule out the hypothesis that the oad 1 mutation affects the site on the microtubules to which the outer arm dyneins bind. Axonemes isolated from mutants grown for one generation at 38 degrees C had a mean of 6.0 outer arm dyneins, and axonemes isolated from mutants grown for two generations at 38 degrees C had a mean of 3.2 outer arm dyneins. Taken together, these results indicate that the oad 1 mutation affects the synthesis of outer arm dyneins in Tetrahymena. 相似文献
20.
《The Journal of cell biology》1993,122(3):653-661
A new allele of the Chlamydomonas oda4 flagellar mutant (oda4-s7) possessing abnormal outer dynein arms was isolated. Unlike the previously described oda4 axoneme lacking all three (alpha, beta, and gamma) outer-arm dynein heavy chains, the oda4-s7 axoneme contains the alpha and gamma heavy chains and a novel peptide with a molecular mass of approximately 160 kD. The peptide reacts with a mAb (18 beta B) that recognizes an epitope on the NH2-terminal part of the beta heavy chain. These observations indicate that this mutant has a truncated beta heavy chain, and that the NH2-terminal part of the beta heavy chain is important for the stable assembly of the outer arms. In averaged electron microscopic images of outer arms from cross sections of axonemes, the mutant outer arm lacks its mid-portion, producing a forked appearance. Together with our previous finding that the mutant oda11 lacks the alpha heavy chain and the outermost portion of the arm (Sakakibara, H., D. R. Mitchell, and R. Kamiya. 1991. J. Cell Biol. 113:615-622), this result defines the approximate locations of the three outer arm heavy chains in the axonemal cross section. The swimming velocity of oda4-s7 is 65 +/- 8 microns/s, close to that of oda4 which lacks the entire outer arm (62 +/- 8 microns/s) but significantly lower than the velocities of wild type (194 +/- 23 microns/s) and oda11 (119 +/- 17 microns/s). Thus, the lack of the beta heavy chain impairs outer-arm function more seriously than does the lack of the alpha heavy chain, suggesting that the alpha and beta chains play different roles in outer arm function. 相似文献