[29] Purification and characterization of Chlamydomonas flagellar dyneins

King, Stephen M.; Otter, Tim; Witman, George B.

doi:10.1016/0076-6879(86)34097-7

Cited by 114 publications

(101 citation statements)

References 34 publications

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“…To identify axonemal components affected by the ptx/mutation, isolated axonemes prepared from wild-type and ptx/ cells were analyzed by gel electrophoresis. No differences in the dynein heavy chains were observed between mutant and wild-type axonemes after separation on 3-5 % acrylamide, 2-8 M urea gels (19) to resolve the high molecular weight proteins (data not shown). Among the over 200 proteins resolved by two-dimensional gel electrophoresis, the only consistent differences observed between wild-type and ptx/were at 75 kD.…”

Section: Identification Of Axonemal Components Correlated With the Ptmentioning

confidence: 99%

“…Lanes cut from the NEPHGE slab gel were covered with equilibration buffer (0.0625 M Tris, pH 6.8, 10% SDS) and transferred immediately to the stacking gel of the second dimension. The SDS-polyacrylamide gel for the second dimension was composed of 5-15% acrylamide and 0-2.4 M glycerol gradients as described by King et al (19). Proteins were revealed by silver stain (24).…”

Section: Two-dimensional Gel Electrophoresismentioning

confidence: 99%

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ptx1, a nonphototactic mutant of Chlamydomonas, lacks control of flagellar dominance.

Horst¹,

Witman²

1993

The Journal of Cell Biology

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Abstract.A new mutant strain of Chlamydomonas, ptxl, 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 wildtype cells, and causes a concentration-dependent shift in flagellar dominance in reactivated, demembranated cell models, ptxl-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.T He unicellular biflagellate alga Chlamydomonas has been used extensively in the study of flagellar motility. This is due, in part, to the ease with which mutant strains displaying aberrant motility can be produced and analyzed. As a result, the structures which generate motility are well characterized, yet the processes which regulate motility remain obscure. To study the regulation of flagellar motility, we have generated mutant strains of Chlamydomonas which are defective in their ability to phototax.The two flagella of Chlamydomonas can be distinguished based on their position relative to a specialized region of the chloroplast referred to as the eyespot (13). During phototaxis, the beat pattern of the cis-flagellum (the flagellum closest to the eyespot) and the trans-flagelium are modified, resulting in a change in swimming direction relative to the light. This requires that the cis-and trans-flagella respond differentially to the signal generated by photoreception. Previous studies have provided evidence that calcium is required for phototaxis (3,4,22,25,37). Moreover, the two flagella are differentially responsive to calcium, such that the cis-flagellum is more effective at force generation at low calcium 00 -9 M), and the trans-flagellum more effective at higher calcium (10 -7 M) (17). This differential sensitivity of the cis-and trans-flagella to calcium has been proposed to mediate ...

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Section: Identification Of Axonemal Components Correlated With the Ptmentioning

confidence: 99%

Section: Two-dimensional Gel Electrophoresismentioning

confidence: 99%

ptx1, a nonphototactic mutant of Chlamydomonas, lacks control of flagellar dominance.

Horst¹,

Witman²

1993

The Journal of Cell Biology

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“…Proteins from sucrose density gradient fractions were separated by electrophoresis in 5-20% polyacrylamide-SDS gels (King et al, 1986). The gels were stained with Coomassie blue and photographed on 35-mm Tech Pan film.…”

Section: Sds-page and Band Quantitationmentioning

confidence: 99%

The Outer Dynein Arm-Docking Complex: Composition and Characterization of a Subunit (Oda1) Necessary for Outer Arm Assembly

Takada

Wilkerson

Wakabayashi

et al. 2002

MBoC

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122

133

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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 ϳ105,000 and ϳ70,000 proteins plus a third protein of M r ϳ25,000, 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 ϳ70,000 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 ϳ70,000 protein. These results demonstrate that the ODA1 gene encodes the M r ϳ70,000 protein, and that the protein is essential for assembly of the ODA-DC and the outer dynein arm onto the doublet microtubule.

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“…No other labeled bands were detectable. These gels resolve proteins ranging from at least 500 to ,x,5 kD (14). The material that does not enter the gel may represent glucosaminoglycans that have incorporated radioactive SO4; the 3 % radiochemical impurities in the [35S]methionine preparations we used include some labeled SO4.…”

Section: Efficacy Of Drug Treatmentmentioning

confidence: 99%

“…If this step was omitted, labeled particulate material damaged the gels by irregularly forcing its way into their upper portions. 80-and 15-p.l aliquots of both samples were then run on 5-15% acrylamide gradient gels as described elsewhere (14). The gels were later stained with Coomassie brilliant blue and autoradiographed on Kodak X Mat XAR-5 film.…”

Section: Gel Analysis Of Protein Synthesismentioning

confidence: 99%

Protein synthesis and the cell cycle: centrosome reproduction in sea urchin eggs is not under translational control

1990

Trends in Genetics

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Abstract. The reproduction, or duplication, of the centrosome is an important event in a cell's preparation for mitosis. We sought to determine if centrosome reproduction is regulated by the synthesis and accumulation of cyclin proteins and/or the synthesis of centrosome-specific proteins at each cell cycle. We continuously treat sea urchin eggs, starting before fertilization, with a combination of emetine and anisomycin, drugs that have separate targets in the protein synthetic pathway. These drugs inhibit the postfertilization incorporation of [35S]methionine into precipitable material by 97.3-100%. Autoradiography of SDS-PAGE gels of drug-treated zygotes reveals that [35S]methionine incorporates exclusively into material that does not enter the gel and material that runs at the dye front; no other labeled bands are detected. Fertilization events and syngamy are normal in drug-treated zygotes, but the cell cycle arrests before first mitosis.The sperm aster doubles once in all zygotes to yield two asters. In a variable but significant percentage of zygotes, the asters continue to double. This continued doubling is slower than normal, asynchronous between zygotes, and sometimes asynchronous within individual zygotes. High voltage electron microscopy of serial semithick sections from drug-treated zygotes reveals that 90% of the daughter centrosomes contain two centrioles of normal appearance. From these results, we conclude that centrosome reproduction in sea urchin zygotes is not controlled by the accumulation of cyclin proteins or the synthesis of centrosomespecific proteins at each cell cycle. New centrosomes are assembled from preexisting pools of ready-to-use subunits. Furthermore, our results indicate that centrosomal and nuclear events are regulated by separate pathways.

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[29] Purification and characterization of Chlamydomonas flagellar dyneins

Cited by 114 publications

References 34 publications

ptx1, a nonphototactic mutant of Chlamydomonas, lacks control of flagellar dominance.

ptx1, a nonphototactic mutant of Chlamydomonas, lacks control of flagellar dominance.

The Outer Dynein Arm-Docking Complex: Composition and Characterization of a Subunit (Oda1) Necessary for Outer Arm Assembly

Protein synthesis and the cell cycle: centrosome reproduction in sea urchin eggs is not under translational control

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