Analyses of 22S Dynein Binding to <i>Tetrahymena</i> Axonemes Lacking Outer Dynein Arms

Sullivan, Jeanell; Ludmann, Susan A.; Hamasaki, Toshikazu; Pennock, David G.

doi:10.1111/j.1550-7408.1996.tb02466.x

Cited by 6 publications

(11 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We calculate that ϳ2% of the molecular weight of outer arm dynein is p34, assuming that there is one p34 for each ␣-heavy chain in the molecule, an assumption consistent with the measurements of Barkalow et al (9) on p29. The temperature-sensitive mutant, oad-1, when deficient in outer arms (15,16) shows no p34 phosphorylation. Further, the in vitro translocation velocity of microtubules by Tetrahymena 22 S outer arm dynein increases by about 70% after thiophosphorylation in the presence of cAMP, an increase that is attributable to cAMP-dependent phosphorylation of an outer arm dynein regulatory light chain leading to faster ciliary beat.…”

Section: Discussionmentioning

confidence: 99%

“…1). In axonemes isolated from cultures grown at 28°C (permissive temperature) where outer arm dyneins are present in this mutant (15,16) 10 M cAMP induced the [␥-…”

Section: Methodsmentioning

confidence: 99%

“…Cell Cultures-The following strains of T. thermophila were used in this study: a mucus-free mutant, SB 255 (14), and oad-1, a temperaturesensitive outer dynein arm-deficient mutant (15,16). SB 255 cells were grown either in 1-liter cultures in 2-liter capacity flat flasks with shaking at 38°C or in 8-liter aerated cultures containing Antifoam C (Sigma) at 27°C.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

A Regulatory Light Chain of Ciliary Outer Arm Dynein inTetrahymena thermophila

Christensen¹,

Guerra²,

Wada³

et al. 2001

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Ciliary beat frequency is primarily regulated by outer arm dyneins (22 S dynein). Chilcote and Johnson (Chilcote, T. J., and Johnson, K. A. (1990) J. Biol. Chem. 256, 17257-17266) previously studied isolated Tetrahymena 22 S dynein, identifying a protein p34, which showed cAMP-dependent phosphorylation. Here, we characterize the molecular biochemistry of p34 further, demonstrating that it is the functional ortholog of the 22 S dynein regulatory light chain, p29, in Paramecium. p34, thiophosphorylated in isolated axonemes in the presence of cAMP, co-purified with 22 S dynein and not with inner arm dynein (14 S dynein). Isolated 22 S dynein containing phosphorylated p34 showed ϳ70% increase in in vitro microtubule translocation velocity compared with its unphosphorylated counterpart. Extracted p34 rebound to isolated 22 S dynein from either Tetrahymena or Paramecium but not to 14 S dynein from either ciliate. Binding of radiolabeled p34 to 22 S dynein was competitive with p29. Phosphorylated p34 was not present in axonemes isolated from a mutant lacking outer arms. Two-dimensional gel electrophoresis followed by phosphorimaging revealed at least five phosphorylated p34-related spots, consistent with multiple phosphorylation sites in p34 or perhaps multiple isoforms of p34. These new features suggest that a class of outer arm dynein light chains including p34 regulates microtubule sliding velocity and consequently ciliary beat frequency through phosphorylation.Cilia are ubiquitous cellular nanomachines, found in protists and multicellular eukaryotes, including man, whose repetitive beat depends on a microtubule-based cytoskeleton, powered by molecular motors, the outer and inner rows of dynein arms (outer arm dynein, 22 S dynein; and inner arm dyneins, 14 S dynein; respectively). The arrangement of the dynein arms along the axonemes is complex (1). Dynein arm mechanochemistry is thought to regulate beat frequency and beat form by signal transduction mechanisms that change the parameters of microtubule sliding within the axoneme, such that the outer arm dyneins principally regulate beat frequency whereas the inner arm dyneins control beat form (cf. Refs. 2 and 3).cAMP specifically increases ciliary beat frequency (4), normally measured by an increase of swimming speed, in the protozoan ciliate, Paramecium tetraurelia. The increase occurs in living cells and in cells that have been permeabilized with Triton X-100 and reactivated with Mg 2ϩ -ATP; it persists in the permeabilized cells even when cAMP is subsequently removed and it is quenched by simultaneous addition of Ca 2ϩ to the medium (5-8). We previously reported on a molecule, p29, whose phosphorylation both in vivo and in vitro correlated with the cAMP-dependent Ca 2ϩ -sensitive increase in swimming speed. Further studies revealed that p29 is a component of outer arm dynein (6,8,9), which specifically binds to one heavy chain isoform of the three-headed 22 S outer arm dynein. Phosphorylation of p29 increases in vitro microtubule translocation velocity by outer ...

show abstract

Section: Discussionmentioning

confidence: 99%

“…1). In axonemes isolated from cultures grown at 28°C (permissive temperature) where outer arm dyneins are present in this mutant (15,16) 10 M cAMP induced the [␥-…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

A Regulatory Light Chain of Ciliary Outer Arm Dynein inTetrahymena thermophila

Christensen¹,

Guerra²,

Wada³

et al. 2001

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Recently, an outer-dynein-arm-deficient mutant of another ciliate, Tetrahymena thermophila, was generated by Pennock and colleagues (Ludman etal. 1993, Sullivan et al 1996. These mutant cells swim with normal ciliary beat form and frequency at a permissive temperature, but swim very slowly, or fail to sustain swimming, at a nonpermissive temperature, due to dramatically decreased ciliary beat activity.…”

Section: Regulation Of Dynein Activity In Light Of Ciliary Beat Controlmentioning

confidence: 95%

Regulation of outer-arm-dynein activity by phosphorylation and control of ciliary beat frequency

Hamasaki

1999

Protoplasma

View full text Add to dashboard Cite

Summary. Ciliary beating is empowered by a mechanochemical enzyme, dynein, which appears as two rows of projections on doublet microtubules. While inner-arm dyneins modulate beat form, outer-arm dynein empowers ciliary beat and sets beat frequency. Beat frequency is controlled via phosphorylation of outer-arm dynein. Using Paramecium tetraurelia as model system, we have previously identified a regulatory light chain of outer-arm dynein (22S dynein), Mr29 (p29), whose phosphorylation is cAMP-dependent. The phosphorylation state of the p29 in 22 S dynein determines in vitro microtubule translocation velocity. Although in vitro phosphorylation of p29 takes place in a short time, the percent change ist significantly less than the percent change in dynein activation, or in ciliary beat frequency. A potential mechanism that explains how a few activated dyneins can change ciliary beating is discussed.

show abstract

“…Historically, Tetrahymena axonemes were thought to contain two dyneins. The 22s dynein is a three-headed dynein particle [23] that makes up the outer arms [5,29,52, 601 and consists of three heavy chains, two intermediate chains, and at least five light chains [ 11,29,31,351. The 14s dyneins appear as a mixture of two different types of single-headed particles in the electron microscope [32] and were thought to contain two heavy chains, one intermediate chain, and an indeterminate number of light chains [32].…”

mentioning

confidence: 99%

New Axonemal Dynein Heavy Chains from Tetrahymena thermophila

Mobberley

Sullivan

Kong

et al. 1999

J Eukaryotic Microbiology

Self Cite

View full text Add to dashboard Cite

Two dyneins can be extracted from Tetrahymena ciliary axonemes. The 22S dynein contains three heavy chains (HC), sediments at 22S in a sucrose gradient, and makes up the outer arms. The 14S dynein contains two to six HCs, sediments at 14S, and is thought to contribute to formation of the inner arms. We have identified two large proteins that are extracted from Tetrahymena axonemes with high salt and that sediment together at approximately 18S. The two large proteins cleave when subjected to UV light in the presence of ATP and vanadate, suggesting both proteins are dynein HC. Antibodies against one of the 18S HCs do not recognize 22S dynein HCs. Antibodies to 22S dynein HC do not bind appreciably to 18S dynein photocleavage fragments. Taken together, these results indicate that the large proteins that sediment at 18S are axonemal dynein heavy chains.

show abstract

Analyses of 22S Dynein Binding to Tetrahymena Axonemes Lacking Outer Dynein Arms

Cited by 6 publications

References 34 publications

A Regulatory Light Chain of Ciliary Outer Arm Dynein inTetrahymena thermophila

A Regulatory Light Chain of Ciliary Outer Arm Dynein inTetrahymena thermophila

Regulation of outer-arm-dynein activity by phosphorylation and control of ciliary beat frequency

New Axonemal Dynein Heavy Chains from Tetrahymena thermophila

Contact Info

Product

Resources

About