In vitro phosphorylation of <i>Paramecium</i> axonemes and permeabilized cells

Hamasaki, Toshikazu; Murtaugh, Timothy J.; Satir, Birgit H.; Satir, Peter

doi:10.1002/cm.970120102

Cited by 78 publications

(56 citation statements)

References 30 publications

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“…In mammalian respiratory cilia (23)(24)(25), Ca 2ϩ also increases ciliary beat frequency synergistically with cAMP, possibly via a similar action on dynein regulatory light chain phosphorylation. This is different from Paramecium, where unusually the effect of Ca 2ϩ on cAMP-dependent phosphorylation of p29 is inhibitory, because Ca 2ϩ inhibits the relevant Paramecium cAMP-dependent protein kinase (5).…”

Section: Discussionmentioning

confidence: 69%

“…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)(6)(7)(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.…”

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confidence: 99%

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A Regulatory Light Chain of Ciliary Outer Arm Dynein inTetrahymena thermophila

Christensen¹,

Guerra²,

Wada³

et al. 2001

Journal of Biological Chemistry

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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 ...

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Section: Discussionmentioning

confidence: 69%

mentioning

confidence: 99%

A Regulatory Light Chain of Ciliary Outer Arm Dynein inTetrahymena thermophila

Christensen¹,

Guerra²,

Wada³

et al. 2001

Journal of Biological Chemistry

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“…In sea urchin sperm flagella, increasing Ca 2+ concentration increases the asymmetry of the waveform and finally induces quiescence (Brokaw, 1979;Brokaw et al, 1974;Gibbons and Gibbons, 1980). For reactivated cell models of Paramecium and Tetrahymena an increase in Ca 2+ induces reversal of swimming direction by changing the direction of the ciliary effective stroke (Bonini et al, 1991;Hamasaki et al, 1989;Izumi, 1985;Naitoh, 1972). For each case, high Ca 2+ -induced changes in motility are predicted to result from modulation of dynein activity on specific doublet microtubules.…”

Section: Discussionmentioning

confidence: 99%

Analysis of microtubule sliding patterns inChlamydomonasflagellar axonemes reveals dynein activity on specific doublet microtubules

Wargo

McPeek

Smith

2004

Journal of Cell Science

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Generating the complex waveforms characteristic of beating eukaryotic cilia and flagella requires spatial regulation of dynein-driven microtubule sliding. To generate bending, one prediction is that dynein arms alternate between active and inactive forms on specific subsets of doublet microtubules. Using an in vitro microtubule sliding assay combined with a structural approach, we determined that ATP induces sliding between specific subsets of doublet microtubules, apparently capturing one phase of the beat cycle. These studies were also conducted using high Ca 2+ conditions. In Chlamydomonas, high Ca 2+ induces changes in waveform which are predicted to result from regulating dynein activity on specific microtubules. Our results demonstrate that microtubule sliding in high Ca 2+ buffer is also induced by dynein arms on specific doublets. However, the pattern of microtubule sliding in high Ca 2+ buffer significantly differs from that in low Ca 2+ . These results are consistent with a 'switching hypothesis' of axonemal bending and provide evidence to indicate that Ca 2+ control of waveform includes modulation of the pattern of microtubule sliding between specific doublets. In addition, analysis of microtubule sliding in mutant axonemes reveals that the control mechanism is disrupted in some mutants.

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“…Diverse experimental systems have revealed that ciliary and flagellar motility is controlled by phosphorylation and that the protein kinases and phosphatases responsible for regulation are anchored in the axoneme (27)(28)(29)(30)(31)(32)(33). Thus, we postulate that phosphorylation regulates dynein activity, and key kinases and phosphatases are anchored in the axoneme near the dynein arms or in the central pair/radial spoke structures.…”

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confidence: 88%

Casein Kinase I Is Anchored on Axonemal Doublet Microtubules and Regulates Flagellar Dynein Phosphorylation and Activity

Yang

Sale

2000

Journal of Biological Chemistry

105

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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.The dynein ATPase is a family of molecular motors responsible for diverse cellular functions including retrograde microtubule-based transport of organelles, assembly and function of the Golgi and mitotic apparatus, and movement of cilia and flagella (1, 2). One of the primary questions is how dynein activity is regulated. The focus of this report is based on a series of studies revealing that the flagellar central pair apparatus and radial spokes play a primary role in regulation of flagellar dynein activity (3, 4). The regulatory mechanism involves a structural network of axonemal kinases and phosphatases that control phosphorylation of key subunits within the inner arm dyneins (5-8). In particular, phosphorylation of a dynein intermediate chain subunit, IC138, 1 correlates with inhibition of flagellar dynein activity (7). This network of enzymes along with the central pair apparatus, radial spokes, and the inner arm dyneins operates to control flagellar waveform (9 -17). These conclusions are based on genetic analysis and in vitro functional studies of flagella from Chlamydomonas reinhardtii. For example, mutations that disrupt either the central pair or the radial spokes result in flagellar paralysis (2,3,18). These paralyzed axonemes undergo dynein-driven microtubule sliding in vitro (19) but at greatly reduced rates of sliding when compared with wild-type axonemes (20). Reconstitution and functional assays demonstrate that the radial spokes are required for wild-type dynein activity, and the velocity of dyneindriven microtubule sliding is mediated by posttranslational modification of the inner dynein arms (20). These results are consistent with other evidence indicating that the central pair/ radial spoke apparatus along with the dynein...

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In vitro phosphorylation of Paramecium axonemes and permeabilized cells

Cited by 78 publications

References 30 publications

A Regulatory Light Chain of Ciliary Outer Arm Dynein inTetrahymena thermophila

A Regulatory Light Chain of Ciliary Outer Arm Dynein inTetrahymena thermophila

Analysis of microtubule sliding patterns inChlamydomonasflagellar axonemes reveals dynein activity on specific doublet microtubules

Casein Kinase I Is Anchored on Axonemal Doublet Microtubules and Regulates Flagellar Dynein Phosphorylation and Activity

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