A Regulatory Light Chain of Ciliary Outer Arm Dynein inTetrahymena thermophila

Abstract

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 ...

The radial spokes and central apparatus: Mechano‐chemical transducers that regulate flagellar motility

“…Modulation of microtubule sliding by localized control of dynein activity may involve the localized phosphorylation of key regulatory proteins including subunits of the dynein arms [Hamasaki et al, 1991;Habermacher and Sale, 1997;King and Dutcher, 1997;Christensen et al, 2001] and possibly components of the DRC. These modifications are not likely to operate on the time scale of a single beat cycle to modulate the inherent oscillatory behavior of beating cilia and flagella.…”

“…For example, in Paramecia, Hamasaki and co-workers have identified one dynein light chain that is phosphorylated in a cAMP-dependent manner, and this phosphorylation correlated with increased swimming velocity [Hamasaki et al, 1991]. And, in Tetrahymena phosphorylation of a dynein light chain resulted in an approximately 70% increase in in vitro microtubule translocation velocity compared with its unphosphorylated counterpart [Christensen et al, 2001].…”

PDGFRβ and oncogenic, mutant PDGFRα D842V promote disassembly of primary cilia by a PLCγ and AURKA dependent mechanism

“…SDS-PAGE and western blotting were performed as described previously (Christensen et al, 2001;Schrøder et al, 2011), except that secondary antibodies were conjugated to horseradish peroxidase, and blots were developed with the FUSION-Fx chemiluminescence system (Vilber Lourmat). Images were processed in Adobe Photoshop CS6.…”

PDGFRα signaling in the primary cilium regulates NHE1-dependent fibroblast migration via coordinated differential activity of MEK1/2-ERK1/2-p90RSK and AKT signaling pathways

“…SDS-PAGE and western blotting (WB) was carried out essentially as previously described (Christensen et al, 2001). Cells were grown in Petri dishes, washed in ice-cold PBS and lysed with SDS lysis buffer.…”