Limits...
Localization of calmodulin and dynein light chain LC8 in flagellar radial spokes.

Yang P, Diener DR, Rosenbaum JL, Sale WS - J. Cell Biol. (2001)

Bottom Line: The isolated radial spokes sediment as 20S complexes that are the size and shape of radial spokes.Extracted radial spokes rescue radial spoke structure when reconstituted with isolated axonemes derived from the radial spoke mutant pf14.Isolated radial spokes are composed of the 17 previously defined spoke proteins as well as at least five additional proteins including calmodulin and the ubiquitous dynein light chain LC8.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Emory University, School of Medicine, Atlanta, Georgia 30322, USA.

ABSTRACT
Genetic and in vitro analyses have revealed that radial spokes play a crucial role in regulation of ciliary and flagellar motility, including control of waveform. However, the mechanisms of regulation are not understood. Here, we developed a novel procedure to isolate intact radial spokes as a step toward understanding the mechanism by which these complexes regulate dynein activity. The isolated radial spokes sediment as 20S complexes that are the size and shape of radial spokes. Extracted radial spokes rescue radial spoke structure when reconstituted with isolated axonemes derived from the radial spoke mutant pf14. Isolated radial spokes are composed of the 17 previously defined spoke proteins as well as at least five additional proteins including calmodulin and the ubiquitous dynein light chain LC8. Analyses of flagellar mutants and chemical cross-linking studies demonstrated calmodulin and LC8 form a complex located in the radial spoke stalk. We postulate that calmodulin, located in the radial spoke stalk, plays a role in calcium control of flagellar bending.

Show MeSH

Related in: MedlinePlus

The 20S radial spoke fraction contains T-shaped particles with the dimensions of intact radial spoke in situ. (A) Longitudinal images of thin section (top) and negatively stained (bottom) doublet microtubules, viewed edge-on, reveals paired radial spoke structures (arrows; compare with Witman et al. 1978). (B) Negative-stained images of the 20S radial spoke particles. A globular structure (arrowhead) was often found at the proximal end of the isolated spokes. Bar, 40 nm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2192029&req=5

Figure 3: The 20S radial spoke fraction contains T-shaped particles with the dimensions of intact radial spoke in situ. (A) Longitudinal images of thin section (top) and negatively stained (bottom) doublet microtubules, viewed edge-on, reveals paired radial spoke structures (arrows; compare with Witman et al. 1978). (B) Negative-stained images of the 20S radial spoke particles. A globular structure (arrowhead) was often found at the proximal end of the isolated spokes. Bar, 40 nm.

Mentions: As a further test, the 20S complexes were directly examined by negative stain electron microscopy. Individual T-shaped particles with the size and shape of radial spokes, as visualized in axonemes, were revealed (Fig. 3). The isolated radial spoke is 40–42 nm long, and a globular structure was often present on the end of the stalk opposite to the T-shaped head (Fig. 3, arrowhead). We conclude that the 20S fraction contains intact radial spokes.


Localization of calmodulin and dynein light chain LC8 in flagellar radial spokes.

Yang P, Diener DR, Rosenbaum JL, Sale WS - J. Cell Biol. (2001)

The 20S radial spoke fraction contains T-shaped particles with the dimensions of intact radial spoke in situ. (A) Longitudinal images of thin section (top) and negatively stained (bottom) doublet microtubules, viewed edge-on, reveals paired radial spoke structures (arrows; compare with Witman et al. 1978). (B) Negative-stained images of the 20S radial spoke particles. A globular structure (arrowhead) was often found at the proximal end of the isolated spokes. Bar, 40 nm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2192029&req=5

Figure 3: The 20S radial spoke fraction contains T-shaped particles with the dimensions of intact radial spoke in situ. (A) Longitudinal images of thin section (top) and negatively stained (bottom) doublet microtubules, viewed edge-on, reveals paired radial spoke structures (arrows; compare with Witman et al. 1978). (B) Negative-stained images of the 20S radial spoke particles. A globular structure (arrowhead) was often found at the proximal end of the isolated spokes. Bar, 40 nm.
Mentions: As a further test, the 20S complexes were directly examined by negative stain electron microscopy. Individual T-shaped particles with the size and shape of radial spokes, as visualized in axonemes, were revealed (Fig. 3). The isolated radial spoke is 40–42 nm long, and a globular structure was often present on the end of the stalk opposite to the T-shaped head (Fig. 3, arrowhead). We conclude that the 20S fraction contains intact radial spokes.

Bottom Line: The isolated radial spokes sediment as 20S complexes that are the size and shape of radial spokes.Extracted radial spokes rescue radial spoke structure when reconstituted with isolated axonemes derived from the radial spoke mutant pf14.Isolated radial spokes are composed of the 17 previously defined spoke proteins as well as at least five additional proteins including calmodulin and the ubiquitous dynein light chain LC8.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Emory University, School of Medicine, Atlanta, Georgia 30322, USA.

ABSTRACT
Genetic and in vitro analyses have revealed that radial spokes play a crucial role in regulation of ciliary and flagellar motility, including control of waveform. However, the mechanisms of regulation are not understood. Here, we developed a novel procedure to isolate intact radial spokes as a step toward understanding the mechanism by which these complexes regulate dynein activity. The isolated radial spokes sediment as 20S complexes that are the size and shape of radial spokes. Extracted radial spokes rescue radial spoke structure when reconstituted with isolated axonemes derived from the radial spoke mutant pf14. Isolated radial spokes are composed of the 17 previously defined spoke proteins as well as at least five additional proteins including calmodulin and the ubiquitous dynein light chain LC8. Analyses of flagellar mutants and chemical cross-linking studies demonstrated calmodulin and LC8 form a complex located in the radial spoke stalk. We postulate that calmodulin, located in the radial spoke stalk, plays a role in calcium control of flagellar bending.

Show MeSH
Related in: MedlinePlus