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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.

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The 20S radial spoke complex contains all 17 previously identified spoke proteins (compare to Piperno et al. 1981). Proteins in one-dimensional (left) and 2D (right) gels of the 20S fraction from pf28pf30 are revealed by silver staining. The 17 radial spoke proteins are numbered. In addition, present in the 20S fraction are the 140- and 210-kD proteins (arrowheads), as well as two unknown proteins (open arrowheads) and tubulin (T). Phosphorylated proteins are labeled (*), and RSP2 distribution, based on Western blots, are indicated by the bracket. Acidic polypeptides are on the right. No polypeptides are present in the basic half of the gel (not shown). A 9% slab gel was used for the second dimension.
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Figure 2: The 20S radial spoke complex contains all 17 previously identified spoke proteins (compare to Piperno et al. 1981). Proteins in one-dimensional (left) and 2D (right) gels of the 20S fraction from pf28pf30 are revealed by silver staining. The 17 radial spoke proteins are numbered. In addition, present in the 20S fraction are the 140- and 210-kD proteins (arrowheads), as well as two unknown proteins (open arrowheads) and tubulin (T). Phosphorylated proteins are labeled (*), and RSP2 distribution, based on Western blots, are indicated by the bracket. Acidic polypeptides are on the right. No polypeptides are present in the basic half of the gel (not shown). A 9% slab gel was used for the second dimension.

Mentions: Genetic and biochemical analyses had revealed that radial spokes are composed of ≥17 proteins with well-defined isoelectric points (pIs) and molecular weights (Piperno et al. 1981). We took advantage of these data to analyze the isolated 20S complex, from pf28pf30, by 2D gels. Using precisely defined 2D map positions, all 17 defined radial spoke proteins were identified (Fig. 2). Moreover, the relative intensity of the silver-stained proteins in the 20S complex qualitatively matched the intensity of the 35S-labeled spoke proteins from axonemes (compare to Figure 1 in Piperno et al. 1981). Thus, analysis of isolated radial spokes by 2D gels and silver staining confirms the relative ratios of spoke proteins predicted from 2D gel analysis of axonemes isolated from wild-type and mutant cells (Piperno et al. 1981). For example, radial spoke proteins 1, 2, 3, 4, 5, 6, 7, 9, 10, and 16 are relatively abundant when analyzed by silver staining or 35S labeling.


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 complex contains all 17 previously identified spoke proteins (compare to Piperno et al. 1981). Proteins in one-dimensional (left) and 2D (right) gels of the 20S fraction from pf28pf30 are revealed by silver staining. The 17 radial spoke proteins are numbered. In addition, present in the 20S fraction are the 140- and 210-kD proteins (arrowheads), as well as two unknown proteins (open arrowheads) and tubulin (T). Phosphorylated proteins are labeled (*), and RSP2 distribution, based on Western blots, are indicated by the bracket. Acidic polypeptides are on the right. No polypeptides are present in the basic half of the gel (not shown). A 9% slab gel was used for the second dimension.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: The 20S radial spoke complex contains all 17 previously identified spoke proteins (compare to Piperno et al. 1981). Proteins in one-dimensional (left) and 2D (right) gels of the 20S fraction from pf28pf30 are revealed by silver staining. The 17 radial spoke proteins are numbered. In addition, present in the 20S fraction are the 140- and 210-kD proteins (arrowheads), as well as two unknown proteins (open arrowheads) and tubulin (T). Phosphorylated proteins are labeled (*), and RSP2 distribution, based on Western blots, are indicated by the bracket. Acidic polypeptides are on the right. No polypeptides are present in the basic half of the gel (not shown). A 9% slab gel was used for the second dimension.
Mentions: Genetic and biochemical analyses had revealed that radial spokes are composed of ≥17 proteins with well-defined isoelectric points (pIs) and molecular weights (Piperno et al. 1981). We took advantage of these data to analyze the isolated 20S complex, from pf28pf30, by 2D gels. Using precisely defined 2D map positions, all 17 defined radial spoke proteins were identified (Fig. 2). Moreover, the relative intensity of the silver-stained proteins in the 20S complex qualitatively matched the intensity of the 35S-labeled spoke proteins from axonemes (compare to Figure 1 in Piperno et al. 1981). Thus, analysis of isolated radial spokes by 2D gels and silver staining confirms the relative ratios of spoke proteins predicted from 2D gel analysis of axonemes isolated from wild-type and mutant cells (Piperno et al. 1981). For example, radial spoke proteins 1, 2, 3, 4, 5, 6, 7, 9, 10, and 16 are relatively abundant when analyzed by silver staining or 35S labeling.

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