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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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Cross-linking analysis reveals dimerization of LC8 and close interaction of calmodulin and LC8 in the radial spoke stalk. The 15S fraction derived from pf17axonemes was treated with DFDNB at concentrations indicated. Samples were then prepared for Western blot analyses using anticalmodulin (left) or anti-LC8 (right) antibodies. A 27-kD cross-linked product reacted with both antibodies (arrowheads). A 20-kD LC8 dimer also results from cross-linking (arrow, right).
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Figure 8: Cross-linking analysis reveals dimerization of LC8 and close interaction of calmodulin and LC8 in the radial spoke stalk. The 15S fraction derived from pf17axonemes was treated with DFDNB at concentrations indicated. Samples were then prepared for Western blot analyses using anticalmodulin (left) or anti-LC8 (right) antibodies. A 27-kD cross-linked product reacted with both antibodies (arrowheads). A 20-kD LC8 dimer also results from cross-linking (arrow, right).

Mentions: Based on cross-linking studies, LC8 is known to form dimers, or higher order structures, in dyneins and myosin V (Benashski et al. 1997). Moreover, based on analysis of pf17 (Fig. 6 and Fig. 7), both LC8 and calmodulin are localized in the spoke stalk. Cross-linking experiments were carried out to (a) determine whether LC8 of the radial spoke fraction forms dimers, (b) confirm the presence of calmodulin in the radial spoke stalk, and (c) test whether LC8 and calmodulin structurally interact in the spoke stalk. Isolated radial spoke stalk fractions from pf17 were treated with increasing concentration of a short cross-linker, DFDNB. The results of cross-linking were analyzed by Western blots using anticalmodulin (Fig. 8, left) and anti-LC8 antibodies (Fig. 8, right). A 27-kD complex (Fig. 8, arrowhead, left) containing calmodulin appears after treatment with DFDNB, suggesting that calmodulin is cross-linked to a protein of ∼10 kD. LC8 is the only 10-kD protein in the spoke stalk fractions. As predicted, LC8 also formed multimeric complexes after the treatment with DFDNB, including bands of 19 and 27 kD (Fig. 8, right). Based on the size and relative intensity, the 19-kD complex must be a LC8 dimer, similar to that found in dynein and myosin V (Benashski et al. 1997). The 27-kD complex containing LC8 coincides with the 27-kD complex containing calmodulin. The complex formed by the 0.3-nm cross-linker further confirms that both calmodulin and LC8 are in the radial spoke stalk and likely assembled in close proximity in the stalk.


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

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

Cross-linking analysis reveals dimerization of LC8 and close interaction of calmodulin and LC8 in the radial spoke stalk. The 15S fraction derived from pf17axonemes was treated with DFDNB at concentrations indicated. Samples were then prepared for Western blot analyses using anticalmodulin (left) or anti-LC8 (right) antibodies. A 27-kD cross-linked product reacted with both antibodies (arrowheads). A 20-kD LC8 dimer also results from cross-linking (arrow, right).
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Related In: Results  -  Collection

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Figure 8: Cross-linking analysis reveals dimerization of LC8 and close interaction of calmodulin and LC8 in the radial spoke stalk. The 15S fraction derived from pf17axonemes was treated with DFDNB at concentrations indicated. Samples were then prepared for Western blot analyses using anticalmodulin (left) or anti-LC8 (right) antibodies. A 27-kD cross-linked product reacted with both antibodies (arrowheads). A 20-kD LC8 dimer also results from cross-linking (arrow, right).
Mentions: Based on cross-linking studies, LC8 is known to form dimers, or higher order structures, in dyneins and myosin V (Benashski et al. 1997). Moreover, based on analysis of pf17 (Fig. 6 and Fig. 7), both LC8 and calmodulin are localized in the spoke stalk. Cross-linking experiments were carried out to (a) determine whether LC8 of the radial spoke fraction forms dimers, (b) confirm the presence of calmodulin in the radial spoke stalk, and (c) test whether LC8 and calmodulin structurally interact in the spoke stalk. Isolated radial spoke stalk fractions from pf17 were treated with increasing concentration of a short cross-linker, DFDNB. The results of cross-linking were analyzed by Western blots using anticalmodulin (Fig. 8, left) and anti-LC8 antibodies (Fig. 8, right). A 27-kD complex (Fig. 8, arrowhead, left) containing calmodulin appears after treatment with DFDNB, suggesting that calmodulin is cross-linked to a protein of ∼10 kD. LC8 is the only 10-kD protein in the spoke stalk fractions. As predicted, LC8 also formed multimeric complexes after the treatment with DFDNB, including bands of 19 and 27 kD (Fig. 8, right). Based on the size and relative intensity, the 19-kD complex must be a LC8 dimer, similar to that found in dynein and myosin V (Benashski et al. 1997). The 27-kD complex containing LC8 coincides with the 27-kD complex containing calmodulin. The complex formed by the 0.3-nm cross-linker further confirms that both calmodulin and LC8 are in the radial spoke stalk and likely assembled in close proximity in the stalk.

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