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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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In vitro reconstitution of radial spokes. Western blots (A) and electron microscopy (B) reveal that extracted radial spokes bind to pf14 axonemes and restore radial spoke structure. Increasing amounts of extract containing radial spokes were incubated with either pf14 axonemes (lanes 1–4) or pf14 axonemes preextracted with 0.6 M NaCl (lanes 5–11). Pellets containing axonemes (lanes 1–4 for pf14 axonemes, and lanes 5–8 for NaCl extracted pf14 axonemes), and the supernatant for the NaCl-extracted pf14 axonemes (lane 9–11) were analyzed by Western analysis using RSP2 antibody. A 1:1 stoichiometry of radial spokes to the binding sites was predicted when 5 μl spoke fraction (arrowheads) was added to the axonemes. Rebinding saturated (*) when 20 μl extract was added to NaCl-extracted axonemes. (B) Cross and longitudinal sections of NaCl extracted pf14 axonemes (top), and the same axonemes reconstituted with extracted radial spokes (bottom). Notably, spoke structures are restored to the A-microtubules (cross section view). Bar, 96 nm.
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Figure 4: In vitro reconstitution of radial spokes. Western blots (A) and electron microscopy (B) reveal that extracted radial spokes bind to pf14 axonemes and restore radial spoke structure. Increasing amounts of extract containing radial spokes were incubated with either pf14 axonemes (lanes 1–4) or pf14 axonemes preextracted with 0.6 M NaCl (lanes 5–11). Pellets containing axonemes (lanes 1–4 for pf14 axonemes, and lanes 5–8 for NaCl extracted pf14 axonemes), and the supernatant for the NaCl-extracted pf14 axonemes (lane 9–11) were analyzed by Western analysis using RSP2 antibody. A 1:1 stoichiometry of radial spokes to the binding sites was predicted when 5 μl spoke fraction (arrowheads) was added to the axonemes. Rebinding saturated (*) when 20 μl extract was added to NaCl-extracted axonemes. (B) Cross and longitudinal sections of NaCl extracted pf14 axonemes (top), and the same axonemes reconstituted with extracted radial spokes (bottom). Notably, spoke structures are restored to the A-microtubules (cross section view). Bar, 96 nm.

Mentions: We tested whether extracted radial spoke complexes could reconstitute radial spokes when added to isolated axonemes lacking the radial spokes. For these experiments, spokes contained in dialyzed KI extracts were added at approximately the original stoichiometry (Fig. 4, Fig. 5-μl extract) to pf14 axonemes or 0.6 M NaCl extracted pf14 axonemes. Based on Western blots using the antibody to RSP2, the radial spokes cosedimented with the axonemes or with extracted axonemes (Fig. 4 A). Binding of spokes was more efficient when pf14 axonemes were first extracted with NaCl, presumably due to the removal of obstructing dynein arms (Fig. 4 A, compare lanes 1–4 with lanes 5–8). Furthermore, binding of spokes saturated when 20 μl of the spoke fraction (fourfold the predicted original stoichiometry of spokes) were added (Fig. 4 A, lanes 7 and 8).


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

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

In vitro reconstitution of radial spokes. Western blots (A) and electron microscopy (B) reveal that extracted radial spokes bind to pf14 axonemes and restore radial spoke structure. Increasing amounts of extract containing radial spokes were incubated with either pf14 axonemes (lanes 1–4) or pf14 axonemes preextracted with 0.6 M NaCl (lanes 5–11). Pellets containing axonemes (lanes 1–4 for pf14 axonemes, and lanes 5–8 for NaCl extracted pf14 axonemes), and the supernatant for the NaCl-extracted pf14 axonemes (lane 9–11) were analyzed by Western analysis using RSP2 antibody. A 1:1 stoichiometry of radial spokes to the binding sites was predicted when 5 μl spoke fraction (arrowheads) was added to the axonemes. Rebinding saturated (*) when 20 μl extract was added to NaCl-extracted axonemes. (B) Cross and longitudinal sections of NaCl extracted pf14 axonemes (top), and the same axonemes reconstituted with extracted radial spokes (bottom). Notably, spoke structures are restored to the A-microtubules (cross section view). Bar, 96 nm.
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Related In: Results  -  Collection

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Figure 4: In vitro reconstitution of radial spokes. Western blots (A) and electron microscopy (B) reveal that extracted radial spokes bind to pf14 axonemes and restore radial spoke structure. Increasing amounts of extract containing radial spokes were incubated with either pf14 axonemes (lanes 1–4) or pf14 axonemes preextracted with 0.6 M NaCl (lanes 5–11). Pellets containing axonemes (lanes 1–4 for pf14 axonemes, and lanes 5–8 for NaCl extracted pf14 axonemes), and the supernatant for the NaCl-extracted pf14 axonemes (lane 9–11) were analyzed by Western analysis using RSP2 antibody. A 1:1 stoichiometry of radial spokes to the binding sites was predicted when 5 μl spoke fraction (arrowheads) was added to the axonemes. Rebinding saturated (*) when 20 μl extract was added to NaCl-extracted axonemes. (B) Cross and longitudinal sections of NaCl extracted pf14 axonemes (top), and the same axonemes reconstituted with extracted radial spokes (bottom). Notably, spoke structures are restored to the A-microtubules (cross section view). Bar, 96 nm.
Mentions: We tested whether extracted radial spoke complexes could reconstitute radial spokes when added to isolated axonemes lacking the radial spokes. For these experiments, spokes contained in dialyzed KI extracts were added at approximately the original stoichiometry (Fig. 4, Fig. 5-μl extract) to pf14 axonemes or 0.6 M NaCl extracted pf14 axonemes. Based on Western blots using the antibody to RSP2, the radial spokes cosedimented with the axonemes or with extracted axonemes (Fig. 4 A). Binding of spokes was more efficient when pf14 axonemes were first extracted with NaCl, presumably due to the removal of obstructing dynein arms (Fig. 4 A, compare lanes 1–4 with lanes 5–8). Furthermore, binding of spokes saturated when 20 μl of the spoke fraction (fourfold the predicted original stoichiometry of spokes) were added (Fig. 4 A, lanes 7 and 8).

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