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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 addition to the radial spoke proteins described above, three small proteins including calmodulin, and dynein light chain LC8 are associated with the isolated radial spoke. (A) The 20S radial spoke fraction was separated by 14% one-dimensional (left) and 2D (right) gels. Silver staining revealed three proteins with masses of ∼18, ∼16, and ∼10 kD (arrowheads). (B) Western blot analyses revealed that the 18-kD protein is recognized by anti–Dictystelium calmodulin monoclonal antibody (top). The 10-kD protein is recognized by anti-LC8 antibody (bottom). The identity of the 16-kD protein is not known.
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Figure 5: In addition to the radial spoke proteins described above, three small proteins including calmodulin, and dynein light chain LC8 are associated with the isolated radial spoke. (A) The 20S radial spoke fraction was separated by 14% one-dimensional (left) and 2D (right) gels. Silver staining revealed three proteins with masses of ∼18, ∼16, and ∼10 kD (arrowheads). (B) Western blot analyses revealed that the 18-kD protein is recognized by anti–Dictystelium calmodulin monoclonal antibody (top). The 10-kD protein is recognized by anti-LC8 antibody (bottom). The identity of the 16-kD protein is not known.

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 addition to the radial spoke proteins described above, three small proteins including calmodulin, and dynein light chain LC8 are associated with the isolated radial spoke. (A) The 20S radial spoke fraction was separated by 14% one-dimensional (left) and 2D (right) gels. Silver staining revealed three proteins with masses of ∼18, ∼16, and ∼10 kD (arrowheads). (B) Western blot analyses revealed that the 18-kD protein is recognized by anti–Dictystelium calmodulin monoclonal antibody (top). The 10-kD protein is recognized by anti-LC8 antibody (bottom). The identity of the 16-kD protein is not known.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: In addition to the radial spoke proteins described above, three small proteins including calmodulin, and dynein light chain LC8 are associated with the isolated radial spoke. (A) The 20S radial spoke fraction was separated by 14% one-dimensional (left) and 2D (right) gels. Silver staining revealed three proteins with masses of ∼18, ∼16, and ∼10 kD (arrowheads). (B) Western blot analyses revealed that the 18-kD protein is recognized by anti–Dictystelium calmodulin monoclonal antibody (top). The 10-kD protein is recognized by anti-LC8 antibody (bottom). The identity of the 16-kD protein is not known.
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