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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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Related in: MedlinePlus

Extraction of flagellar radial spoke proteins and isolation of a 20S radial spoke complex and a 15S radial spoke stalk complex. (A, left) Diagram illustrating the predicted location of radial spoke proteins and the gene products of pf14, pf17, and pf24 in the radial spoke stalk and head (Curry and Rosenbaum 1993). (middle) Western blot analyses of isolated axonemes and axonemal fractions, using antibodies to RSP2 and -3 (lane 1, wild-type axonemes after extraction in 0.6 M NaCl; (lanes 2–5) axonemes from pf14, pf17, pf24, and pf27; (lane 6) wild-type axonemes after 0.6 M KI extraction; (lane 7) KI extract from wild-type axonemes. Notably, RSP2 and -3 are extracted with 0.6 M KI (middle, compare lanes 6 and 7) and cosediment at ∼20S in the 5–20% sucrose gradient (right, fraction 3). Protein loads and transfers were controlled by Ponceau red staining of blots (not shown). (right) Sucrose gradient fractions of the KI extract from wild-type cells probed with antibodies to RSP2 and -3. (B) Coomassie-stained 5% gels of sucrose gradient fractions reveal cosedimentation of several proteins in addition to RSP2 and -3. RSP1, -2, and -3 are indicated (•; left panel, fraction 3). Extraction of pf17 axonemes yields a diffuse 15S peak containing a subset of the proteins found in the 20S complex. Arrowheads indicate the 140- and 210-kD proteins that cosediment with both the 20S radial spoke and 15S radial spoke stalk complexes. RSP1 (gray arrow), a spoke head protein, is missing in the 15S complex. (C) Silver-stained 8% gels of sucrose gradient fractions of the axonemal extract from pf28pf30, a mutant lacking outer arm dynein and inner arm dynein I1. The 20S radial spoke complex is composed of several proteins (left), that are missing in the 20S fractions from a mutant lacking the radial spokes, pf14 (right panel). Samples analyzed in B and C were derived from 0.5 M KI (B) or 0.6 M NaBr (C) extract of axonemes first extracted in 0.6 M NaCl. Molecular weights are indicated on the left.
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Figure 1: Extraction of flagellar radial spoke proteins and isolation of a 20S radial spoke complex and a 15S radial spoke stalk complex. (A, left) Diagram illustrating the predicted location of radial spoke proteins and the gene products of pf14, pf17, and pf24 in the radial spoke stalk and head (Curry and Rosenbaum 1993). (middle) Western blot analyses of isolated axonemes and axonemal fractions, using antibodies to RSP2 and -3 (lane 1, wild-type axonemes after extraction in 0.6 M NaCl; (lanes 2–5) axonemes from pf14, pf17, pf24, and pf27; (lane 6) wild-type axonemes after 0.6 M KI extraction; (lane 7) KI extract from wild-type axonemes. Notably, RSP2 and -3 are extracted with 0.6 M KI (middle, compare lanes 6 and 7) and cosediment at ∼20S in the 5–20% sucrose gradient (right, fraction 3). Protein loads and transfers were controlled by Ponceau red staining of blots (not shown). (right) Sucrose gradient fractions of the KI extract from wild-type cells probed with antibodies to RSP2 and -3. (B) Coomassie-stained 5% gels of sucrose gradient fractions reveal cosedimentation of several proteins in addition to RSP2 and -3. RSP1, -2, and -3 are indicated (•; left panel, fraction 3). Extraction of pf17 axonemes yields a diffuse 15S peak containing a subset of the proteins found in the 20S complex. Arrowheads indicate the 140- and 210-kD proteins that cosediment with both the 20S radial spoke and 15S radial spoke stalk complexes. RSP1 (gray arrow), a spoke head protein, is missing in the 15S complex. (C) Silver-stained 8% gels of sucrose gradient fractions of the axonemal extract from pf28pf30, a mutant lacking outer arm dynein and inner arm dynein I1. The 20S radial spoke complex is composed of several proteins (left), that are missing in the 20S fractions from a mutant lacking the radial spokes, pf14 (right panel). Samples analyzed in B and C were derived from 0.5 M KI (B) or 0.6 M NaBr (C) extract of axonemes first extracted in 0.6 M NaCl. Molecular weights are indicated on the left.

Mentions: The radial spokes are T-shaped structures anchored on the A-microtubule of each outer doublet, adjacent to the inner dynein arms (Warner and Satir 1974; Witman et al. 1978; Goodenough and Heuser 1985, Goodenough and Heuser 1989). In cross sections, the spokes project toward the central pair apparatus, where the spoke heads transiently interact with the central pair projections (Warner and Satir 1974; Goodenough and Heuser 1985, Goodenough and Heuser 1989; Smith and Lefebvre 1997; Mitchell and Sale 1999; Omoto et al. 1999). In the long axis, radial spokes repeat in either pairs or triplet groups every 96 nm along each doublet microtubule, in exact register with the inner dynein arms (Warner and Satir 1974; Goodenough and Heuser 1985, Goodenough and Heuser 1989; Piperno et al. 1990; Mastronarde et al. 1992; Gardner et al. 1994; Porter and Sale 2000). Biochemical analysis of Chlamydomonas axonemes has demonstrated the radial spokes are composed of ≥17 proteins, 5 located in the spoke head and at least ≥12 located in the spoke stalk (Fig. 1 A; Piperno et al. 1981). Five of the stalk proteins are phosphorylated in vivo (Piperno et al. 1981), and several have been cloned and characterized (for review see Curry and Rosenbaum 1993). In particular, radial spoke protein 3, RSP3, is located at the proximal end of the spoke stalk in position to target and anchor the spoke to the doublet microtubule (Huang et al. 1981; Diener et al. 1993). Recently, it has also been determined that RSP3 is an A-kinase anchor protein, predicted to anchor protein kinase A (PKA) in position near the inner dynein arms (Gaillard et al. 2001). However, little more is known about the nature of radial spoke proteins or the mechanism for their control of motility.


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

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

Extraction of flagellar radial spoke proteins and isolation of a 20S radial spoke complex and a 15S radial spoke stalk complex. (A, left) Diagram illustrating the predicted location of radial spoke proteins and the gene products of pf14, pf17, and pf24 in the radial spoke stalk and head (Curry and Rosenbaum 1993). (middle) Western blot analyses of isolated axonemes and axonemal fractions, using antibodies to RSP2 and -3 (lane 1, wild-type axonemes after extraction in 0.6 M NaCl; (lanes 2–5) axonemes from pf14, pf17, pf24, and pf27; (lane 6) wild-type axonemes after 0.6 M KI extraction; (lane 7) KI extract from wild-type axonemes. Notably, RSP2 and -3 are extracted with 0.6 M KI (middle, compare lanes 6 and 7) and cosediment at ∼20S in the 5–20% sucrose gradient (right, fraction 3). Protein loads and transfers were controlled by Ponceau red staining of blots (not shown). (right) Sucrose gradient fractions of the KI extract from wild-type cells probed with antibodies to RSP2 and -3. (B) Coomassie-stained 5% gels of sucrose gradient fractions reveal cosedimentation of several proteins in addition to RSP2 and -3. RSP1, -2, and -3 are indicated (•; left panel, fraction 3). Extraction of pf17 axonemes yields a diffuse 15S peak containing a subset of the proteins found in the 20S complex. Arrowheads indicate the 140- and 210-kD proteins that cosediment with both the 20S radial spoke and 15S radial spoke stalk complexes. RSP1 (gray arrow), a spoke head protein, is missing in the 15S complex. (C) Silver-stained 8% gels of sucrose gradient fractions of the axonemal extract from pf28pf30, a mutant lacking outer arm dynein and inner arm dynein I1. The 20S radial spoke complex is composed of several proteins (left), that are missing in the 20S fractions from a mutant lacking the radial spokes, pf14 (right panel). Samples analyzed in B and C were derived from 0.5 M KI (B) or 0.6 M NaBr (C) extract of axonemes first extracted in 0.6 M NaCl. Molecular weights are indicated on the left.
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Figure 1: Extraction of flagellar radial spoke proteins and isolation of a 20S radial spoke complex and a 15S radial spoke stalk complex. (A, left) Diagram illustrating the predicted location of radial spoke proteins and the gene products of pf14, pf17, and pf24 in the radial spoke stalk and head (Curry and Rosenbaum 1993). (middle) Western blot analyses of isolated axonemes and axonemal fractions, using antibodies to RSP2 and -3 (lane 1, wild-type axonemes after extraction in 0.6 M NaCl; (lanes 2–5) axonemes from pf14, pf17, pf24, and pf27; (lane 6) wild-type axonemes after 0.6 M KI extraction; (lane 7) KI extract from wild-type axonemes. Notably, RSP2 and -3 are extracted with 0.6 M KI (middle, compare lanes 6 and 7) and cosediment at ∼20S in the 5–20% sucrose gradient (right, fraction 3). Protein loads and transfers were controlled by Ponceau red staining of blots (not shown). (right) Sucrose gradient fractions of the KI extract from wild-type cells probed with antibodies to RSP2 and -3. (B) Coomassie-stained 5% gels of sucrose gradient fractions reveal cosedimentation of several proteins in addition to RSP2 and -3. RSP1, -2, and -3 are indicated (•; left panel, fraction 3). Extraction of pf17 axonemes yields a diffuse 15S peak containing a subset of the proteins found in the 20S complex. Arrowheads indicate the 140- and 210-kD proteins that cosediment with both the 20S radial spoke and 15S radial spoke stalk complexes. RSP1 (gray arrow), a spoke head protein, is missing in the 15S complex. (C) Silver-stained 8% gels of sucrose gradient fractions of the axonemal extract from pf28pf30, a mutant lacking outer arm dynein and inner arm dynein I1. The 20S radial spoke complex is composed of several proteins (left), that are missing in the 20S fractions from a mutant lacking the radial spokes, pf14 (right panel). Samples analyzed in B and C were derived from 0.5 M KI (B) or 0.6 M NaBr (C) extract of axonemes first extracted in 0.6 M NaCl. Molecular weights are indicated on the left.
Mentions: The radial spokes are T-shaped structures anchored on the A-microtubule of each outer doublet, adjacent to the inner dynein arms (Warner and Satir 1974; Witman et al. 1978; Goodenough and Heuser 1985, Goodenough and Heuser 1989). In cross sections, the spokes project toward the central pair apparatus, where the spoke heads transiently interact with the central pair projections (Warner and Satir 1974; Goodenough and Heuser 1985, Goodenough and Heuser 1989; Smith and Lefebvre 1997; Mitchell and Sale 1999; Omoto et al. 1999). In the long axis, radial spokes repeat in either pairs or triplet groups every 96 nm along each doublet microtubule, in exact register with the inner dynein arms (Warner and Satir 1974; Goodenough and Heuser 1985, Goodenough and Heuser 1989; Piperno et al. 1990; Mastronarde et al. 1992; Gardner et al. 1994; Porter and Sale 2000). Biochemical analysis of Chlamydomonas axonemes has demonstrated the radial spokes are composed of ≥17 proteins, 5 located in the spoke head and at least ≥12 located in the spoke stalk (Fig. 1 A; Piperno et al. 1981). Five of the stalk proteins are phosphorylated in vivo (Piperno et al. 1981), and several have been cloned and characterized (for review see Curry and Rosenbaum 1993). In particular, radial spoke protein 3, RSP3, is located at the proximal end of the spoke stalk in position to target and anchor the spoke to the doublet microtubule (Huang et al. 1981; Diener et al. 1993). Recently, it has also been determined that RSP3 is an A-kinase anchor protein, predicted to anchor protein kinase A (PKA) in position near the inner dynein arms (Gaillard et al. 2001). However, little more is known about the nature of radial spoke proteins or the mechanism for their control of motility.

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