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Drosophila roadblock and Chlamydomonas LC7: a conserved family of dynein-associated proteins involved in axonal transport, flagellar motility, and mitosis.

Bowman AB, Patel-King RS, Benashski SE, McCaffery JM, Goldstein LS, King SM - J. Cell Biol. (1999)

Bottom Line: The gene identified by robl encodes a 97-amino acid polypeptide that is 57% identical (70% similar) to the 105-amino acid Chlamydomonas outer arm dynein-associated protein LC7, also reported here.Furthermore, we demonstrate that members of this family of proteins are associated with both flagellar outer arm dynein and Drosophila and rat brain cytoplasmic dynein.We propose that roadblock/LC7 family members may modulate specific dynein functions.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Division of Cellular and Molecular Medicine, Department of Pharmacology, University of California San Diego, La Jolla, California 92093-0683, USA.

ABSTRACT
Eukaryotic organisms utilize microtubule-dependent motors of the kinesin and dynein superfamilies to generate intracellular movement. To identify new genes involved in the regulation of axonal transport in Drosophila melanogaster, we undertook a screen based upon the sluggish larval phenotype of known motor mutants. One of the mutants identified in this screen, roadblock (robl), exhibits diverse defects in intracellular transport including axonal transport and mitosis. These defects include intra-axonal accumulations of cargoes, severe axonal degeneration, and aberrant chromosome segregation. The gene identified by robl encodes a 97-amino acid polypeptide that is 57% identical (70% similar) to the 105-amino acid Chlamydomonas outer arm dynein-associated protein LC7, also reported here. Both robl and LC7 have homology to several other genes from fruit fly, nematode, and mammals, but not Saccharomyces cerevisiae. Furthermore, we demonstrate that members of this family of proteins are associated with both flagellar outer arm dynein and Drosophila and rat brain cytoplasmic dynein. We propose that roadblock/LC7 family members may modulate specific dynein functions.

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LC7 is a component of the outer dynein arm. (A) Flagellar axonemes were prepared from wild-type Chlamydomonas (WT) and from mutants lacking the outer arm (oda9), inner arm I1 (ida1, ida2, and ida3), and inner arms I2/3 (ida4). Samples were electrophoresed in a 5–15% acrylamide gradient gel and either stained with Coomassie blue (upper panel) or blotted to nitrocellulose and probed with the R7178 antibody to detect LC7. The LC7 protein is highly reduced only in the strain lacking outer dynein arms. (B) A high salt extract of wild-type axonemes was sedimented through a 5–20% sucrose gradient and fractions electrophoresed in 5–15% acrylamide gels. Fraction 1 is the bottom of the gradient. The upper panel shows the gel stained with Coomassie blue, the lower panel is an immunoblot probed with R7178 to detect the LC7 protein. All the salt extractable LC7 protein precisely comigrates at ∼18 S with components of the outer dynein arm (e.g., IC1 and IC2).
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Figure 3: LC7 is a component of the outer dynein arm. (A) Flagellar axonemes were prepared from wild-type Chlamydomonas (WT) and from mutants lacking the outer arm (oda9), inner arm I1 (ida1, ida2, and ida3), and inner arms I2/3 (ida4). Samples were electrophoresed in a 5–15% acrylamide gradient gel and either stained with Coomassie blue (upper panel) or blotted to nitrocellulose and probed with the R7178 antibody to detect LC7. The LC7 protein is highly reduced only in the strain lacking outer dynein arms. (B) A high salt extract of wild-type axonemes was sedimented through a 5–20% sucrose gradient and fractions electrophoresed in 5–15% acrylamide gels. Fraction 1 is the bottom of the gradient. The upper panel shows the gel stained with Coomassie blue, the lower panel is an immunoblot probed with R7178 to detect the LC7 protein. All the salt extractable LC7 protein precisely comigrates at ∼18 S with components of the outer dynein arm (e.g., IC1 and IC2).

Mentions: The outer arm dynein samples used to obtain LC7 peptide sequences also contained inner dynein arm I1. This dynein partially cofractionates with the outer arm and is now known to contain light chain components (Harrison et al. 1998). To confirm that the LC7 protein is a component of the outer arm, axonemes were prepared from mutants lacking specific dynein structures including the outer arm (oda9), inner arm I1 (ida1, ida2, and ida3), and a subset of inner arms I2/3 (ida4). Immunoblot analysis of these samples using a polyclonal LC7 antiserum revealed that the LC7 polypeptide was present in the mutants lacking inner arms, but was drastically reduced in the strain lacking outer arms (Fig. 3 A). Upon overexposure of the blot, a very small amount of LC7 could be detected in the outer armless axonemes. The origin of this minor fraction remains unclear as the LC7 protein could not be detected in sucrose gradient profiles of high salt extracts from outer armless strains (data not shown). Furthermore, sucrose gradient analysis of extracts from wild-type axonemes revealed that all the extracted LC7 comigrated with the outer arm at ∼18 S (Fig. 3 B).


Drosophila roadblock and Chlamydomonas LC7: a conserved family of dynein-associated proteins involved in axonal transport, flagellar motility, and mitosis.

Bowman AB, Patel-King RS, Benashski SE, McCaffery JM, Goldstein LS, King SM - J. Cell Biol. (1999)

LC7 is a component of the outer dynein arm. (A) Flagellar axonemes were prepared from wild-type Chlamydomonas (WT) and from mutants lacking the outer arm (oda9), inner arm I1 (ida1, ida2, and ida3), and inner arms I2/3 (ida4). Samples were electrophoresed in a 5–15% acrylamide gradient gel and either stained with Coomassie blue (upper panel) or blotted to nitrocellulose and probed with the R7178 antibody to detect LC7. The LC7 protein is highly reduced only in the strain lacking outer dynein arms. (B) A high salt extract of wild-type axonemes was sedimented through a 5–20% sucrose gradient and fractions electrophoresed in 5–15% acrylamide gels. Fraction 1 is the bottom of the gradient. The upper panel shows the gel stained with Coomassie blue, the lower panel is an immunoblot probed with R7178 to detect the LC7 protein. All the salt extractable LC7 protein precisely comigrates at ∼18 S with components of the outer dynein arm (e.g., IC1 and IC2).
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Related In: Results  -  Collection

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Figure 3: LC7 is a component of the outer dynein arm. (A) Flagellar axonemes were prepared from wild-type Chlamydomonas (WT) and from mutants lacking the outer arm (oda9), inner arm I1 (ida1, ida2, and ida3), and inner arms I2/3 (ida4). Samples were electrophoresed in a 5–15% acrylamide gradient gel and either stained with Coomassie blue (upper panel) or blotted to nitrocellulose and probed with the R7178 antibody to detect LC7. The LC7 protein is highly reduced only in the strain lacking outer dynein arms. (B) A high salt extract of wild-type axonemes was sedimented through a 5–20% sucrose gradient and fractions electrophoresed in 5–15% acrylamide gels. Fraction 1 is the bottom of the gradient. The upper panel shows the gel stained with Coomassie blue, the lower panel is an immunoblot probed with R7178 to detect the LC7 protein. All the salt extractable LC7 protein precisely comigrates at ∼18 S with components of the outer dynein arm (e.g., IC1 and IC2).
Mentions: The outer arm dynein samples used to obtain LC7 peptide sequences also contained inner dynein arm I1. This dynein partially cofractionates with the outer arm and is now known to contain light chain components (Harrison et al. 1998). To confirm that the LC7 protein is a component of the outer arm, axonemes were prepared from mutants lacking specific dynein structures including the outer arm (oda9), inner arm I1 (ida1, ida2, and ida3), and a subset of inner arms I2/3 (ida4). Immunoblot analysis of these samples using a polyclonal LC7 antiserum revealed that the LC7 polypeptide was present in the mutants lacking inner arms, but was drastically reduced in the strain lacking outer arms (Fig. 3 A). Upon overexposure of the blot, a very small amount of LC7 could be detected in the outer armless axonemes. The origin of this minor fraction remains unclear as the LC7 protein could not be detected in sucrose gradient profiles of high salt extracts from outer armless strains (data not shown). Furthermore, sucrose gradient analysis of extracts from wild-type axonemes revealed that all the extracted LC7 comigrated with the outer arm at ∼18 S (Fig. 3 B).

Bottom Line: The gene identified by robl encodes a 97-amino acid polypeptide that is 57% identical (70% similar) to the 105-amino acid Chlamydomonas outer arm dynein-associated protein LC7, also reported here.Furthermore, we demonstrate that members of this family of proteins are associated with both flagellar outer arm dynein and Drosophila and rat brain cytoplasmic dynein.We propose that roadblock/LC7 family members may modulate specific dynein functions.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Division of Cellular and Molecular Medicine, Department of Pharmacology, University of California San Diego, La Jolla, California 92093-0683, USA.

ABSTRACT
Eukaryotic organisms utilize microtubule-dependent motors of the kinesin and dynein superfamilies to generate intracellular movement. To identify new genes involved in the regulation of axonal transport in Drosophila melanogaster, we undertook a screen based upon the sluggish larval phenotype of known motor mutants. One of the mutants identified in this screen, roadblock (robl), exhibits diverse defects in intracellular transport including axonal transport and mitosis. These defects include intra-axonal accumulations of cargoes, severe axonal degeneration, and aberrant chromosome segregation. The gene identified by robl encodes a 97-amino acid polypeptide that is 57% identical (70% similar) to the 105-amino acid Chlamydomonas outer arm dynein-associated protein LC7, also reported here. Both robl and LC7 have homology to several other genes from fruit fly, nematode, and mammals, but not Saccharomyces cerevisiae. Furthermore, we demonstrate that members of this family of proteins are associated with both flagellar outer arm dynein and Drosophila and rat brain cytoplasmic dynein. We propose that roadblock/LC7 family members may modulate specific dynein functions.

Show MeSH
Related in: MedlinePlus