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The DHC1b (DHC2) isoform of cytoplasmic dynein is required for flagellar assembly.

Pazour GJ, Dickert BL, Witman GB - J. Cell Biol. (1999)

Bottom Line: The deletion also results in a massive redistribution of raft subunits from a peri-basal body pool (Cole, D.G., D.R.Natl.These results indicate that DHC1b is a cytoplasmic dynein essential for flagellar assembly, probably because it is the motor for retrograde IFT.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.

ABSTRACT
Dyneins are microtubule-based molecular motors involved in many different types of cell movement. Most dynein heavy chains (DHCs) clearly group into cytoplasmic or axonemal isoforms. However, DHC1b has been enigmatic. To learn more about this isoform, we isolated Chlamydomonas cDNA clones encoding a portion of DHC1b, and used these clones to identify a Chlamydomonas cell line with a deletion mutation in DHC1b. The mutant grows normally and appears to have a normal Golgi apparatus, but has very short flagella. The deletion also results in a massive redistribution of raft subunits from a peri-basal body pool (Cole, D.G., D.R. Diener, A.L. Himelblau, P.L. Beech, J.C. Fuster, and J.L. Rosenbaum. 1998. J. Cell Biol. 141:993-1008) to the flagella. Rafts are particles that normally move up and down the flagella in a process known as intraflagellar transport (IFT) (Kozminski, K.G., K.A. Johnson, P. Forscher, and J.L. Rosenbaum. 1993. Proc. Natl. Acad. Sci. USA. 90:5519-5523), which is essential for assembly and maintenance of flagella. The redistribution of raft subunits apparently occurs due to a defect in the retrograde component of IFT, suggesting that DHC1b is the motor for retrograde IFT. Consistent with this, Western blots indicate that DHC1b is present in the flagellum, predominantly in the detergent- and ATP-soluble fractions. These results indicate that DHC1b is a cytoplasmic dynein essential for flagellar assembly, probably because it is the motor for retrograde IFT.

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The Chlamydomonas reinhardtii DHC1b gene groups  with DHC1b sequences from other organisms and is induced by  deflagellation. (a) Partial sequence of the C. reinhardtii gene encoding DHC1b. P-loops 1 and 2 are underlined. Sequence data  are available from GenBank/EMBL/DDBJ under accession  number AF096277. (b) Alignment of the Chlamydomonas  DHC1b and pcr4 peptides with other cytoplasmic dynein heavy  chains. A representative selection of DHC sequences from GenBank were aligned with CLUSTAL W and the residues identical  to Chlamydomonas DHC1b were shaded in black. Species names  are abbreviated as described in c. A longer sequence for pcr4 is  available under accession number AF106079. (c) Phylogenetic  tree showing the relationship of the Chlamydomonas DHC1b  and pcr4 sequences (arrowheads) to other DHC sequences. The  predicted peptide sequences of the Chlamydomonas DHC1b  cDNA (starting just upstream of P-loop 1 at the sequence  CYLTLT and ending with the sequence FVTLNP) and pcr4  cDNA were aligned with a subset of DHC sequences in GenBank using CLUSTAL W, and a phylogenetic tree drawn with  PHYLIP using the UPGMA method. Ce, C. elegans; Cr, C. reinhardtii; Dd, Dictyostelium discoideum; Dm, Drosophila melanogaster; Hs, Homo sapiens; Mm, Mus musculus; Nc, Neurospora  crassa; Pt, Paramecium tetraurelia; Rn, Rattus novegicus; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; Tg,  Tripneustes gratilla; Tt, Tetrahymena thermophila. (d) The  Chlamydomonas DHC1b gene is induced by deflagellation.  mRNA was isolated from control, nondeflagellated cells (C), and  from cells 30 min after deflagellation (D). The mRNA was analyzed by Northern blot using probes for the DHC1b, ODA3, and  PTX2 genes. The latter two genes served as standards; transcription of ODA3 is induced by deflagellation (Koutoulis et al.,  1997), whereas that of PTX2, a gene involved in phototaxis, is not  (Pazour, G., and G. Witman, unpublished observations).
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Figure 1: The Chlamydomonas reinhardtii DHC1b gene groups with DHC1b sequences from other organisms and is induced by deflagellation. (a) Partial sequence of the C. reinhardtii gene encoding DHC1b. P-loops 1 and 2 are underlined. Sequence data are available from GenBank/EMBL/DDBJ under accession number AF096277. (b) Alignment of the Chlamydomonas DHC1b and pcr4 peptides with other cytoplasmic dynein heavy chains. A representative selection of DHC sequences from GenBank were aligned with CLUSTAL W and the residues identical to Chlamydomonas DHC1b were shaded in black. Species names are abbreviated as described in c. A longer sequence for pcr4 is available under accession number AF106079. (c) Phylogenetic tree showing the relationship of the Chlamydomonas DHC1b and pcr4 sequences (arrowheads) to other DHC sequences. The predicted peptide sequences of the Chlamydomonas DHC1b cDNA (starting just upstream of P-loop 1 at the sequence CYLTLT and ending with the sequence FVTLNP) and pcr4 cDNA were aligned with a subset of DHC sequences in GenBank using CLUSTAL W, and a phylogenetic tree drawn with PHYLIP using the UPGMA method. Ce, C. elegans; Cr, C. reinhardtii; Dd, Dictyostelium discoideum; Dm, Drosophila melanogaster; Hs, Homo sapiens; Mm, Mus musculus; Nc, Neurospora crassa; Pt, Paramecium tetraurelia; Rn, Rattus novegicus; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; Tg, Tripneustes gratilla; Tt, Tetrahymena thermophila. (d) The Chlamydomonas DHC1b gene is induced by deflagellation. mRNA was isolated from control, nondeflagellated cells (C), and from cells 30 min after deflagellation (D). The mRNA was analyzed by Northern blot using probes for the DHC1b, ODA3, and PTX2 genes. The latter two genes served as standards; transcription of ODA3 is induced by deflagellation (Koutoulis et al., 1997), whereas that of PTX2, a gene involved in phototaxis, is not (Pazour, G., and G. Witman, unpublished observations).

Mentions: The Chlamydomonas DHC1b cDNA was cloned by PCR using primers based on conserved regions of DHC1b from other species. Peptide sequences of the dynein isoforms in GenBank were aligned by CLUSTAL W (Thompson et al., 1994) and examined to identify regions that are highly conserved in the DHC1b isoform, but not conserved in other isoforms. The peptide, NPAGKGYG, which is highly specific for the DHC1b isoforms, was used to design a gene-specific antisense primer using Chlamydomonas codon bias. Similarly, three dynein-specific sense primers (based on CYLTLTQ, FNCDEG, and WGCFDEFNR peptides) were designed. Three sets of PCR reactions using Elongase (GIBCO BRL) were carried out on a cDNA library enriched for DHC sequences (Wilkerson et al., 1994). All three produced bands of the predicted size and all three PCR products were found by sequencing to be highly homologous to DHC1b from other species. One of these products, encoding amino acids Phe-776 to Gly-862 in Fig. 1 a, was used to screen the same cDNA library by hybridization. Eight positive phage clones were identified and the ends were sequenced. The sequences were compared to the C. elegans DHC1b gene to determine the relationship between the individual clones. The two overlapping clones (pGP638 and pGP639) that extended out the farthest were sequenced on both strands by the Iowa State University Sequencing Facility.


The DHC1b (DHC2) isoform of cytoplasmic dynein is required for flagellar assembly.

Pazour GJ, Dickert BL, Witman GB - J. Cell Biol. (1999)

The Chlamydomonas reinhardtii DHC1b gene groups  with DHC1b sequences from other organisms and is induced by  deflagellation. (a) Partial sequence of the C. reinhardtii gene encoding DHC1b. P-loops 1 and 2 are underlined. Sequence data  are available from GenBank/EMBL/DDBJ under accession  number AF096277. (b) Alignment of the Chlamydomonas  DHC1b and pcr4 peptides with other cytoplasmic dynein heavy  chains. A representative selection of DHC sequences from GenBank were aligned with CLUSTAL W and the residues identical  to Chlamydomonas DHC1b were shaded in black. Species names  are abbreviated as described in c. A longer sequence for pcr4 is  available under accession number AF106079. (c) Phylogenetic  tree showing the relationship of the Chlamydomonas DHC1b  and pcr4 sequences (arrowheads) to other DHC sequences. The  predicted peptide sequences of the Chlamydomonas DHC1b  cDNA (starting just upstream of P-loop 1 at the sequence  CYLTLT and ending with the sequence FVTLNP) and pcr4  cDNA were aligned with a subset of DHC sequences in GenBank using CLUSTAL W, and a phylogenetic tree drawn with  PHYLIP using the UPGMA method. Ce, C. elegans; Cr, C. reinhardtii; Dd, Dictyostelium discoideum; Dm, Drosophila melanogaster; Hs, Homo sapiens; Mm, Mus musculus; Nc, Neurospora  crassa; Pt, Paramecium tetraurelia; Rn, Rattus novegicus; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; Tg,  Tripneustes gratilla; Tt, Tetrahymena thermophila. (d) The  Chlamydomonas DHC1b gene is induced by deflagellation.  mRNA was isolated from control, nondeflagellated cells (C), and  from cells 30 min after deflagellation (D). The mRNA was analyzed by Northern blot using probes for the DHC1b, ODA3, and  PTX2 genes. The latter two genes served as standards; transcription of ODA3 is induced by deflagellation (Koutoulis et al.,  1997), whereas that of PTX2, a gene involved in phototaxis, is not  (Pazour, G., and G. Witman, unpublished observations).
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Related In: Results  -  Collection

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Figure 1: The Chlamydomonas reinhardtii DHC1b gene groups with DHC1b sequences from other organisms and is induced by deflagellation. (a) Partial sequence of the C. reinhardtii gene encoding DHC1b. P-loops 1 and 2 are underlined. Sequence data are available from GenBank/EMBL/DDBJ under accession number AF096277. (b) Alignment of the Chlamydomonas DHC1b and pcr4 peptides with other cytoplasmic dynein heavy chains. A representative selection of DHC sequences from GenBank were aligned with CLUSTAL W and the residues identical to Chlamydomonas DHC1b were shaded in black. Species names are abbreviated as described in c. A longer sequence for pcr4 is available under accession number AF106079. (c) Phylogenetic tree showing the relationship of the Chlamydomonas DHC1b and pcr4 sequences (arrowheads) to other DHC sequences. The predicted peptide sequences of the Chlamydomonas DHC1b cDNA (starting just upstream of P-loop 1 at the sequence CYLTLT and ending with the sequence FVTLNP) and pcr4 cDNA were aligned with a subset of DHC sequences in GenBank using CLUSTAL W, and a phylogenetic tree drawn with PHYLIP using the UPGMA method. Ce, C. elegans; Cr, C. reinhardtii; Dd, Dictyostelium discoideum; Dm, Drosophila melanogaster; Hs, Homo sapiens; Mm, Mus musculus; Nc, Neurospora crassa; Pt, Paramecium tetraurelia; Rn, Rattus novegicus; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; Tg, Tripneustes gratilla; Tt, Tetrahymena thermophila. (d) The Chlamydomonas DHC1b gene is induced by deflagellation. mRNA was isolated from control, nondeflagellated cells (C), and from cells 30 min after deflagellation (D). The mRNA was analyzed by Northern blot using probes for the DHC1b, ODA3, and PTX2 genes. The latter two genes served as standards; transcription of ODA3 is induced by deflagellation (Koutoulis et al., 1997), whereas that of PTX2, a gene involved in phototaxis, is not (Pazour, G., and G. Witman, unpublished observations).
Mentions: The Chlamydomonas DHC1b cDNA was cloned by PCR using primers based on conserved regions of DHC1b from other species. Peptide sequences of the dynein isoforms in GenBank were aligned by CLUSTAL W (Thompson et al., 1994) and examined to identify regions that are highly conserved in the DHC1b isoform, but not conserved in other isoforms. The peptide, NPAGKGYG, which is highly specific for the DHC1b isoforms, was used to design a gene-specific antisense primer using Chlamydomonas codon bias. Similarly, three dynein-specific sense primers (based on CYLTLTQ, FNCDEG, and WGCFDEFNR peptides) were designed. Three sets of PCR reactions using Elongase (GIBCO BRL) were carried out on a cDNA library enriched for DHC sequences (Wilkerson et al., 1994). All three produced bands of the predicted size and all three PCR products were found by sequencing to be highly homologous to DHC1b from other species. One of these products, encoding amino acids Phe-776 to Gly-862 in Fig. 1 a, was used to screen the same cDNA library by hybridization. Eight positive phage clones were identified and the ends were sequenced. The sequences were compared to the C. elegans DHC1b gene to determine the relationship between the individual clones. The two overlapping clones (pGP638 and pGP639) that extended out the farthest were sequenced on both strands by the Iowa State University Sequencing Facility.

Bottom Line: The deletion also results in a massive redistribution of raft subunits from a peri-basal body pool (Cole, D.G., D.R.Natl.These results indicate that DHC1b is a cytoplasmic dynein essential for flagellar assembly, probably because it is the motor for retrograde IFT.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.

ABSTRACT
Dyneins are microtubule-based molecular motors involved in many different types of cell movement. Most dynein heavy chains (DHCs) clearly group into cytoplasmic or axonemal isoforms. However, DHC1b has been enigmatic. To learn more about this isoform, we isolated Chlamydomonas cDNA clones encoding a portion of DHC1b, and used these clones to identify a Chlamydomonas cell line with a deletion mutation in DHC1b. The mutant grows normally and appears to have a normal Golgi apparatus, but has very short flagella. The deletion also results in a massive redistribution of raft subunits from a peri-basal body pool (Cole, D.G., D.R. Diener, A.L. Himelblau, P.L. Beech, J.C. Fuster, and J.L. Rosenbaum. 1998. J. Cell Biol. 141:993-1008) to the flagella. Rafts are particles that normally move up and down the flagella in a process known as intraflagellar transport (IFT) (Kozminski, K.G., K.A. Johnson, P. Forscher, and J.L. Rosenbaum. 1993. Proc. Natl. Acad. Sci. USA. 90:5519-5523), which is essential for assembly and maintenance of flagella. The redistribution of raft subunits apparently occurs due to a defect in the retrograde component of IFT, suggesting that DHC1b is the motor for retrograde IFT. Consistent with this, Western blots indicate that DHC1b is present in the flagellum, predominantly in the detergent- and ATP-soluble fractions. These results indicate that DHC1b is a cytoplasmic dynein essential for flagellar assembly, probably because it is the motor for retrograde IFT.

Show MeSH
Related in: MedlinePlus