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. 1999 Feb 8;144(3):473-81.
doi: 10.1083/jcb.144.3.473.

The DHC1b (DHC2) Isoform of Cytoplasmic Dynein Is Required for Flagellar Assembly

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Free PMC article

The DHC1b (DHC2) Isoform of Cytoplasmic Dynein Is Required for Flagellar Assembly

G J Pazour et al. J Cell Biol. .
Free PMC article

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.

Figures

Figure 1
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).
Figure 1
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).
Figure 2
Figure 2
Phenotype of the dhc1b deletion mutant. (a) Identification of the dhc1b deletion mutant. DNA was isolated from >300 Chlamydomonas insertional mutants (Pazour et al., 1995; Koutoulis et al., 1997), cleaved with PstI, and analyzed by Southern blotting using a 0.3-kbp fragment of a DHC1b cDNA as probe. The DNA hybridized to a single band in wild-type (Wild type) and all strains except V92.2 (DHC1bΔ), which had no hybridizing band. (b) Deletion of the DHC1b gene does not affect growth rate. Growth of wild-type or mutant cells (V92.2 and 3088.4) in liquid medium was monitored as described previously (Pazour et al., 1998). On day 3, a second set of cultures was inoculated by diluting cells from the first series to 105 cells/ml (arrow). (c) Flagella are much shorter in the dhc1b deletion mutant (DHC1bΔ) than in wild-type cells. Cells were recorded by differential interference-contrast microscopy as described in Pazour et al. (1998). (d) The dhc1b deletion segregates with the flagellar defect. Strains V92.2 (DHC1bΔ) and CC124 (Wild type) were mated, tetrads were dissected, and the offspring were scored for motility by light microscopy. DNA was isolated from a single product of 49 independent tetrads and analyzed by Southern blotting as in panel a. The results for the parents and 12 progeny are shown. In all cases, progeny with the motility defect (−) lacked the DHC1b gene, whereas those with normal motility (+) had the DHC1b gene.
Figure 3
Figure 3
Ultrastructure of dhc1b deletion mutant flagella. In wild-type cells (a) the space between the flagellar membrane and doublet microtubules is usually devoid of material. In contrast, some dhc1b mutant cells (b, d, and e) have an apparently normal axoneme but the space between the doublet microtubules and the flagellar membrane is filled with electron-dense material identical in appearance to the rafts of IFT (Kozminski et al., 1993, 1995). The flagella of other dhc1b mutant cells (c) lack some or all of the axonemal microtubules and are completely filled with rafts. Cells were fixed as described previously (Pazour et al., 1998).
Figure 4
Figure 4
Localization of an IFT raft protein, FLA10, and DHC1b in wild-type and dhc1b mutant cells by indirect immunofluorescence. The antigens of interest are shown in green whereas autofluorescence of the cell body is shown in red. Antibody specific for the p172 subunit of the IFT rafts (Cole et al., 1998) shows that in wild-type cells, the raft proteins are located primarily in the cell body at the base of the flagella, with some punctate staining along the length of the flagella (a). In contrast, in the dhc1b deletion mutant (DHC1bΔ), almost no staining is seen in the peri-basal body region, but there is a very intense staining of the flagellar stubs (b). FLA10, a subunit of the anterograde IFT motor, is localized primarily in the peri-basal body region of wild-type cells with some punctate staining along the flagella (c). In the dhc1b deletion mutant (DHC1bΔ), FLA10 staining similarly is observed in the cell body at the base of the flagella; it is also present in the flagellar stubs (d). In wild-type cells, DHC1b is localized in the peri-basal body region with some punctate staining along the flagella (e). The dhc1b deletion mutant (DHC1bΔ), which lacks this antigen, shows only a small amount of nonspecific punctate staining in the cell body (f). No flagellar staining is detected.
Figure 5
Figure 5
The location and morphology of the Golgi complex is the same in wild-type (a) and dhc1b (b) cells.
Figure 6
Figure 6
DHC1b is found in the flagella. (a) Whole cell extracts were made from wild-type and dhc1b (DHC1bΔ) cells and probed with affinity-purified DHC1b antibodies or the 12γC mAb to the γ DHC of the outer arm dynein. The DHC1b antibodies detect a single high molecular weight band in wild-type cells but not in the mutant, whereas the γ DHC antibody detects a similarly sized band in both samples. 12γC also detects a smaller unidentified band in both cell types. (b) Flagella were isolated from wild-type cells and separated into the following fractions: detergent-soluble membrane proteins and soluble proteins of the flagellar matrix (M + M); proteins released from the axoneme by a first, second, or third rinse with ATP (ATP1, ATP2, and ATP3, respectively); proteins released from the ATP-rinsed axoneme by 0.6 M KCl (Salt); and the axonemal proteins remaining after treatment with detergent, ATP and KCl (Axo). Gels were loaded with extracts from equivalent numbers of flagella and analyzed by Western blotting with antibodies to DHC1b, FLA10 (FLA10N; Cole et al., 1998), p139 [Raft (p139); Cole et al., 1998], and DHCγ (12γC; King et al., 1985).
Figure 7
Figure 7
In wild-type cells, IFT components are continuously removed from the peri-basal body pool for IFT, and returned to the pool (modified from Fig. 13 of Cole et al., 1998). In dhc1b cells, IFT components are likewise transported from the peri-basal body pool into the flagella, but they then accumulate there, presumably due to a defect in retrograde IFT. As a result, the peri-basal body pool is depleted.

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