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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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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.
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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.

Mentions: In an effort to identify a Chlamydomonas strain with a defect in the DHC1b gene, a portion of one of the DHC1b cDNAs was used to screen a collection of insertional mutants that had been generated by transforming cells with DNA containing a selectable marker. Transformation of Chlamydomonas occurs by the nonhomologous integration of DNA into the genome (Kindle, 1990; Tam and Lefebvre, 1993), resulting in the disruption or deletion of genes at the site of insertion. Such mutations can be detected as restriction fragment length polymorphisms in Southern blots probed with DNAs encoding the affected genes, so that cell lines in which a cloned gene has been disrupted can be readily identified (Wilkerson et al., 1995; Koutoulis et al., 1997; Pazour et al., 1998). From a screen of >300 insertional mutants with a wide range of phenotypes (Pazour et al., 1995; Koutoulis et al., 1997), one strain (V92.2) was found with a disrupted DHC1b gene (Fig. 2 a). Further Southern blot analysis of this strain using the two longer DHC1b cDNAs as probes indicated that at least 3.6 kb of DHC1b coding region was deleted. Because the deleted region includes P-loops 1 and 2, this deletion allele, termed dhc1b-1, is likely to be a loss-of-function allele.


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

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

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132917&req=5

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.
Mentions: In an effort to identify a Chlamydomonas strain with a defect in the DHC1b gene, a portion of one of the DHC1b cDNAs was used to screen a collection of insertional mutants that had been generated by transforming cells with DNA containing a selectable marker. Transformation of Chlamydomonas occurs by the nonhomologous integration of DNA into the genome (Kindle, 1990; Tam and Lefebvre, 1993), resulting in the disruption or deletion of genes at the site of insertion. Such mutations can be detected as restriction fragment length polymorphisms in Southern blots probed with DNAs encoding the affected genes, so that cell lines in which a cloned gene has been disrupted can be readily identified (Wilkerson et al., 1995; Koutoulis et al., 1997; Pazour et al., 1998). From a screen of >300 insertional mutants with a wide range of phenotypes (Pazour et al., 1995; Koutoulis et al., 1997), one strain (V92.2) was found with a disrupted DHC1b gene (Fig. 2 a). Further Southern blot analysis of this strain using the two longer DHC1b cDNAs as probes indicated that at least 3.6 kb of DHC1b coding region was deleted. Because the deleted region includes P-loops 1 and 2, this deletion allele, termed dhc1b-1, is likely to be a loss-of-function allele.

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