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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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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.
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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.

Mentions: To determine if the distribution of IFT proteins in dhc1b cells differed in any other way from that in wild-type cells, we examined both cell types by immunofluorescence microscopy. Antibodies to raft subunits and to the FLA10 subunit of the anterograde IFT motor stain wild-type cells with very similar patterns. The antibodies localize primarily to the peri-basal body region within the cell body, and to punctate spots along the flagella (Cole et al., 1998; Fig. 4, a and c). In contrast, dhc1b mutant cells, stained with an antibody specific for the p172 raft subunit, lacked the peri-basal body staining but showed dramatically stronger staining of the flagella (Fig. 4 b). The latter result confirms that the electron-dense particles accumulated in the dhc1b flagella are IFT rafts. Antibodies to the FLA10 subunit of the anterograde motor also stain dhc1b flagella more strongly than they stain wild-type flagella (Fig. 4 d). However, in contrast to the raft subunit, FLA10 is retained in the peri-basal body region of dhc1b cells. There is little staining of the general cytoplasm or of other cell structures by these antibodies in either wild-type or mutant cells.


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

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

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.
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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.
Mentions: To determine if the distribution of IFT proteins in dhc1b cells differed in any other way from that in wild-type cells, we examined both cell types by immunofluorescence microscopy. Antibodies to raft subunits and to the FLA10 subunit of the anterograde IFT motor stain wild-type cells with very similar patterns. The antibodies localize primarily to the peri-basal body region within the cell body, and to punctate spots along the flagella (Cole et al., 1998; Fig. 4, a and c). In contrast, dhc1b mutant cells, stained with an antibody specific for the p172 raft subunit, lacked the peri-basal body staining but showed dramatically stronger staining of the flagella (Fig. 4 b). The latter result confirms that the electron-dense particles accumulated in the dhc1b flagella are IFT rafts. Antibodies to the FLA10 subunit of the anterograde motor also stain dhc1b flagella more strongly than they stain wild-type flagella (Fig. 4 d). However, in contrast to the raft subunit, FLA10 is retained in the peri-basal body region of dhc1b cells. There is little staining of the general cytoplasm or of other cell structures by these antibodies in either wild-type or mutant cells.

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