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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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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).
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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).

Mentions: If DHC1b powers retrograde IFT, then this isoform of cytoplasmic dynein should be present in wild-type flagella. To test this, we generated an antibody against a bacterially expressed fragment of Chlamydomonas DHC1b. The peptide used for antibody production constitutes ∼50 kD near the NH2-terminal end of the region that we have cloned; this region is not highly conserved among DHCs (Wilkerson et al., 1994; Gibbons, 1995; Pazour, G.J., and G.B. Witman, unpublished results), so an antibody to it is likely to be isoform specific. To check this, we examined the ability of the antibody to react with DHCs in Western blots of extracts of wild-type vs. dhc1b whole cells. The antibody detected a single high molecular weight band in wild-type cells, but did not recognize any band in the cells lacking DHC1b (Fig. 6 a, DHC1b). Antibody to the outer dynein arm γ DHC detected bands in both cell lines (Fig. 6 a, DHCγ), confirming that other DHCs are present in the dhc1b cells. Therefore, the antibody is isoform specific.


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

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

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).
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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).
Mentions: If DHC1b powers retrograde IFT, then this isoform of cytoplasmic dynein should be present in wild-type flagella. To test this, we generated an antibody against a bacterially expressed fragment of Chlamydomonas DHC1b. The peptide used for antibody production constitutes ∼50 kD near the NH2-terminal end of the region that we have cloned; this region is not highly conserved among DHCs (Wilkerson et al., 1994; Gibbons, 1995; Pazour, G.J., and G.B. Witman, unpublished results), so an antibody to it is likely to be isoform specific. To check this, we examined the ability of the antibody to react with DHCs in Western blots of extracts of wild-type vs. dhc1b whole cells. The antibody detected a single high molecular weight band in wild-type cells, but did not recognize any band in the cells lacking DHC1b (Fig. 6 a, DHC1b). Antibody to the outer dynein arm γ DHC detected bands in both cell lines (Fig. 6 a, DHCγ), confirming that other DHCs are present in the dhc1b cells. Therefore, the antibody is isoform specific.

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