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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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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).
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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).

Mentions: Electron microscopic analysis showed that the dhc1b flagella have a very aberrant structure (Fig. 3). The flagella barely extend beyond the flagellar collars (Fig. 3, d and e), and their microtubules are often disorganized. In some cross-sections, the axoneme has a normal looking 9 + 2 arrangement of microtubules (Fig. 3 b), whereas in others no microtubules are seen (Fig. 3 c). At least some inner and outer arms are present on those dhc1b axonemes (Fig. 3 b) that extend beyond the point where arms first appear on the doublet microtubules of wild-type axonemes (Hoops and Witman, 1983). The most distinctive feature of the dhc1b flagellar cross-sections is that they all contain an unusual abundance of electron-dense material that is identical in appearance to the IFT raft particles (Kozminski et al., 1993, 1995; Pazour et al., 1998). In cross-sections of wild-type flagella, rafts are occasionally seen between the doublet microtubules and the flagellar membrane (Kozminski et al., 1993, 1995), but in the dhc1b mutant this space is completely filled with rafts. For example, in the flagellum shown in Fig. 3 b, the axoneme is surrounded by two or three concentric rings of tightly packed rafts. In the flagellum shown in Fig. 3 c, the axoneme is missing and the entire space within the flagellar membrane is filled with rafts. In longitudinal sections (Fig. 3, d and e), the rafts have the typical appearance of a linear array of subunits (Kozminski et al., 1993).


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

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

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).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132917&req=5

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).
Mentions: Electron microscopic analysis showed that the dhc1b flagella have a very aberrant structure (Fig. 3). The flagella barely extend beyond the flagellar collars (Fig. 3, d and e), and their microtubules are often disorganized. In some cross-sections, the axoneme has a normal looking 9 + 2 arrangement of microtubules (Fig. 3 b), whereas in others no microtubules are seen (Fig. 3 c). At least some inner and outer arms are present on those dhc1b axonemes (Fig. 3 b) that extend beyond the point where arms first appear on the doublet microtubules of wild-type axonemes (Hoops and Witman, 1983). The most distinctive feature of the dhc1b flagellar cross-sections is that they all contain an unusual abundance of electron-dense material that is identical in appearance to the IFT raft particles (Kozminski et al., 1993, 1995; Pazour et al., 1998). In cross-sections of wild-type flagella, rafts are occasionally seen between the doublet microtubules and the flagellar membrane (Kozminski et al., 1993, 1995), but in the dhc1b mutant this space is completely filled with rafts. For example, in the flagellum shown in Fig. 3 b, the axoneme is surrounded by two or three concentric rings of tightly packed rafts. In the flagellum shown in Fig. 3 c, the axoneme is missing and the entire space within the flagellar membrane is filled with rafts. In longitudinal sections (Fig. 3, d and e), the rafts have the typical appearance of a linear array of subunits (Kozminski et al., 1993).

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