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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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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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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.

Mentions: We reported previously that an insertional mutant of Chlamydomonas lacking the dynein light chain LC8 was defective in retrograde IFT (Pazour et al., 1998). This mutant accumulated large numbers of rafts at the tips of short flagella, apparently because the rafts were transported into the flagella but could not readily return to the base due to the defect in retrograde IFT. The dhc1b mutant has even shorter flagella filled with even more rafts. In this mutant the raft proteins are completely redistributed from the peri-basal body pool, where they are predominantly located in wild-type and the LC8 mutant (results not shown), into the flagella. Although the flagella of dhc1b cells are too short for IFT to be observed directly in them, the similar accumulation of rafts in the flagella of the LC8 and dhc1b mutants suggests that the latter also is defective in retrograde IFT. It seems likely that in wild-type cells, rafts continuously move from the peri-basal body pool up the flagella and then back down, returning to the pool (Fig. 7). In the dhc1b mutant, rafts presumably move into the flagella as in wild-type cells, but then accumulate there because there is no retrograde IFT to return them to the peri-basal body pool. As a result, the peri-basal body pool becomes depleted. The fact that the phenotype is stronger in the dhc1b mutant than in the LC8 mutant suggests that some retrograde transport of rafts remains in the absence of the light chain. However, it is possible that loss of DHC1b and LC8 affect flagellar assembly and IFT through different mechanisms. Currently, we are carrying out studies to determine if LC8 is a component of DHC1b dynein, which should clarify this matter.


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

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

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132917&req=5

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.
Mentions: We reported previously that an insertional mutant of Chlamydomonas lacking the dynein light chain LC8 was defective in retrograde IFT (Pazour et al., 1998). This mutant accumulated large numbers of rafts at the tips of short flagella, apparently because the rafts were transported into the flagella but could not readily return to the base due to the defect in retrograde IFT. The dhc1b mutant has even shorter flagella filled with even more rafts. In this mutant the raft proteins are completely redistributed from the peri-basal body pool, where they are predominantly located in wild-type and the LC8 mutant (results not shown), into the flagella. Although the flagella of dhc1b cells are too short for IFT to be observed directly in them, the similar accumulation of rafts in the flagella of the LC8 and dhc1b mutants suggests that the latter also is defective in retrograde IFT. It seems likely that in wild-type cells, rafts continuously move from the peri-basal body pool up the flagella and then back down, returning to the pool (Fig. 7). In the dhc1b mutant, rafts presumably move into the flagella as in wild-type cells, but then accumulate there because there is no retrograde IFT to return them to the peri-basal body pool. As a result, the peri-basal body pool becomes depleted. The fact that the phenotype is stronger in the dhc1b mutant than in the LC8 mutant suggests that some retrograde transport of rafts remains in the absence of the light chain. However, it is possible that loss of DHC1b and LC8 affect flagellar assembly and IFT through different mechanisms. Currently, we are carrying out studies to determine if LC8 is a component of DHC1b dynein, which should clarify this matter.

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