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A dynein light chain is essential for the retrograde particle movement of intraflagellar transport (IFT)
G J Pazour1, C G Wilkerson, G B Witman
1Department of Cell Biology, University of Massachusetts Medical Center (UMMC), Worcester Foundation Campus, Shrewsbury, Massachusetts 01545, USA.
Abstract:
Several enzymes, including cytoplasmic and flagellar outer arm dynein, share an Mr 8,000 light chain termed LC8. The function of this chain is unknown, but it is highly conserved between a wide variety of organisms. We have identified deletion alleles of the gene (fla14) encoding this protein in Chlamydomonas reinhardtii. These mutants have short, immotile flagella with deficiencies in radial spokes, in the inner and outer arms, and in the beak-like projections in the B tubule of the outer doublet microtubules. Most dramatically, the space between the doublet microtubules and the flagellar membrane contains an unusually high number of rafts, the particles translocated by intraflagellar transport (IFT) (Kozminski, K.G., P.L. Beech, and J.L. Rosenbaum. 1995. J. Cell Biol. 131:1517-1527). IFT is a rapid bidirectional movement of rafts under the flagellar membrane along axonemal microtubules. Anterograde IFT is dependent on a kinesin whereas the motor for retrograde IFT is unknown. Anterograde IFT is normal in the LC8 mutants but retrograde IFT is absent; this undoubtedly accounts for the accumulation of rafts in the flagellum. This is the first mutation shown to specifically affect retrograde IFT; the fact that LC8 loss affects retrograde IFT strongly suggests that cytoplasmic dynein is the motor that drives this process. Concomitant with the accumulation of rafts, LC8 mutants accumulate proteins that are components of the 15-16S IFT complexes (Cole, D.G., D.R. Deiner, A.L. Himelblau, P.L. Beech, J.C. Fuster, and J.L. Rosenbaum. 1998. J. Cell Biol. 141:993-1008), confirming that these complexes are subunits of the rafts. Polystyrene microbeads are still translocated on the surface of the flagella of LC8 mutants, indicating that the motor for flagellar surface motility is different than the motor for retrograde IFT.
Insights
Loss of the conserved LC8 light chain in Chlamydomonas reinhardtii flagella disrupts retrograde intraflagellar transport (IFT). This study identifies LC8 as essential for retrograde IFT, suggesting cytoplasmic dynein as the motor protein.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The Mr 8,000 light chain, LC8, is a conserved protein component of several dynein enzymes.
- The precise function of LC8 remains largely unknown despite its widespread conservation across organisms.
- Intraflagellar transport (IFT) is crucial for flagellar assembly and maintenance, involving bidirectional movement of protein complexes (rafts) along axonemal microtubules.
Purpose of the Study:
- To investigate the function of the LC8 light chain in flagellar structure and motility.
- To identify the molecular motor responsible for retrograde IFT.
Main Methods:
- Generation and analysis of deletion alleles (fla14 mutants) in the gene encoding LC8 in Chlamydomonas reinhardtii.
- Phenotypic characterization of flagellar structure and motility in wild-type versus fla14 mutant cells.
- Assessment of intraflagellar transport (IFT) dynamics, including anterograde and retrograde transport, in fla14 mutants.
Main Results:
- LC8 mutants exhibit short, immotile flagella with defects in flagellar ultrastructure, including radial spokes and dynein arms.
- A significant accumulation of IFT rafts is observed within the flagella of LC8 mutants.
- Anterograde IFT is unaffected, while retrograde IFT is completely abolished in LC8 mutants, implicating LC8 in this process.
Conclusions:
- This study provides the first genetic evidence that LC8 is essential for retrograde IFT.
- The findings strongly suggest that cytoplasmic dynein serves as the motor for retrograde IFT.
- LC8 is critical for maintaining flagellar integrity and function by regulating retrograde transport of IFT components.