Benjamin Engel, Max Planck Institute of Biochemistry
6 November 2013
The differences between the fla24 and dhc1b-3 phenotypes are quite interesting and may shed more light on the role of retrograde IFT in the maintenance of flagellar length.
At 21C, fla24 cellular DHC1b and D1bLIC levels are dramatically decreased by 18 and 16-fold, respectively, although immunofluorescence shows that some of this dynein protein still localizes to flagella (Figs 4 and 5). This decrease in dynein levels is correlated with a strong reduction in retrograde IFT speed (29% of WT) and frequency (61% of WT) at 21C (Iomini et al., 2001, Table 2). Despite this retrograde IFT defect, anterograde IFT is hardly affected (speed is 88% of WT, frequency is 106% of WT; Iomini et al., 2001, Table 2) and fla24 flagella maintain a length that is similar to WT (Fig. 2D). However, once shifted to 34C, fla24 flagella shorten and are lost within 6 hours, similar to anterograde mutants such as fla10.
In contrast, the flagellar levels of DHC1b and D1bLIC in dhc1b-3 cells at 21C are only reduced by about 2-fold (Engel et al., 2012, Fig. 5B), retrograde speed and frequency are decreased to only 75% of WT, and anterograde IFT is unaffected (Engel et al., 2012, Fig. 2). After shifting to 34C for 12-18 hours, the flagellar levels of DHC1b and D1bLIC proteins are greatly reduced, retrograde IFT is strongly decreased (speed is 40% of WT, frequency is 20% of WT), coupled with a more moderate reduction in anterograde IFT (speed is 89% of WT, frequency is 58% of WT). Flagella are maintained at close-to-WT length for much longer than typical anterograde mutants, but are eventually lost between 18 and 48 hours after the shift to restrictive temperature.
Comparing these two mutants, it seems that the IFT conditions in fla24 flagella at 21C are similar to the conditions in dhc1b-3 flagella following 6-18 hours at 34C. In both cases, there is a dramatic reduction in dynein protein levels, which is coupled to a strong decrease in retrograde IFT and only a minor affect on anterograde IFT. In both cases, wildtype-length flagella are maintained, indicating that fully-functioning retrograde IFT is not required for maintenance of flagellar length. The loose coupling between retrograde and anterograde IFT seen in dhc1b-3 (Engel et al., 2012, Table 2) indicates that when retrograde IFT function is reduced below a certain threshold, then anterograde IFT begins to be affected. In the case of fla24 flagella at 21C, this threshold is never reached and flagella are maintained indefinitely. However, the shift to 34C likely inhibits fla24 dynein well below this threshold and flagella are lost due to a coupled reduction in anterograde IFT. In the case of dhc1b-3 cells at 34C, crossing this threshold occurs more gradually, but retrograde IFT is eventually inhibited to such an extent that anterograde IFT drops to a level where flagella cannot be maintained (or cannot be newly assembled following cell division).
In summary, the fla24 mutation clearly has a more severe phenotype than dhc1b-3, but the two phenotypes may tell a consistent story about the coupling between retrograde and anterograde IFT, and the impact of this relationship on flagellar length. The difference in phenotype severity is likely caused by the different regions of the DHC1b protein affected by the two mutations. While fla24 is in the AAA5 domain, which extends into the strut/buttress (Fig. 8), dhc1b-3 is in the C-sequence (Engel et al., 2012, Fig. S3) and thus may be less detrimental to protein function.
Comparison of fla24 and dhc1b-3 phenotypes
6 November 2013
The differences between the fla24 and dhc1b-3 phenotypes are quite interesting and may shed more light on the role of retrograde IFT in the maintenance of flagellar length.
At 21C, fla24 cellular DHC1b and D1bLIC levels are dramatically decreased by 18 and 16-fold, respectively, although immunofluorescence shows that some of this dynein protein still localizes to flagella (Figs 4 and 5). This decrease in dynein levels is correlated with a strong reduction in retrograde IFT speed (29% of WT) and frequency (61% of WT) at 21C (Iomini et al., 2001, Table 2). Despite this retrograde IFT defect, anterograde IFT is hardly affected (speed is 88% of WT, frequency is 106% of WT; Iomini et al., 2001, Table 2) and fla24 flagella maintain a length that is similar to WT (Fig. 2D). However, once shifted to 34C, fla24 flagella shorten and are lost within 6 hours, similar to anterograde mutants such as fla10.
In contrast, the flagellar levels of DHC1b and D1bLIC in dhc1b-3 cells at 21C are only reduced by about 2-fold (Engel et al., 2012, Fig. 5B), retrograde speed and frequency are decreased to only 75% of WT, and anterograde IFT is unaffected (Engel et al., 2012, Fig. 2). After shifting to 34C for 12-18 hours, the flagellar levels of DHC1b and D1bLIC proteins are greatly reduced, retrograde IFT is strongly decreased (speed is 40% of WT, frequency is 20% of WT), coupled with a more moderate reduction in anterograde IFT (speed is 89% of WT, frequency is 58% of WT). Flagella are maintained at close-to-WT length for much longer than typical anterograde mutants, but are eventually lost between 18 and 48 hours after the shift to restrictive temperature.
Comparing these two mutants, it seems that the IFT conditions in fla24 flagella at 21C are similar to the conditions in dhc1b-3 flagella following 6-18 hours at 34C. In both cases, there is a dramatic reduction in dynein protein levels, which is coupled to a strong decrease in retrograde IFT and only a minor affect on anterograde IFT. In both cases, wildtype-length flagella are maintained, indicating that fully-functioning retrograde IFT is not required for maintenance of flagellar length. The loose coupling between retrograde and anterograde IFT seen in dhc1b-3 (Engel et al., 2012, Table 2) indicates that when retrograde IFT function is reduced below a certain threshold, then anterograde IFT begins to be affected. In the case of fla24 flagella at 21C, this threshold is never reached and flagella are maintained indefinitely. However, the shift to 34C likely inhibits fla24 dynein well below this threshold and flagella are lost due to a coupled reduction in anterograde IFT. In the case of dhc1b-3 cells at 34C, crossing this threshold occurs more gradually, but retrograde IFT is eventually inhibited to such an extent that anterograde IFT drops to a level where flagella cannot be maintained (or cannot be newly assembled following cell division).
In summary, the fla24 mutation clearly has a more severe phenotype than dhc1b-3, but the two phenotypes may tell a consistent story about the coupling between retrograde and anterograde IFT, and the impact of this relationship on flagellar length. The difference in phenotype severity is likely caused by the different regions of the DHC1b protein affected by the two mutations. While fla24 is in the AAA5 domain, which extends into the strut/buttress (Fig. 8), dhc1b-3 is in the C-sequence (Engel et al., 2012, Fig. S3) and thus may be less detrimental to protein function.
Very thought-provoking study!
Competing interests
No competing interest