Both strains had the same kinetics of flagellar shortening and loss, which was far more rapid than flagellar loss in the dhc1b-3 single mutant. Immunofluorescence was performed essentially as described in Keller et al. This anterograde transport is driven by kinesin-2, a heterotrimeric complex composed of the FLA10 and FLA8 motor subunits Walther et al. Because fla10 abrogates both anterograde and retrograde transport, this result does not reveal which phase of IFT is involved. This A-to-T substitution results in a single amino acid switch from isoleucine to phenylalanine at position of the amino acid sequence Fig. Molecular map of the Chlamydomonas reinhardtii nuclear genome.
Here, we describe a new conditional mutation in dynein, dhc1b-3 , which enables the inducible decoupling of retrograde IFT from anterograde transport. Flagellar beat frequency was measured as described previously Kamiya, ; Wakabayashi and King, IFT particles contain proteins required for ciliary assembly in Caenorhabditis elegans sensory neurons”. Thus, our understanding of anterograde IFT has resulted in large part from the availability of a conditional mutation in the anterograde motor that allows inducible shutoff of motor function. Abstract The maintenance of flagellar length is believed to require both anterograde and retrograde intraflagellar transport IFT. Thus, it appears that retrograde IFT helps maintain both physical beat frequency and signaling phototaxis sign functions of Chlamydomonas flagella.
Flagellar regeneration and shortening Flagellar regeneration was initiated by pH shock, as described previously Lefebvre, Abbreviations used in this paper: Thus, the increased rate of flagellar shortening in the presence of IFT is likely due to anterograde transport, which has been shown to carry the microtubule-depolymerizing factor kinesin into flagella in response to NaPPi treatment Piao et al.
Noncomplementation in the dhc1b-1 dhc1b-3 diploid strain proved that the dhc1b-3 phenotype was due to a mutation in DHC1b Fig. Positives were restruck onto new plates and retested by PCR. The maximum intensity value for TIRF or average intensity for DIC for each traneport along the length of the line was plotted for each frame in the movie.
S4 B of dhc1b -3 flagellar regeneration were comparable to wild-type. Novel heterotrimeric kinesin-related protein purified from sea urchin eggs.
Live-cell microscopy of IFT and kymograph analysis were performed as detailed previously Engel et al. Bar graphs depict the percentage of deflagellated cells, and line graphs show the average flagellar length of flagellated cells. The Chlamydomonas FLA10 gene encodes a novel kinesin-homologous protein.
To expand our investigation to a wider scope of proteins and time points, we performed Western blot analysis of isolated flagella from wild-type and dhc1b-3 cells Fig. Witman4 Ritsu Kamiya3 and Wallace F. Cilia intraflagellxr flagella are dynamic structures whose assembly and maintenance require an active transport process known as intraflagellar transport IFT; Kozminski et al. Engelco-funded by the Japan Society for the Promotion of Science. However, the only ts dynein mutant that has been isolated up to this point dhc1b -2, Pazour et al.
Anterograde train speed was 2. Flagellar beat frequency was measured as described previously Kamiya, ; Wakabayashi and King, For each condition wild-type 21C, dhc1b -3 21C, dhc1b -3 34C 18 htwo liters of cells were grown in TAP with constant light and aeration.
Flagellar length control system: The starting flagellar lengths of both mutant and wild-type strains were slightly shorter than normal, likely due to unknown variations in the culture conditions.
Analysis inttraflagellar cell vibration for assessing axonemal motility in Chlamydomonas. Thus, it appears that retrograde IFT helps maintain both physical beat frequency and signaling phototaxis sign functions of Chlamydomonas flagella.
The role of retrograde intraflagellar transport in flagellar assembly, maintenance, and function
The relationship between anterograde and retrograde IFT, and implications for flagellar length control One clear conclusion that can be drawn from our data is that anterograde and retrograde IFT are coupled, but it is not a 1: Instead of switching the sign of phototaxis, bbs cells swam in random directions, irrespective of temperature.
Colonies were picked, struck onto 1. Isolation and electrophoretic analysis of microtubules, matrix, membranes, and mastigonemes. Summary of the dhc1b -3 IFT and flagellar phenotypes compared to wild-type cells at the same condition. Swimming velocities were calculated from the tracks left by swimming cells during the extended exposure of images, obtained using Image Hyper software. Intraflagellar transport particle size scales inversely with flagellar length: Crystal structure of the dynein motor domain.
IFT has been shown to be necessary for the movement of other signaling proteins within the cilia, and therefore may play a role in many different signaling pathways. This A-to-T substitution results in a single amino acid switch from isoleucine to phenylalanine at position of the amino acid sequence Fig.
However, this difference may be due to the fact that the bbs mutant was generated from an agg1 strain.
Intraflagellar transport motors in cilia: These two proteins are exclusively found in the membrane plus matrix fraction and may rely on DHC1b activity for entry into flagella. IFT train speed and frequency were quantified from the kymographs by measuring the slope and number of traces, respectively.
Intraflagellar transport – Wikipedia
Thus, the replacement of Ile with the bulkier Phe residue could presumably impede protein stability, a defect that would be exacerbated by the temperature shift. Previous studies have indicated a role for IFT in active disassembly of flagella, as inttraflagellar rate of sodium pyrophosphate NaPPi —induced flagellar shortening is decreased in fla10 cells at the nonpermissive temperature Pan transoprt Snell,