Distinct mutants of retrograde intraflagellar transport (IFT) share similar morphological and molecular defects

Abstract

A microtubule-based transport of protein complexes, which is bidirectional and occurs between the space surrounding the basal bodies and the distal part of Chlamydomonas flagella, is referred to as intraflagellar transport (IFT). The IFT involves molecular motors and particles that consist of 17S protein complexes. To identify the function of different components of the IFT machinery, we isolated and characterized four temperature-sensitive (ts) mutants of flagellar assembly that represent the loci FLA15, FLA16, and FLA17. These mutants were selected among other ts mutants of flagellar assembly because they displayed a characteristic bulge of the flagellar membrane as a nonconditional phenotype. Each of these mutants was significantly defective for the retrograde velocity of particles and the frequency of bidirectional transport but not for the anterograde velocity of particles, as revealed by a novel method of analysis of IFT that allows tracking of single particles in a sequence of video images. Furthermore, each mutant was defective for the same four subunits of a 17S complex that was identified earlier as the IFT complex A. The occurrence of the same set of phenotypes, as the result of a mutation in any one of three loci, suggests the hypothesis that complex A is a portion of the IFT particles specifically involved in retrograde intraflagellar movement.

Figure 4

Representative illustration of intraflagellar transport of particles occurring at permissive temperature. Composite plots of longitudinal linescans of light intensity along flagella of (a) pf15 and (b) fla15pf15. One linescan from the proximal to the distal part of the flagellum was measured for each successive image in a video sequence obtained at a rate of 30 frames/s. The ensemble of linescans was then subjected to singular value decomposition and reconstructed as described in the text. The processed linescans were stacked and displayed so that the origin of the x axis corresponds to both the first linescan of a sequence and the proximal part of the flagellum. The distance on the y axis was measured relative to the proximal part of the flagellum. Particles undergoing anterograde or retrograde transport are identifiable as ridges with rightward and leftward slopes, respectively. (a and b) Examples of red and green ridges represent particles undergoing anterograde and retrograde transport, respectively.

Figure 1

Flagellar assembly of temperature-sensitive mutants fla15, fla16, and fla17-1 was defective at permissive temperature. A wild-type strain and fla15, fla16, and fla17-1 were cultured, deflagellated by pH shock, and analyzed by phase contrast microscopy during the regeneration of flagella at permissive temperature. The length of flagella was measured after fixation. Vertical bars represent the standard error of the mean of 25 determinations. Squares, wild-type; diamonds, fla17-1; triangles, fla16; X, fla15.

Figure 2

fla15 caused a bulge of the flagellar membrane. Micrographs of fla15 cultured and fixed at permissive temperature. (a) Differential interference contrast micrographs. (b–d) Phase contrast micrographs. Bar, 10 μm.

Figure 3

The deformation of the flagellar shape caused by fla15 was correlated with the accumulation of cytoplasmic matrix. (a and b) Electron micrographs. (a) Longitudinal section cut through the central pair microtubules. The cytoplasm accumulated between the outer doublet microtubules and the flagellar membrane in a section of the axoneme. (b) Longitudinal section cut through the cytoplasm accumulated in the bulge of the flagellar membrane. Bar, 0.1 μm.

Figure 5

fla15 was defective for two polypeptides of the 17S sedimenting fractions from the cytoplasmic matrix of flagella. Autoradiograms of ³⁵S-labeled polypeptides contained in sucrose gradient fractions 6–16 after separation by gel electrophoresis. Molecular weight standards are indicated on the left. (a) Proteins from a wild-type strain. Lines between lanes 9 and 10 indicate the presence of components 1–13 of the two 17S complexes. (b) Proteins from fla15. Asterisks between lanes 9 and 10 indicate the position of the two polypeptides that are deficient in fla15. Lines indicate the rest of the subunits of 17S complexes.

Figure 6

fla15, fla16 and fla17-1 were defective to different extents in the same three polypeptides. Autoradiograms of ³⁵S-labeled polypeptides of 17S sedimenting fractions from flagella of wild-type and mutant strains following one-dimensional electrophoresis at high resolution. First lane in a and b, proteins from a wild-type strain. Second lane in a, proteins from fla15. Second and third lane in b, proteins from fla16 and fla17-1. Equal amounts of cpm were analyzed in each lane. Numbers and lines at the left of the figure refer to 13 subunits of 17S complexes. Lines between the two panels indicate the electrophoretic bands that are deficient in each mutant and represent putative subunits of complex A. A dot between the second and third lane in b refers to a polypeptide that is present only in 17S sedimenting fractions from fla16 and fla17-1. Molecular weights of standards are indicated on the right side.

Figure 7

17S sedimenting fractions from fla15 and fla17-1 were deficient for the same four polypeptides to a different extent. Autoradiographs of two-dimensional maps of ³⁵S-labeled polypeptides contained in 17S sedimenting fractions from flagella of (a) wild-type, (b) fla15, and (c) fla17-1. Numbers and lines at the left side of a refer to the position of subunits of the 17S complexes, as determined by one-dimensional electrophoresis. Oblique lines in each panel indicate the four polypeptides that are deficient in fla15 and fla17-1. The new polypeptide present in the 17S sedimenting fractions from fla17-1 is indicated by an arrowhead in c. Polypeptides appear in the maps in increasing order of acidity from left to right.

Figure 8

The four polypeptides that are deficient from flagella of fla15, fla16, and fla17-1 behaved as subunits of a complex. Autoradiography of a two-dimensional map of ³⁵S-labeled polypeptides from flagella of a wild-type strain after subsequent sedimentation in sucrose gradient and chromatography in a DEAE-Sepharose column. Oblique lines indicate the four polypeptides that are deficient in fla15, fla16, and fla17-1. Polypeptides appear in the map in increasing order of acidity from left to right.