Protein particles in Chlamydomonas flagella undergo a transport cycle consisting of four phases

Abstract

We used an improved procedure to analyze the intraflagellar transport (IFT) of protein particles in Chlamydomonas and found that the frequency of the particles, not only the velocity, changes at each end of the flagella. Thus, particles undergo structural remodeling at both flagellar locations. Therefore, we propose that the IFT consists of a cycle composed of at least four phases: phases II and IV, in which particles undergo anterograde and retrograde transport, respectively, and phases I and III, in which particles are remodeled/exchanged at the proximal and distal end of the flagellum, respectively. In support of our model, we also identified 13 distinct mutants of flagellar assembly (fla), each defective in one or two consecutive phases of the IFT cycle. The phase I-II mutant fla10-1 revealed that cytoplasmic dynein requires the function of kinesin II to participate in the cycle. Phase I and II mutants accumulate complex A, a particle component, near the basal bodies. In contrast, phase III and IV mutants accumulate complex B, a second particle component, in flagellar bulges. Thus, fla mutations affect the function of each complex at different phases of the cycle.

Figure 1

Digital kymography identifies particles undergoing IFT along one flagellum of a Chlamydomonas pf15 mutant cell. Light intensity profiles, or linescans, were measured along the flagellum in a sequence of VE-DIC images. The same data were processed and plotted as described either in Piperno et al. 1998 (a) or in Materials and Methods (b). Vertical and horizontal axes report distance from the cell body in micrometers and time of observation, respectively, 1 s being equivalent to 30 video frames or linescans. IFT particles are seen as streaks (a) and shaded relief-like traces (b). Anterograde and retrograde particles form acute and obtuse angles, respectively, with the bottom horizontal axis. Black and white arrows indicate traces formed by the same anterograde or retrograde IFT particles, respectively, in both panels. The sudden appearance (marked by a black asterisk) or disappearance of a trace is probably due to a particle moving in or out, respectively, of the microscope focal plane. The two white asterisks mark points where two traces appear to merge and separate again. This event probably occurs when a faster particle (lower trace at early times) reaches a slower one (upper trace at early time) (lower left, white asterisk) and then surpasses it (upper right, white asterisk).

Figure 2

The results of the cluster analysis using the PAM program are displayed; the values of anterograde-to-retrograde particle frequency ratio are plotted as a function of anterograde and retrograde velocity. The members of each of the six clusters of mutants are identified by color. A filled circle indicates the medoid, or most representative member, of each cluster.

Figure 3

Kymograms of flagella of pf15, pf15fla27, pf15fla18, pf15fla24, and pf15fla9. Each recombinant is defective in one or two phases of the IFT cycle, as listed in Table . Black and white arrows indicate traces formed by anterograde or retrograde IFT particles, respectively. Anterograde particles in pf15fla18 and retrograde particles in pf15fla24 are slower than in pf15. The apparent size of these slower particles is larger than that of the corresponding particles in pf15.

Figure 4

IFT particles accumulate at one of the flagellar extremities as a consequence of specific fla mutations. Left and right panels display cells that were incubated at the permissive temperature of 21°C or for 90 min at the restrictive temperature of 32°C. Each panel shows the same field observed by immunofluorescence, left, and phase microscopy, right. A polyclonal Ab to subunits p148 and p127 of the IFT complex A was applied to cells of fla1-2 and fla10-1; an mAb to subunit p81 of the IFT complex B was applied to cells of fla15 and fla17-1. Bar, 5 μm.

Figure 5

Complex B accumulates in flagella of phase III and IV mutants. Gels were loaded with equal amounts of flagellar proteins from a wild-type (wt) strain, fla8, fla1-3, fla11, and fla21. (a) The autoradiogram of ³⁵S-labeled flagellar proteins. (b) Western blot obtained with two mAbs, one against subunit p148 of complex A and the other against p81 of complex B. Open circle, p148; filled circle, p81.

Figure 6

The accumulation of complex B in flagella of phase III and IV mutants does not necessarily correlate with that of cytoplasmic dynein. (a) The autoradiogram of ³⁵S-labeled proteins resolved by gel electrophoresis. (b and c) Western blots of equal amounts of flagellar proteins from pf28, pf28fla15, and pf28fla24 that were processed in parallel. (b) Same gel as in panel a after exposure to Abs to p148 of complex A and p81 of complex B. Open circle, p148; filled circle, p81. (c) Western blot of a gel processed in parallel and developed with Abs to KHP1 and LC7. Open square, KHP1; filled square, LC7. (d–f) Each panel displays the same field observed by immunofluorescence (top) and phase microscopy (bottom). An Ab to DHC1b was applied to cells of pf28, pf28fla15, and pf28fla24. Bar, 2 μm.

Figure 7

IFT is inhibited in flagella of pf15fla10-1 exposed to the restrictive temperature. Kymograms of flagella of pf15fla10-1 at 21°C (top) and after incubation for 1 or 2 h at 32°C (middle and bottom, respectively). No IFT particles are visible in flagella exposed to 32°C for 2 h.

Figure 8

The depletion of particles from flagella of pf28fla10-1 cells as a function of time of exposure to the restrictive temperature of 32°C correlates with the depletion of complexes A and B as well as of kinesin II and cytoplasmic dynein. (b and c) Western blots of equal amounts of flagellar proteins that were processed in parallel. (a) Autoradiogram of ³⁵S-labeled proteins resolved by gel electrophoresis. (b) Same gel as in panel a after exposure to mAbs against subunits p148 of complex A and p81 of complex B. Open circle, p148; filled circle, p81. (c) Western blot developed using Abs to KHP1 and LC7. Open square, KHP1; filled square, LC7. (d–f) Immunofluorescence signal from an anti-DHC1b Ab applied to pf28fla10-1 cells incubated at 21°C or at 32°C for 1 or 2 h, respectively. In each panel, the same cells are displayed as viewed by immunofluorescence (top) and phase microscopy (bottom). Bar, 2 μm.