Testing the time-of-flight model for flagellar length sensing

Abstract

Cilia and flagella are microtubule-based organelles that protrude from the surface of most cells, are important to the sensing of extracellular signals, and make a driving force for fluid flow. Maintenance of flagellar length requires an active transport process known as intraflagellar transport (IFT). Recent studies reveal that the amount of IFT injection negatively correlates with the length of flagella. These observations suggest that a length-dependent feedback regulates IFT. However, it is unknown how cells recognize the length of flagella and control IFT. Several theoretical models try to explain this feedback system. We focused on one of the models, the "time-of-flight" model, which measures the length of flagella on the basis of the travel time of IFT protein in the flagellar compartment. We tested the time-of-flight model using Chlamydomonas dynein mutant cells, which show slower retrograde transport speed. The amount of IFT injection in dynein mutant cells was higher than that in control cells. This observation does not support the prediction of the time-of-flight model and suggests that Chlamydomonas uses another length-control feedback system rather than that described by the time-of-flight model.

FIGURE 1:

Schematic of the time-of-flight model. See the main text for details.

FIGURE 2:

Modeling the time-of-flight length-sensing mechanism. (A) The time-of-flight model can produce stable length control and recapitulates decelerating growth kinetics seen in Chlamydomonas flagella. Plot shows a numerical solution of flagellar length vs. time according to Eq. 8 with the following parameter values: A = 0.0015 s^–1, D = 0.0003 µm/s (based on shortening rate of flagella in absence of IFT; see Marshall and Rosenbaum, 2001), lifetime = 3 s, v_a = 2.5 µm/s (Engel et al., 2012), τ = 3 s (Chien et al., 2017), v_r = 3.5 µm/s, and P = 40 (Marshall et al., 2005). (B) IFT injection rate is a decreasing function of flagellar length. Plot is based on the numerical solution of panel A and depicts injection rate as a function of length. (C) Dependence of injection rate on retrograde IFT velocity. Numerical solutions were determined for a range of retrograde velocities, keeping all other parameters the same as in the solution shown in A. Plot demonstrates that a reduction in retrograde IFT velocity is predicted to yield a reduction in the IFT injection rate.

FIGURE 3:

IFT speed during flagella regeneration in dynein mutant strains. (A) Representative DIC kymographs of Chlamydomonas flagella in control (fla3 KAP-GFP) and fla24 strains. These kymographs were assembled from Supplemental Videos S1 and S2. The horizontal axis is time, and the vertical axis is position along the flagellum. Horizontal bar: 5 s; vertical bar: 5 µm. (B, D) Average speeds of anterograde and retrograde IFT were measured from each kymograph and plotted against their flagellar length. Control (blue circles, n = 76 flagella), dhc1b-3 (red squares, n = 52 flagella), and fla24 (orange triangles, n = 47 flagella). (C, E) The differences of anterograde and retrograde IFT speed from the control regression line. The distances from the control regression line to each data point for each sample were calculated and shown as a box-and-whisker plot. Significance was determined by unpaired two-tailed t test (***P < 0.001 and ****P < 0.0001).

FIGURE 4:

Prediction of the time-of-flight model for dynein mutant flagella. (A) Estimation of travel time of IFT in control and dynein mutant cells. The speeds of anterograde and retrograde IFT were based on our observation (Figure 3). Remodeling time at the tip of flagella was estimated as 3 s on the basis of our photobleaching assay and published results (Chien et al., 2017). Longer flagella still show longer IFT travel time. (B) Prediction for the time-of-flight model of dynein mutant flagella. Dynein mutant cells show slower retrograde IFT speed than control. When dynein mutant cells have same flagella length as control cells, dynein mutant cells should inject a lesser amount of IFT into flagella than control cells because slower retrograde IFT mimics the effect of longer flagella.

FIGURE 5:

IFT injection intensities were increased in dynein mutant cells. (A) Representative kymographs of Chlamydomonas flagella in control (fla3 KAP-GFP) and dhc1b-3 (dhc1b-3 fla3 KAP-GFP) strains. These kymographs were assembled from Supplemental Videos S3 and S4. Horizontal bar: 5 s; vertical bar: 5 µm. (B) The mean injection intensity of each flagellum was calculated from kymographs and plotted against flagella length. Control (blue circles, n = 103 flagella), dhc1b-3 (red squares, n = 85 flagella), and fla24 (orange triangles, n = 37 flagella). (C) A box-and-whisker plot shows the mean difference of injection intensity from the control regression line. Both dynein mutant cells inject more IFT into flagella than control cells. Significance was determined by unpaired two-tailed t test (****P < 0.0001). (D) Relationship between injection rate and retrograde speed, derived by taking all flagella in the length range 4–6 µm for all three genotypes and plotting their average injection intensity (as in B) on the vertical axis and their average retrograde speed (as in Figure 3D) on the horizontal axis as a two-dimensional box-and-whisker plot. Control (blue, n = 18 flagella), dhc1b-3 (red, n = 13 flagella), and fla24 (orange, n = 12 flagella).

FIGURE 6:

The kinetics of flagella regeneration of control (wild-type CC-125, blue circles) and dynein mutant (dhc1b-3 and fla24, red squares and orange triangles, respectively) cells. n = 20 cells per strain and time point.