Intraflagellar transport particle size scales inversely with flagellar length: revisiting the balance-point length control model

Abstract

The assembly and maintenance of eukaryotic flagella are regulated by intraflagellar transport (IFT), the bidirectional traffic of IFT particles (recently renamed IFT trains) within the flagellum. We previously proposed the balance-point length control model, which predicted that the frequency of train transport should decrease as a function of flagellar length, thus modulating the length-dependent flagellar assembly rate. However, this model was challenged by the differential interference contrast microscopy observation that IFT frequency is length independent. Using total internal reflection fluorescence microscopy to quantify protein traffic during the regeneration of Chlamydomonas reinhardtii flagella, we determined that anterograde IFT trains in short flagella are composed of more kinesin-associated protein and IFT27 proteins than trains in long flagella. This length-dependent remodeling of train size is consistent with the kinetics of flagellar regeneration and supports a revised balance-point model of flagellar length control in which the size of anterograde IFT trains tunes the rate of flagellar assembly.

Figure 1.

During flagellar regeneration, there is an inverse relationship between IFT train size and flagellar length. (A) The kinetics of regeneration after pH shock. n for all time points combined = 2,026 flagella. (B) The growth rate of regenerating flagella decreases as a function of length. (C) The balance-point model predicts that equilibrium will be reached between a length-independent disassembly rate and an assembly rate that decreases as a function of length. (D) The setup for imaging C. reinhardtii flagella via TIRF. (E and F) Kymographs of KAP-GFP (E) and IFT27-GFP (F) cells undergoing regeneration after pH shock, generated from Videos 1 and 2. Bars: (horizontal) 5 µm; (vertical) 1 s. (G–I) The frequency (G), speed (H), and average (I) intensity of IFT trains during pH shock regeneration, binned by flagellar length. The intensities of IFT traces were normalized by either camera noise (dark blue and red bars) or background flagellar intensity (light blue bars). n for all time points = 113 KAP-GFP flagella, 97 IFT27-GFP flagella, and 97 wild-type (wt) flagella imaged by DIC. Error bars indicate standard deviation.

Figure 2.

Quantitative photobleaching confirms that IFT trains in short flagella contain more KAP protein. (A) At different time points after pH shock, KAP-GFP cells were for fixed and photobleached under constant TIRF illumination. The mean intensity was measured from 0.4 × 0.4 µm ROIs (red squares) centered on IFT trains. Bar, 2 µm. (B) Three examples of intensity plots of ROIs from flagella of different lengths. The raw data were filtered with a Chung-Kennedy edge-preserving algorithm to enhance the detection of stepwise GFP bleaching events. (C) Histograms of the number of KAP-GFP proteins measured in ROIs from long, mid-length, and short flagella (yellow vertical bars denote the mean, yellow horizontal bars show the standard deviation). n = 230 ROIs from 90 flagella.

Figure 3.

During long-zero regeneration, there is a linear correlation between IFT train size and flagellar length. (A) Three examples of KAP-GFP cells undergoing long-zero regeneration, showing brighter IFT trains in the shorter of the two flagella (cell dimensions are diagramed above the kymographs). As the length disparity between the long and short flagella increases (kymographs left to right), the difference in average train intensity also increases. Kymographs were generated from Videos 3–5. (B) Example of an IFT27-GFP long-zero cell. Kymograph was generated from Video 6. (A and B) Bars: (horizontal) 5 µm; (vertical) 1 s. (C) Plotting data from all of the KAP-GFP and IFT27-GFP long-zero cells together reveals a linear relationship (r² = 0.86) between the ratio of flagellar lengths and the ratio of IFT train intensities. Train intensities were corrected for the flagellar background and normalized by the ratio of integrated flagellar intensities (Fig. S3 B) to control for variability in total IFT content. (D and E) Despite this correlation between length and train intensity, the speed (D) and frequency (E) of IFT is the same in both the long and short flagella. (C–E) Data are plotted on a ratio scale. S, short flagella; L, long flagella, C, corrected for camera noise; F, corrected for flagellar background.

Figure 4.

The original and revised balance-point models of flagellar length control and possible mechanisms of IFT train size modulation. (A) The original balance-point model predicted that flagella contain a fixed number of IFT trains. Thus, as flagella regenerate, the length-dependent assembly rate would be driven by the decreasing frequency of train arrival at the flagellar tip. This model was refuted by the DIC observation that IFT frequency is constant (Dentler, 2005). In the revised model, the length-dependent assembly rate is mediated by IFT train size that scales inversely with flagellar length. Although the total amount of kinesin-2 and IFT protein in a flagellum is length independent, these proteins are redistributed into a greater number of smaller trains as the flagellum regenerates, reducing the rate of flagellar assembly. (B) Two possible models of train size control. In a closed system, at least one essential component of the IFT machinery does not exchange freely with the cytoplasmic pool. As the flagellum lengthens, lower concentrations of this key protein arrive at the flagellar base via retrograde transport, which results in the production of smaller anterograde IFT trains. In an open system, there is always a high availability of IFT material at the flagellar base, as proteins freely exchange with the large cytoplasmic pool. Thus, an additional length-sensor mechanism is required to modulate the size of trains that enter the flagellum. Blue arrows indicate high IFT protein concentration, whereas red arrows indicate low concentration.