Flagellar length control system: testing a simple model based on intraflagellar transport and turnover

Abstract

Flagellar length regulation provides a simple model system for addressing the general problem of organelle size control. Based on a systems-level analysis of flagellar dynamics, we have proposed a mechanism for flagellar length control in which length is set by the balance of continuous flagellar assembly and disassembly. The model proposes that the assembly rate is length dependent due to the inherent length dependence of intraflagellar transport, whereas disassembly is length independent, such that the two rates can only reach a balance point at a single length. In this report, we test this theoretical model by using three different measurements: 1) the quantity of intraflagellar transport machinery as a function of length, 2) the variation of flagellar length as a function of flagellar number, and 3) the rate of flagellar growth as a function of length. We find that the quantity of intraflagellar transport machinery is independent of length, that flagellar length is a decreasing function of flagellar number, and that flagellar growth rate in regenerating flagella depends on length and not on the time since regeneration began. These results are consistent with the balance-point model for length control. The three strategies used here are not limited to flagella and can in principle be adapted to probe size control systems for any organelle.

Figure 1.

Balance-point model for flagellar length control. (A) Intraflagellar transport and turnover. IFT particles move cargo such as tubulin from the cell body, where all protein is synthesized, out to the flagellar tip, where all assembly occurs. Simultaneously, axonemal proteins are continuously disassembled at the tip and returned to the cell body by retrograde IFT. Length maintenance requires that the rates of assembly and disassembly at the tip must be equal. + and - indicate microtubule plus and minus ends. Green indicates flagellar basal body located at cell surface. (B) Rates of assembly (blue curve) and disassembly (red curve) as a function of length. Disassembly is length independent, but transport-limited assembly is length dependent. The two curves only intersect at a single steady-state length.

Figure 2.

IFT protein content within the flagellum is independent of flagellar length. (A) Flagellar length plotted versus time in cells growing flagella after a shift from solid to liquid medium. (B) Western blot analysis of IFT proteins in flagella isolated from cells plotted in A in which an equal number of flagella were loaded per well. Radial spoke protein RspI content increases proportional to flagellar length during regeneration, whereas IFT protein content remains constant. (C) IFT proteins in flagella during regeneration in which an equal quantity of protein was loaded per well. (D) Number of IFT aggregates (rafts) plotted versus flagellar length. Each black circle represents data from a single cell. Variation of number versus length was insignificant over the length range examined. Inset, example immunofluorescence staining of IFT52. (E) Flagellar elongation rate calculated from flagellar regeneration data. Curve indicates a best fit to Eq. 3, indicating that elongation rate is proportional to 1/L as suggested by the balance-point model. (F) Reduction of IFT protein levels in fla10 temperature-sensitive mutant grown at 28°C, conditions which result in half-length flagella at steady-state, confirming a partial reduction in IFT protein content.

Figure 3.

Flagellar disassembly rate is length independent. (A) Flagellar length versus time after shift from 21°C (permissive temperature) to 32°C (nonpermissive temperature) in wild-type and IFT motor mutants. Wild type (○), fla1 mutant in motor subunit of heterotrimeric kinesin (•), fla10-1 mutant in motor subunit of heterotrimeric kinesin-II (▪), and fla3 mutant in nonmotor KAP subunit of heterotrimeric kinesin-II (▴). Data points show average length measured for 30 cells per time point for fla3, 12 per time point for fla1, and 15 cells per time point for fla10-1 and wild type. Best-fit lines are indicated as follows wild type (-----), fla1 mutant (―), fla3 mutant (······), and fla10-1 mutant (—·—·—). Error bars denote SEM. Only cells that had flagella were counted to avoid effects due to spontaneous deflagellation (Parker and Quarmby, 2003). In all three mutants, flagella shorten at a constant rate indicating disassembly in the absence of IFT is length independent. (B) Flagellar shortening before mitosis. Synchronized wild-type (cc-124) cells embedded in agarose were observed using 4D microscopy. Three-dimensional length measurement software was used to measure flagellar lengths at 10-min time points. Four individual flagella from different cells are shown in figure. In all cases, flagella shorten at a constant rate, indicating that predivision disassembly is length independent.

Figure 5.

Flagellar growth rate is length dependent. (A) Strategy for measuring flagellar elongation rate of partial-length flagella. Gametes with partial-length flagella are produced either by taking shf1 mutants that constitutively form short flagella, or by inducing flagellar regeneration in wild-type gametes in the absence of protein synthesis, under which conditions cells grow half-length flagella. Once cells with partial-length flagella are obtained, these are mated to wild-type cells of the opposite mating type having full-length flagella. Immediately after mating, the lengths of all four flagella are measured at 5- to 10-min time points. (B) Flagellar elongation in shf1 cells mated to wild type. Red circles, average length of short flagella after mating to wild-type cells (n = 56 flagella measured per time point). Green circles, average length of wild-type flagella regenerating in a control experiment (n = 30 flagella measured per time point). Error bars indicate SE of the mean length. (C) Flagellar elongation after mating of wild-type cells with partial-length flagella regenerated in cycloheximide (red circles; n = 10 flagella measured per time point) to wild-type cells with full-length flagella, compared with regeneration of flagella in completely deflagellated cells (green circles; n = 20 flagella measured per time point). Control regeneration kinetics and extent are different than in B because the mating experiment as well as the wild-type control were both done in the presence of cycloheximide.

Figure 4.

Variation of flagellar length with flagellar number. Lengths were measured in vfl1 gametes having different numbers of flagella (see data from Table 2). Average length indicated by circles: cells fixed in glutaraldehyde (○); cells fixed with Lugol's iodine (•). Error bars indicate SEM. Lines represent best-fit curves from Eq. 4. Fitting parameters for glutaraldehyde fixed cells: T = 76.7, D/A = 5.7, rms fitting error = 0.36 μm. Fitting parameters for iodine fixed cells: T = 93.9, D/A = 8.5, rms fitting error 0.22 μm.