Abstract
The singlecelled green algae Chlamydomonas reinhardtii with its two flagella—microtubulebased structures of equal and constant lengths—is the canonical model organism for studying size control of organelles. Experiments have identified motordriven transport of tubulin to the flagella tips as a key component of their length control. Here we consider a class of models whose key assumption is that proteins responsible for the intraflagellar transport (IFT) of tubulin are present in limiting amounts. We show that the limitingpool assumption is insufficient to describe the results of severing experiments, in which a flagellum is regenerated after it has been severed. Next, we consider an extension of the limitingpool model that incorporates proteins that depolymerize microtubules. We show that this ‘active disassembly’ model of flagellar length control explains in quantitative detail the results of severing experiments and use it to make predictions that can be tested in experiments.
Introduction
The size regulation of cellular organelles is a fundamental problem in biology (Marshall, 2016; Milo and Phillips, 2015). For example, nuclear size is tightly coupled with cell size across a wide range of species (Hara and Merten, 2015) and the loss of this coupling mechanism is implicated in various types of cancer (Zink et al., 2004).
A striking example of organelle size control in eukaryotes is the singlecelled algae Chlamydomonas reinhardtii (Figure 1), which uses two flagella to move through its aqueous environment. The backbone of each flagellum is an assembly known as the axoneme that consists of nine microtubule doublets arranged in a ring around a central pair of microtubules (Fawcett and Porter, 1954; Witman et al., 1972). Unlike the dynamic instability of cytoplasmic microtubules, which can alternate between rapidly shortening ‘catastrophe’ and stable ‘rescue’ states depending on whether or not the tip is bound to GTP, microtubules in the axoneme exist in a highly stable state (Behnke and Forer, 1967; Orbach and Howard, 2019). This stability reflects a tight control over flagellar lengths, the loss of which has dramatic physiological consequences; mutants with longer flagella have decreased swimming velocities and beat frequencies (Khona et al., 2013) compared to wild type cells and mutants with unequal flagellar lengths are observed to spin around in circles (Tam et al., 2003).
A key process contributing to the assembly of flagella is the continual transport of proteins from the flagellar base to tip and back. The original evidence for this intraflagellar transport (IFT) was provided around 25 years ago by experimental observations in Chlamydomonas of particles moving processively along the flagellum at constant speed (Kozminski et al., 1993). In the time since, a significant body of work has revealed the many proteins and biochemical pathways that coordinate this complex process in Chlamydomonas and other organisms such as C. elegans, as described in several review articles (Prevo et al., 2017; Scholey, 2003; Scholey, 2008; Cole, 2003; Rosenbaum et al., 1999; Rosenbaum and Witman, 2002; Rosenbaum et al., 1999).
Already at the time of its discovery, IFT was hypothesized to play a role in flagellar length control by transporting building blocks to the tip of the flagellum (Kozminski et al., 1993). IFT particles containing tubulin are transported along the flagellum by two different motor proteins: kinesin2 transports IFT particles from the flagellar base to tip (the anterograde direction) whereas dynein carries IFT particles from the tip to the base (the retrograde direction). As shown in subsequent work (Song and Dentler, 2001; Marshall and Rosenbaum, 2001; Buisson et al., 2013), these flagellar proteins are continually exchanged with those localized to the basal body of the flagellum, represented schematically in Figure 2a as a basal pool.
These observations motivated the development of mathematical models of flagellar length dynamics such as the balance point model (Marshall and Rosenbaum, 2001; Marshall et al., 2005). In the balance point model, there is a continual competition between assembly and disassembly, and either the rate of assembly, the rate of disassembly, or both, may be lengthdependent. The steadystate length is determined by the point at which the assembly and disassembly processes come into balance.
Up to now, the balance point model has been considered primarily for a single flagellum and the focus has been on assembly being the length dependent process that leads to length control (Marshall and Rosenbaum, 2001). However, other formulations of the balance point model, such as the case in which the disassembly rate is also lengthdependent, have received considerably less attention. In this work, we revisit the balance point model and particularly the assumption of a constant rate of disassembly in the context of simultaneous length control of multiple flagella assembled from a shared pool of biomolecules.
We limit our theoretical exploration to the space of models defined by the following processes: IFT particle assembly and injection at the flagellar base, motion of IFT proteins along the flagellum, and tubulin polymerization and depolymerization at the flagellar tip (see schematic Figure 2a). We further assume that IFT particle injection satisfies firstorder chemical kinetics and allow for a control mechanism that regulates protein levels in the basal pool. Note that this model space does not include all possibilities. In particular, it does not include the timeofflight model (Wren et al., 2013), in which additional reactions affect protein state inside the flagellum (e.g. proteins enter in an activated state and deactivate at some rate).
Nevertheless, our work retains a high degree of generality. For example, it allows for different modes of coupling between proteins in basal pools (shared or separate), different modes of IFT motion (ballistic or diffusive and processive or nonprocessive), and general depolymerizing activity. Within the model space outlined, our main results hold independent of these details. Notably, we find that lengthindependent disassembly of microtubules cannot account for the experimental results, whereas incorporating lengthdependent disassembly (e.g. through the ballistictodiffusive motion of a depolymerizing protein) leads to reasonable agreement with the experiments.
We analyze these models at two levels of detail, both using detailed agentbased stochastic simulations and a reduced description in terms of ordinary differential equations (ODEs) and compare the results of simulations to experimental data. It is known from experiments on Chlamydomonas that (i) its two flagella reach steadystate lengths of about 10 µm, and that (ii) the flagellar lengths are correlated since, if one flagellum is severed, the remaining flagellum shortens until it reaches the length of the growing, previously severed flagellum (Rosenbaum et al., 1969). This latter protocol is typically referred to as a ‘severing’ or ‘longzero’ experiment.
As shown in Figure 1b, the severing experiments contain two separate timescales. There is an initial fast timescale of 10–20 min over which the flagellar lengths first equalize, followed by a slower timescale of 60–90 min over which the lengths increase simultaneously to their original lengths. With respect to the short time behavior, we find that none of the candidate models assuming constant disassembly capture the rapid length equalization observed experimentally. Moreover, if an external control mechanism is added that replenishes proteins lost due to severing over a slower timescale, the constant disassembly models within our model space fail to control lengths at all.
Motivated by experimental observations of microtubuledepolymerizing proteins within the flagellum (Piao et al., 2009; Luo et al., 2011; Hilton et al., 2013), we subsequently consider a model in which the disassembly rate is dependent on the local concentration of a depolymerizing protein at the flagellar tips. This gives rise to a lengthdependent disassembly rate, which is a departure from existing models that assume constant disassembly. We find that this model is consistent with the rapid length equalization observed in experiments. Further, upon adding a control mechanism on protein levels in the basal protein pool, the model is able to capture the slow return to the original steadystate lengths observed experimentally. In the Discussion, we examine this model in light of the currently available experimental data and discuss possible candidates for the depolymerizing protein.
Results
Limiting motor pool gives rise to lengthdependent injection rate
It is known from experiments that the injection rate of IFT particles in Chlamydomonas is lengthdependent (Dentler, 2005). In this section, we show how lengthdependent injection is a consequence of mass action kinetics of proteins available in limiting amounts.
We first consider the flagellar length dynamics of a single flagellum. Our approach initially closely follows Hendel et al. (2018), although important differences will appear later on in the case of two flagella. The key biochemical variables are the tubulin dimers that make up the flagellar axoneme and the molecular motors kinesin2 and dynein that transport IFT particles from the base to the tip and back. IFT particles combine in the basal pool with kinesin2, tubulin and dynein to form a complex that is injected into the flagellum (Cole et al., 1998); see the schematic in Figure 2a.
Here we consider the case that the ratelimiting molecule is kinesin2, although similar results would apply to any other protein being ratelimiting for IFT assembly. Denoting the number of free molecular motors in the pool at the flagellar base by ${M}_{f}$, the injection flux ${J}_{i}$ of IFT particles into the flagellum satisfies
Note that we consider the cell volume to be fixed, in which case biomolecule numbers and concentrations may be used interchangeably.
First we consider the case in which the total number of motors is conserved. Later on we will also consider the case in which motor concentrations in basal pools are regulated by an external control mechanism, motivated by the severing experiments described previously in which length recovery indicates replenishment of protein levels.
Kinesin2 has been shown to undergo ballistic transport in the anterograde direction and diffusive motion in the retrograde direction (Chien et al., 2017). The total number of motors $M$ satisfies $M={M}_{f}+{M}_{b}+{M}_{d}$, where ${M}_{b}$ is the number of motors moving ballistically on the flagellum in IFT particles and ${M}_{d}$ is the number of motors moving diffusively. Therefore we may rewrite Equation 1 as
The intraflagellar dynamics are fast compared to changes in length. The timescale of flagellar length dynamics, for example the recovery time after severing, is of the order of 10 min, whereas the molecular motors involved in IFT take at most tens of seconds to traverse the length of the flagellum by either ballistic or diffusive motion. Based on this separation of timescales, we treat IFT as a quasisteady state process in which the injection flux ${J}_{i}$, diffusive flux ${J}_{b}$, and ballistic flux ${J}_{d}$ are balanced, that is
As shown in Materials and methods, the quasisteady state assumption of flux balance together with mass action kinetics of injection expressed in Equation 1 imply that
where ${k}_{\text{on}}$ is the rate constant of motor injection, $M$ is the total number of motors, $v$ is the ballistic motor speed in IFT, and $D$ is the diffusion coefficient of motors in the flagellum.
We have validated the quasisteady state assumption used to derive Equation 4 by comparing the IFT particle flux and the concentration of diffusing motors in the flagellum to the results of stochastic simulations (see Appendix 1), in which the dynamics of motors as well as the dynamics of microtubule assembly are taken into account explicitly; see Figure 2b. Parameter values used in simulations are provided in Table 1. (Note that in Equation 4 and throughout the manuscript, terms written in the form $x/yz$ are shorthand for $x/(yz)$, for example ${k}_{\text{on}}{L}^{2}/2D$ is to be read as ${k}_{\text{on}}{L}^{2}/(2D)$.)
IFT particle injection arising from a finite number of motors shared between the flagellum and the basal pool therefore leads to a lengthdependent flux. This result holds true regardless of the identity of the ratelimiting IFT protein. However, the scaling of formula Equation 4 with length depends on whether ballistic or diffusive transport dominates. In the limit $D\gg Lv$, the $1/L$ scaling of Marshall and Rosenbaum (2001) is recovered whereas in the limit $D\ll Lv$, we recover the $1/{L}^{2}$ scaling of Hendel et al. (2018). Note that a distinction between our model and these previous works is the presence of a constant term in the denominator of Equation 4, which implies that in our formulation the flux does not blow up at $L=0$.
In our model the assembly rate is determined by the rate of tubulin transport to the flagellar tip, as in Marshall and Rosenbaum (2001) and Hendel et al. (2018). Given massaction kinetics of IFT particle assembly in the basal pool, this is simply the flux $J$ of IFT particles times the amount of free tubulin ${T}_{f}$, so that the growth rate is given by the following ODE:
where $d$ is the disassembly speed (assumed constant for now) and $\gamma $ is a constant. The total amount of tubulin $T$ is assumed to be conserved for now so that $T={T}_{f}+L$, where tubulin is measured in units of corresponding flagellar length. As previously mentioned, later on we will consider the case in which protein levels in basal pools are not conserved and are instead monitored and regulated by an external control mechanism.
Substituting the expression Equation 4 for the flux into the above growth rate results in
As shown in Materials and methods, this equation yields a stable steadystate length ${L}_{ss}$ for the single flagellum.
Limitingpool mechanisms alone cannot account for the rapid length equalization observed in severing experiments
Whereas for a single flagellum the limitingpool mechanism leads to a stable steadystate length, we show next that this mechanism is insufficient for the simultaneous length control of two flagella. A general limitingpool model for the dynamics of two flagella having lengths ${L}_{1}(t)$ and ${L}_{2}(t)$ is given by the following ODEs:
where ${J}_{i}$ and ${T}_{f,i}$ for $i=1,\mathrm{\hspace{0.17em}2}$ denote the fluxes of IFT particles into the two flagella and the amounts of free tubulin in their basal pools, respectively. As we will show next, the particular forms of ${J}_{i}$ and ${T}_{f,i}$ depend on how the pools are coupled. In particular, the fluxes and free tubulin will be equal for the two flagella if the basal proteins are held in a common shared pool.
Severing experiments illustrate that the flagella are coupled. We consider various modes of coupling, that is shared or separate motor pools and shared or separate tubulin pools, as depicted in Figure 3a, that give rise to different forms of ${J}_{i}$ and ${T}_{f,i}$ within our model. We investigate their consequences for length control by focusing on the solutions to the steadystate equations
Length control implies that these steadystate equations must yield a unique steadystate solution for ${L}_{1}$ and ${L}_{2}$. We consider the steadystate lengths before and after severing. It is known from experiments (Figure 1b) that initially the two flagella have equal lengths, and that after severing, which is accompanied by a loss of material (e.g. tubulin and motors lost from the severed flagellum), there is a rapid equalization of flagellar lengths. This initial, fast equalization of lengths leads to flagella that are shorter than they were before the severing. These experimental observations may be used to reject candidate models. Here we focus on the shorttime dynamics after severing and therefore do not account for protein replenishment; however, as we show in the next section, incorporating protein replenishment is incompatible with the constant disassembly models considered here.
As we shall see, the constant disassembly models within our model space do not yield the rapid length equalization observed experimentally. As shown in Figure 3b and considered next on a casebycase basis, if the lengths are in steadystate prior to severing, they are also in steadystate immediately after severing as well so that no length equalization occurs. This is because in these models the assembly and disassembly rates depend only on free protein levels in the basal protein pools, which are unaffected by severing. To be more precise, we may rewrite the governing equations Equations 7 and 8 in terms of the number of free motors ${M}_{f,1}$ and ${M}_{f,2}$ in the following form:
In order to simulate a severing experiment, we first allow the two flagella to reach steadystate. Prior to severing, there is an amount of tubulin ${L}_{1}$ and a number of motors ${M}_{1,b}+{M}_{1,d}$ loaded on the first flagellum corresponding to the numbers of motors undergoing ballistic or diffusive motion (see Materials and methods). Severing is modeled by setting ${L}_{1}\to 0$, ${T}_{1}\to ({T}_{1}{L}_{1})$, and ${M}_{1}\to ({M}_{1}{M}_{1,b}{M}_{1,d})$. Because severing does not change any of the protein levels in the basal pools, ${M}_{f,i}$ and ${T}_{f,i}$ for $i=1,\mathrm{\hspace{0.17em}2}$ are the same before and after severing. Moreover, the linear concentration profile of diffusing motors remains linear in the truncated flagellum; no redistribution of motors is required to maintain quasisteady state. As a consequence the constant disassembly models predict no length recovery of the severed flagellum, contrary to what is observed.
Tubulin separate, motors separate
The case of separate tubulin pools and individual motor pools leads to two uncoupled instances of (6). This is fundamentally inconsistent with the coupling observed in severing experiments, in particular the significant decrease in the length of the unsevered flagellum (Figure 3b(i)). In this model, the unsevered flagellum does not change length after severing, and therefore it may be ruled out.
Tubulin separate, motors shared
When motors are shared through a common pool, IFT particles are injected into either flagellum with equal probability. Therefore ${J}_{1}={J}_{2}=:J$ and it can be shown by a straightforward generalization of Equation 4 (see Materials and methods) that the flux satisfies
Initially the tubulin pools are equal, as are the flagellar lengths. However, after the loss of material due to severing, ${T}_{1}\ne {T}_{2}$ and the lengths do not equalize (see Figure 3b(ii) and Figure 3—video 1). Because of the separate tubulin pools, severing leads to asymmetrical tubulin depletion and unequal steadystate lengths after severing. Therefore, we may rule out the model.
Tubulin shared, motors separate
This case is analogous to the previous model in which only motors are shared, but this time only the tubulin pools are shared so that ${T}_{f,1}={T}_{f,2}=:{T}_{f}$, with
As shown in Figure 3b(iii) and Figure 3—video 2, this model does not capture the length equalization observed in severing experiments and therefore may be ruled out. See Materials and methods for details.
Tubulin shared, motors shared
The flux resulting from the shared motor assumption is the same as in Equation 13, and we are left with the steadystate equations
Because the two steadystate equations are identical, that is $\mathrm{d}{L}_{1}/\mathrm{d}t=\mathrm{d}{L}_{2}/\mathrm{d}t$, this model does not account for the simultaneous positive and negative growth rates for the two flagella observed in the severing experiment.
More strikingly, subtracting the steadystate equations yields
so that the difference in lengths is not controlled at all. Note that this result is independent of the parameters. Indeed, a similar conclusion was reached in the context of actin filaments (Mohapatra et al., 2017), in which it was observed that sharing all biomolecules between filaments does not yield simultaneous length control. In the context of the full stochastic simulations, this degeneracy is manifested by the difference in lengths undergoing a random walk (inset to Figure 3b(iv) and Figure 3—video 3).
The above analysis shows that, regardless of the manner in which tubulin and motors are shared between flagella, the constant disassembly models we have considered are unable to explain the results of severing experiments. Although sharing either tubulin or motors, but not both, yields a unique steadystate, these models do not agree with the rapid length equalization observed in severing experiments. This motivates us to extend our study beyond the models considered thus far.
Controlling protein levels in the basal pool is incompatible with the constant disassembly models considered
So far, we have assumed that the tubulin pool $T$ and motor pool $M$ are fixed throughout the simulation. While this assumption is reasonable for the fast initial phase of the severing experiment in which the flagellar lengths rapidly equalize, the slower second phase of recovery to the original steady state lengths requires replenishment of proteins back to their original levels. This was shown experimentally by using cycloheximide at the time of severing to block the synthesis of new proteins (Rosenbaum et al., 1969), resulting in shorter flagella that did not recover to their original lengths.
It may seem plausible that adding an external control mechanism that replenishes protein levels would lead to length equalization, thus resolving the issue of unequal steadystate lengths after severing. However, as we next show, adding such a control mechanism on free proteins levels in the basal pool does not lead to length equalization. Instead, in this case the constant disassembly models we have considered completely fail to control lengths.
To incorporate control on protein levels into our model, we assume that the total levels $T$ and $M$ of tubulin and motors are replenished over a timescale ${\tau}_{r}$ as the cell synthesizes new protein to achieve target protein levels ${\overline{T}}_{f}$ and ${\overline{M}}_{f}$ in the basal pool:
In steadystate, the above equations become ${T}_{f}={\overline{T}}_{f}$ and ${M}_{f}={\overline{M}}_{f}$. The steadystate equation for length then becomes $0=\gamma {k}_{\text{on}}{\overline{M}}_{f}{\overline{T}}_{f}d$, that is length drops out of the assembly term completely! Therefore, this external control mechanism actually destabilizes the flagellar lengths (see Figure 3—videos 4—6 to observe this destabilization for various modes of coupling). Note that although we have used the simplest case of linear feedback Equations 18 and 19 to illustrate the point, this argument is general and does not depend on the details of the control mechanism. The only requirement is that the free protein levels ${T}_{f}$ and ${M}_{f}$ are driven to their target values ${\overline{T}}_{f}$ and ${\overline{M}}_{f}$ in steady state. This argument gives another compelling reason to look beyond the constant disassembly models considered thus far.
Tubulin shared, motors shared and concentrationdependent disassembly
We next consider a model that allows for full exchange of IFT components between basal protein pools and replaces the constant disassembly assumption with a concentrationdependent disassembly rate. The assumption of a constant disassembly rate was based on experiments on mutants in which IFT was disabled (Marshall et al., 2005). However, subsequent experiments in organisms with intact IFT led to 50fold greater disassembly rates than those measured in the absence of IFT (Ludington et al., 2012).
Experimental observations that some kinesin species (e.g. kinesin13) participate in microtubule disassembly (Piao et al., 2009) provide a potential biochemical basis for IFTdependent disassembly. In what follows we take the disassembly rate to depend on the concentration of a depolymerizing protein. This is a reasonable model for a depolymerizer that is nonprocessive in its depolymerization activity in that it removes at most a few tubulin subunits before falling off into a deactivated state. (The case of processive depolymerizers is investigated in Appendix 3).
We will assume that the depolymerizer has the same motion as kinesin2—uninterrupted ballistic motion to the tip followed by diffusive motion to the base—resulting in a linear concentration profile. This would be the case for any nonmotile protein that is transported ballistically to the flagellar tip as IFT cargo and diffuses back to the flagellar base. Note however that the ballistictodiffusive assumption is not essential for the model; so long as there is a gradient in the depolymerizer concentration, the conclusion of simultaneous length control holds. The essential ingredient is the nonconstant concentration along the length of the flagellum, which in this case is achieved by ballistic anterograde motion and diffusive return.
Given that the formulas for the steadystate flux apply for any ratelimiting IFT protein undergoing ballistictodiffusive motion along the flagella, for convenience we assume in what follows that the depolymerizer is the ratelimiting protein. However, this assumption is made only for convenience; in the more general case that the depolymerizer and the ratelimiting IFT protein are different, the same results are obtained with suitably rescaled parameters, as shown in Appendix 3.
We replace the assumption of constant disassembly by a disassembly speed of the form ${d}_{0}+{d}_{1}{c}_{d}(L)$, where ${c}_{d}(L)$ is the concentration of diffusing motors at the tip of the flagellum and ${d}_{1}>0$. In general the disassembly rate may be an arbitrary function of concentration, in which case this model may be viewed as a firstorder Taylor series expansion valid near steadystate. The flux and concentration at the flagellar tip are related by ${c}_{d}(L)=JL/D$ (Equation 31 in Materials and methods and Figure 2b (inset)), so that we may rewrite the governing equations as
where as before in the case of shared motors
This model yields simultaneous length control and length equalization after severing (Figure 4 and Figure 4—video 1). Subtracting Equation 21 from Equation 20, it follows immediately that ${L}_{1,ss}={L}_{2,ss}=:{L}_{ss}$, and solving for the steadystate length results in
In Appendix 3 we show that this solution is stable using linear stability analysis. Therefore, concentrationdependent disassembly yields simultaneous length control when all biomolecules are shared between flagella and is consistent with the rapid length equalization observed after severing Figure 4b. The presence of a concentration gradient is a critical ingredient in this model and here it is achieved by ballistic transport to the flagellar tip with diffusive return. The concentration gradient makes the disassembly rates lengthdependent and yields independent equations for the steadystate lengths.
Unlike the constant disassembly models we have considered, for which a limitingpool mechanism is essential for length control, concentrationdependent disassembly yields length control under mild assumptions including the case that all biomolecules are in excess. Nevertheless, limitingpools of biomolecules are needed to capture the depletion effects observed in severing experiments on Chlamydomonas, for example the shortening of the unsevered flagellum and the previouslymentioned absence of length recovery after cyclohexamide treatment (Rosenbaum et al., 1969). In Appendix 3 we explain that limiting pools of IFT motors are necessary for agreement with data whereas tubulin may either be limited or in excess. The presence of a limitingpool once again raises the question, now in the context of concentrationdependent disassembly, of how biomolecules may be shared between flagella. In Appendix 3 we show that all relevant biomolecule pools must be shared for the concentrationdependent disassembly model to capture the rapid length equalization observed.
Another feature of the concentrationdependent disassembly model is that it allows for an external control mechanism on protein levels in the basal pool, unlike the constant disassembly models we have considered. As shown in Figure 4c and Figure 4—video 2, upon including protein replenishment via Equations 18 and 19 on a timescale of ${\tau}_{r}=10$ mins, the recovery of the flagella back to their original lengths is in reasonable agreement with experimental data.
Generalization to $N>2$ flagella
The concentrationdependent disassembly model may be generalized to arbitrary flagellar number $N$, and here we demonstrate simultaneous length control in the case of $N=8$ flagella (Figure 5).
Because motors are shared, the injection fluxes are equal and ${J}_{i}=J$ for all $i=1\mathrm{\dots}N$, with $J$ satisfying
and length dynamics given by
where we have applied the boundary condition ${c}_{d,i}(0)=0$ as before. Taking any pairwise difference between the $i$^{th} and $j$^{th} equations at steadystate yields immediately ${L}_{i,ss}={L}_{j,ss}$, so that the steadystate lengths are equal to
for all $i=1,\mathrm{\dots},N$. Stability follows from analyzing the linearized equations, as shown in Appendix 3.
Discussion
In this work, we capture the aspects of IFT essential for length control, that is the motordriven transport of tubulin across the flagellum, to explore models of flagellar length dynamics. Our theoretical framework makes it possible to investigate the consequences of biomolecule exchange between flagella on length control. In our initial exploration, in which we take the disassembly rate to be constant, we find that sharing both tubulin and motors leads to an indeterminate system of equations regardless of the details of the model, whereas sharing either tubulin or motors, but not both, results in simultaneous length control of both flagella. However, by examining the steadystate lengths immediately before and after severing and accounting for depletion in both the tubulin and motor pools, we observe that none of the constant disassembly models we have considered are able to capture the length equalization observed in experiments.
Given that the constant disassembly models we have considered are unable to explain the experiments, we have proposed a model in which disassembly depends on the local concentration of a depolymerizing protein at the tip of the flagellum (Figure 4). In this ‘active disassembly’ model, a lengthdependent concentration at the tip is achieved by assuming that the depolymerizer undergoes ballistic motion to the tip and returns diffusively. This model agrees with the results of severing experiments. As suggested by the title of Hendel et al. (2018), the linear concentration gradient generated by diffusion acts as a ruler. However, in our model diffusion must be combined with a mechanism such as concentrationdependent disassembly; the constant disassembly models we have considered are inconsistent with severing experiments regardless of whether the motor dynamics are ballistic, diffusive, or some combination.
We remark on the differences between our model and Hendel et al. (2018), in which simultaneous length control was obtained using a balance point model with constant disassembly. As we describe in Materials and methods, the formulation of Hendel et al. (2018) is very similar to Equations 56 and 57 derived in the case of shared tubulin pools and separate motor pools with constant disassembly. Whereas these equations do not lead to length equalization after severing in the case of no protein replenishment (Figure 3b(iii)), the model of Hendel et al. (2018) achieves length equalization by replenishing the total number of motors on the flagellum, that is through an additional control mechanism that instantaneously adds the motors lost through severing back into the basal pool. The importance of replenishment for the model appears to be inconsistent with severing experiments that show length equalization occurs even when protein synthesis is blocked using cyclohexamide (Rosenbaum et al., 1969). In contrast, the concentrationdependent disassembly model achieves length equalization with or without replenishment (Figure 4b and Figure 4c). Note further that controlling motor number in the flagellum is not equivalent to controlling protein concentrations in the basal pool. As shown in Results, when basal pool concentrations are controlled according to Equations 18 and 19, there is a breakdown of length control for the constant disassembly models.
Our model predicts that the tubulin and depolymerizer pools must both be shared for the concentrationdependent disassembly model to capture the rapid length equalization observed (Appendix 3). This illustrates the dramatic consequences in behavior that can occur when biomolecules are shared between compartments, and highlights the importance of knowing which proteins are exchanged between the basal pools in the context of flagellar length control. In particular, having a protein that is not exchanged can provide simultaneous length control by a limitingpool mechanism, but it introduces asymmetries that contradict the length equalization observed in severing experiments.
In addition to our claim that the depolymerization rate is nonconstant and dependent on length, our model leads to testable predictions that may be useful in identifying candidate depolymerizers. For example, according to our model the depolymerizer active in length control is not uniformly distributed along the flagellum; its concentration increases toward the flagellar tip. This could be tested experimentally by fluorescently labeling candidate depolymerizers and studying their concentration profiles along the flagellum as recently done to characterize the concentration profile of kinesin13 in Giardia (McInally et al., 2019).
In our model the essential ingredient that leads to simultaneous length control is the presence of a depolymerizing protein with a concentration gradient along the flagellum. How proteins can develop and maintain such concentration profiles is therefore one of the key questions raised by the model. Indeed, concentration gradients (unassociated with depolymerizing activity) were observed already in Hendel et al. (2018) as the result of ballistictodiffusive motion. Although here as proof of principle this concentration gradient is achieved by the mechanism of ballistictodiffusive motion, our main results are independent of the detailed form of this concentration gradient and how it is generated. As shown in Appendix 3—figure 3b, our model allows for depolymerizers with nonlinear concentration profiles and different patterns of motion, for example exponential concentration distributions such as those recently observed in Giardia (McInally et al., 2019) and those generated by motile proteins that bind and unbind to cytoskeletal filaments, as theorized in the context of actinmyosin systems (Naoz et al., 2008; Orly et al., 2014; Pinkoviezky and Gov, 2014; Pinkoviezky and Gov, 2017; Yochelis et al., 2015).
Our main results do not rely on many of the details of the model. Although here we have taken depolymerase activity to depend linearly on concentration, the model generalizes to the nonlinear case in a straightforward manner. For a local depolymerization rate that is an arbitrary function of concentration, a Taylor series expansion may be performed as in Klein et al. (2005) to obtain the corresponding linearized system discussed in Appendix 3. Further, whereas here we have explored the case of nonmotile depolymerizers transported to the flagellar tip by IFT, motile proteins could in principle aggregate at the flagellar tip independent of IFT. Although we are not aware of any such examples in Chlamydomonas, interestingly in budding yeast the motile protein Kip3p has been shown to depolymerize microtubules in a lengthdependent manner (Varga et al., 2009). The aggregation of motile depolymerizing proteins has also been demonstrated in previous theoretical studies of microtubule length control (Klein et al., 2005; Johann et al., 2012; Reese et al., 2014); note however that these previous works differed from our model of flagellar IFT in that they considered isolated microtubules surrounded by a constant concentration bath and/or significant steric interactions between motile proteins.
Finally, although in principle concentrationdependent disassembly yields length control even the case that all biomolecules are in excess, in Chlamydomonas severing experiments have shown that depletion effects are important (e.g. cyclohexamide treatment yields short flagella that do not return to their original steadystate lengths [Rosenbaum et al., 1969]). Within our model such depletion effects arise through limitingpools of proteins, and in Appendix 3 we explain that limiting pools of IFT motors are necessary for agreement with data whereas tubulin may either be limited or in excess.
Although our results suggest that having a depolymerizer—one which is ballistically transported to the tip and then diffuses back—provides an appealing model for simultaneous length control, such a depolymeriser has yet to be identified in Chlamydomonas. Further experiments such as the single molecule turnaround experiments pioneered recently in C. elegans (Mijalkovic et al., 2018) are needed to establish the identity of the hypothesized depolymerizer. While recent experiments have shown kinesin13 to be involved in length control in Giardia (McInally et al., 2019), the observation that only negligible amounts of flagellar kinesin13 are present at steadystate (Wang et al., 2013) appears to preclude it from being the candidate depolymerizer of our model. Other candidates include auroralike kinase CALK, which has been shown to influence disassembly through its state of phosphorylation (Luo et al., 2011; Cao et al., 2013), and CNK2, a NIMArelated protein kinase known to localize to flagella (Bradley and Quarmby, 2005) whose absence yields Chlamydomonas with abnormally long flagella and decreased disassembly rates (Hilton et al., 2013).
On the side of theory, a promising avenue to further test the active disassembly model and discriminate between different possibilities is to study length fluctuations about steadystate. This could be done using agentbased stochastic simulations, stochastic differential equations, or a combination of the two. The fluctuation spectra of each model provides a signature that can be used to assess both the general model framework and to test the autocorrelation timescales predicted by each model (Amir and Balaban, 2018).
Materials and methods
Single flagellum
We first consider a single flagellum with timedependent length $L(t)$. Given the highly regular structure of the axoneme revealed by cryoelectron microscopy (Bui et al., 2008; Barber et al., 2012), we assume a constant crosssectional area in which case the flagellar geometry is fully described by its length. In our model the flagellar assembly rate is proportional to the flux $J$ of IFT particles times the tubulin carried per particle. The tubulin carried is proportional to the amount of free tubulin ${T}_{f}$, assuming mass action kinetics in the basal pool (i.e. constant probability per time of tubulin binding to an IFT particle). For now we take the disassembly speed to be equal to a constant $d$. This yields the growth rate
As described in Results, we assume that motors are conserved having total number $M={M}_{f}+{M}_{b}+{M}_{d}$, where ${M}_{f}$ is the number of motors freely available in the basal protein pool, ${M}_{b}$ is the number of motors moving ballistically on the flagellum in IFT particles, and ${M}_{d}$ is the number of motors moving diffusively.
We assume a limiting pool of tubulin in addition to the limiting pool of motors, that is tubulin is conserved with total amount $T={T}_{f}+L$. As the flagellum grows it incorporates more tubulin and the size of the free tubulin pool decreases. (In reality $T={T}_{b}+{T}_{f}+L$, where ${T}_{b}$ is the amount of tubulin undergoing IFT, but this correction is negligible; the amount of tubulin moving ballistically in IFT satisfies ${T}_{b}/{T}_{f}<2\gamma {k}_{\text{on}}M{L}_{ss}/v$, and consequently ${T}_{b}/{T}_{f}<2.6\times {10}^{3}$ for the parameters contained in Table 1).
Flux balance
Request a detailed protocolIn our model the flux, or injection rate, is proportional to the number of free molecular motors ${M}_{f}$ so that
according to mass action kinetics with firstorder rate constant ${k}_{\text{on}}$. By mass action and conservation of motors, the flux of motors may be expressed as
The ballistic flux is related to concentration in a simple manner. It satisfies $J=\overline{{c}_{a}}v$, where $\overline{{c}_{a}}$ is the average concentration of motors moving in the anterograde direction and $v$ is the anterograde velocity. (As mentioned previously, it follows from the quasisteady state assumption that the injection flux, anterograde flux, and retrograde flux are equal so that there is a single flux $J$.) Therefore
In quasisteady state, the diffusive flux $D\partial {c}_{d}/\partial x$ must equal the injection rate $J$. By Fick’s law, for constant $J$ the concentration profile ${c}_{d}(x)$ of the diffusing motors is linear, that is
in which $\overline{{c}_{d}}$ is the average concentration along the flagellum and $D$ is the diffusion constant (Figure 2b (inset)). We treat the flagellar base as a diffusive sink by fixing the boundary condition ${c}_{d}(0)=0$, which assumes that motors in the basal pool cannot leak diffusively into the flagellum; instead they attach to the microtubules in the axoneme and move directionally toward the tip (More general boundary conditions are discussed in Appendix 2.) This implies that
Therefore
for the diffusivelymoving motors. Substituting the expressions Equation 30 and Equation 33 for ${M}_{b}$ and ${M}_{d}$ into Equation 29 results in
The denominator is a quadratic function in length, and it is interesting to note that the flux has a similar functional form to the familiar substrate production rate in MichaelisMenten enzyme kinetics (Fall, 2002); this is because of the separation of timescales assumption invoked in both derivations.
Using the above expression for the flux in the growth rate Equation 27 together with the relation ${T}_{f}=TL$ gives
Solving Equation 35 for the steadystate results in a quadratic equation for ${L}_{ss}$. One root is always negative, leaving the solution
which is positive provided that $T>d/\gamma kM$. (We remind the reader that terms such as $d/\gamma {k}_{\text{on}}M$ are to be interpreted as $d/(\gamma {k}_{\text{on}}M)$.) This inequality provides a theoretical lower limit on the product of total motors and tubulin needed to obtain a positive steadystate length. When the inequality is not satisfied, that is $T\le d/\gamma {k}_{\text{on}}M$, the disassembly term dominates and the length shrinks to zero.
We evaluate the stability of this solution by linearizing about ${L}_{ss}$. Expanding to first order in $\mathrm{\Delta}L:=L{L}_{ss}$, we find that the steadystate is stable, that is $\mathrm{d}(\mathrm{\Delta}L)/\mathrm{d}t=\lambda (\mathrm{\Delta}L)$ with $\lambda $ a positive constant given by
Based on the parameters estimated in Appendix 2, the associated timescale $\tau :=1/\lambda $ is approximately 15 min, which is consistent with experiment. This timescale is long compared to the few tens of seconds needed for molecular motors to traverse the flagellum in IFT, which justifies a posteriori our approximation of IFT as a quasisteady state process.
We next consider the parameter space associated with the length dynamics. Introducing the nondimensional length $\stackrel{~}{L}=L/{L}_{ss}$ and nondimensional time $\stackrel{~}{t}=t\gamma {k}_{\text{on}}MT/{L}_{ss}$, we may rewrite Equation 35 in terms of the dimensionless parameters ${\pi}_{1}=d/\gamma {k}_{\text{on}}MT$, ${\pi}_{2}={L}_{ss}/T$, ${\pi}_{3}={k}_{\text{on}}{L}_{ss}/v$, and ${\pi}_{4}={k}_{\text{on}}{L}_{ss}^{2}/2D$ as
We interpret these parameters as follows: ${\pi}_{1}$ is the ratio of disassembly and assembly rates, ${\pi}_{2}$ is the fraction of the tubulin pool taken up by the flagellum at steadystate, ${\pi}_{3}={\tau}_{b}/{\tau}_{i}$ is the ratio of the ballistic timescale ${\tau}_{b}:={L}_{ss}/v$ of IFT transport to the injection timescale ${\tau}_{i}:={k}_{\text{on}}^{1}$, and ${\pi}_{4}={\tau}_{d}/{\tau}_{i}$ is an analogous ratio of the diffusive timescale ${\tau}_{d}={L}_{ss}^{2}/2D$ to the injection timescale. (We could equivalently think of ${\pi}_{3}$ and ${\pi}_{4}$ as ratios of lengthscales related to the same physical processes.)
In terms of the experimentally measured parameters and those estimated in Appendix 2, we find ${\pi}_{1}\approx 0.4$, ${\pi}_{2}\approx 0.2$, ${\pi}_{3}\approx 0.1$, and ${\pi}_{4}\approx 0.8$. The relatively small values of ${\pi}_{2}$ and ${\pi}_{3}$ lead us to consider the limit ${\pi}_{2}\to 0$ (i.e. no tubulin depletion) and ${\pi}_{3}\to 0$ (i.e. instantaneous ballistic motion). In this limit, we have
nearly recovering the model of Hendel et al. (2018) with the distinction that, as mentioned above, in our model there is an additional constant term in the denominator. Note however that the essential difference between our model and Hendel et al. (2018) lies in their effective control mechanism on the number of motors loaded on the flagella, which is not captured by any differences in these formulas (see Discussion).
Two flagella
In the case of two flagella with lengths ${L}_{1}(t)$ and ${L}_{2}(t)$ the length dynamics are given by
where ${J}_{i}$ and ${T}_{f,i}$ for $i=1,\mathrm{\hspace{0.17em}2}$ denote the fluxes and free amounts of tubulin for the two flagella, which may be equal when the biomolecule pools are shared. We consider various modes of coupling between the flagella giving rise to different forms of ${J}_{i}$ and ${T}_{f,i}$ and their consequences for length control. To assess whether a model achieves simultaneous length control we analyze the stability of solutions to the following steadystate equations:
Here, we focus on shorttime behavior, that is whether a candidate model yields rapid length equalization, and do not include the protein replenishment that takes place over a longer timescale.
Tubulin separate, motors separate
Request a detailed protocolThe presence of separate tubulin pools and separate motor pools leads to two uncoupled instances of the single flagellum dynamics, that is
Setting ${M}_{1}={M}_{2}=M$ and ${T}_{1}={T}_{2}=T$ leads to steady state lengths given by Equation 36.
Tubulin separate, motors shared
Request a detailed protocolIn the case of separate tubulin pools, we have ${T}_{f,1}={T}_{1}{L}_{1}$ and ${T}_{f,2}={T}_{2}{L}_{2}$. The flux may be calculated according to
where the factor of onehalf comes from assuming equal injection probability into either flagellum. Further, ${M}_{b}=J({L}_{1}+{L}_{2})/v$ and
so that
This yields the flagellar length dynamics
The steadystate equations are given by
When ${T}_{1}={T}_{2}$ it follows from subtracting the above equations that ${L}_{1,ss}={L}_{2,ss}$. The steadystate equations are identical to the corresponding steadystate Equation 35 for a single flagellum with $M$ replaced by $M/2$. Therefore the steadystate length satisfies Equation 36 upon rescaling $M\to M/2$:
The steady state lengths are only equal if ${T}_{1}={T}_{2}$, which is not the case after asymmetrical depletion of tubulin pools by severing (see Figure 3b(ii)).
Tubulin shared, motors separate
Request a detailed protocolWe next consider the case in which tubulin is shared but the motor pools are separate. The separate motor pools yield decoupled fluxes identical to Equations 44 and 45:
which leads to the systems of equations
This system of equations is similar to the case of no sharing given by Equations 4445, with the notable exception that the equations are coupled through the shared tubulin pool term $T{L}_{1}{L}_{2}$. The resulting steadystate equations are identical to those of Equation 35 for a single flagellum, with $T$ replaced by $T/2$ and $\gamma $ replaced by $2\gamma $. Therefore the steadystate lengths satisfy Equation 36 upon rescaling $T\to T/2$ and $\gamma \to 2\gamma $. This model yields simultaneous length control, and the resulting steadystate lengths satisfy ${L}_{1,ss}={L}_{2,ss}$ only if ${M}_{1}={M}_{2}=M$. It follows that the steadystate lengths are unequal after severing because one of the two motor pools is depleted.
The model equations have a similar form to existing models (Marshall et al., 2005; Hendel et al., 2018), in which the assembly rates involve a factor of $T{L}_{1}{L}_{2}$ and either a $1/{L}_{i}$ or $1/{L}_{i}^{2}$dependence in the denominator, for $i=1,\mathrm{\hspace{0.17em}2}$ as discussed earlier in the context of a single growing flagellum. Although the equations are similar, the absence of length equalization in our model (Figure 3biii) contrasts with the length equalization achieved in Hendel et al. (2018) by an additional control mechanism that instantaneously replenishes the number of motors on the flagellum after severing. As noted in the Discussion, the importance of protein replenishment for the model appears to be inconsistent with experimental results (Rosenbaum et al., 1969), which show that length equalization occurs even in the absence of new protein synthesis.
Tubulin shared, motors shared
Request a detailed protocolWe finally consider the case in which both tubulin and motors are shared through a common pool. By the shared tubulin pool assumption ${T}_{f,1}={T}_{f,2}=T{L}_{1}{L}_{2}$. Further, by the shared motor pool assumption the injection rates satisfy ${J}_{1}={J}_{2}\equiv J$. Therefore
in which $J$ satisfies Equation 48. We are left with the steadystate equations
These equations are identical, so that there is only a single equation for the two unknowns ${L}_{1,ss}$ and ${L}_{2,ss}$ and the steadystate lengths are indeterminate provided that the disassembly rate is constant. Note that this conclusion holds regardless of the particular form of the flux.
We next use linear stability analysis to demonstrate this breakdown of simultaneous length in greater detail. Let ${L}_{1,ss}$ and ${L}_{2,ss}$ denote any one of the infinitelymany possible solutions to Equations 60 and 61. Letting $\mathrm{\Delta}{L}_{1}$ and $\mathrm{\Delta}{L}_{2}$ be the deviations from steadystate such that ${L}_{1}={L}_{1,ss}+\mathrm{\Delta}{L}_{1}$ and ${L}_{2}={L}_{2,ss}+\mathrm{\Delta}{L}_{2}$, linearizing about any one of these solutions yields a matrix equation of the form
for $a>0$. The 2 × 2 matrix above has an vanishing eigenvalue, as we now show. Diagonalizing in terms of the sum $\mathrm{\Sigma}=\mathrm{\Delta}{L}_{1}+\mathrm{\Delta}{L}_{2}$ and difference $\mathrm{\Gamma}=\mathrm{\Delta}{L}_{1}\mathrm{\Delta}{L}_{2}$ gives
There is a vanishing eigenvalue associated to the difference of lengths, that is perturbations from steadystate in the difference of lengths do not decay on a finite timescale. This is consistent with previous results from stochastic simulations that the model with constant disassembly in which all biomolecules are shared does not yield simultaneous length control (Mohapatra et al., 2017). Noise must be included to observe this result; if fluctuations are not included, as in the deterministic ODE, any initial state with the correct sum in lengths appears stable. This is because the zero eigenvalue causes such states to be marginally stable.
Appendix 1
Stochastic simulation
The simulations are done using an agentbased model in which individual motors are tracked at each point of time, similar to that used in Hendel et al. (2018).
The simulations are run for a fixed amount of time discretized into small time steps of size $\mathrm{\Delta}t$. Parameters such as the diffusion constant $D$, velocity $v$, disassembly rate $d$, and injection rate ${k}_{on}$ are fixed. Four different variables are tracked for each motor:
A variable indicating whether motors are located in the basal pool (value 0) or the flagellum (value 1). In the case of multiple flagella with shared motors, each flagellum is assigned a different positive integer.
A Boolean variable indicating whether motors are undergoing ballistic or diffusive motion.
The position ${x}_{j}(t)$ of motor $j$ for $j=1,\mathrm{\dots},M$.
The amount of tubulin ${t}_{j}(t)$ carried by the motor (${t}_{j}=\gamma {T}_{f}$ at the time of injection).
The following processes take place at each time step:
The length of each flagellum ${L}_{i}(t)$ for $i=1,\mathrm{\hspace{0.17em}2}$ is decreased by a constant amount $d\mathrm{\Delta}t$ (in the case of constant disassembly) or by an amount $({d}_{0}+{d}_{1}{c}_{d}(L))\mathrm{\Delta}t$ (in the case of concentrationdependent disassembly), where the concentration ${c}_{d}(L)$ is computed by counting the number of diffusing motors within 1 µm of the flagellar tip.
Motors are injected into the flagella with a probability ${k}_{\text{on}}{M}_{f}\mathrm{\Delta}t$, where ${M}_{f}$ is the number of motors in the basal pool.
Motors undergoing ballistic motion are advanced along the flagellum by a constant amount $v\mathrm{\Delta}t$.
Motors undergoing diffusive motion are advanced by a random amount drawn from a normal distribution with mean 0 and variance $2D\mathrm{\Delta}t$.
If a motor undergoing ballistic motion reaches the tip of the flagellum, the flagellar length is increased by the amount of tubulin ${t}_{j}$ carried by the motor. The motion of the motor is changed to diffusive and its position is reset to $L(t)$, that is the location of the flagellar tip.
If the position of a motor moving diffusively exceeds the length of flagella, that is ${x}_{j}(t)>L(t)$, it is reflected according to ${x}_{j}(t)\to L(t)\left({x}_{j}(t)L(t)\right)$.
If a diffusive motor reaches the base of the flagellum, it is taken up into the basal pool and stops diffusing. This enforces the absorbing boundary condition ${c}_{d}(0)=0$.
By advancing the above processes in time, we obtain the length profiles ${L}_{i}(t)$.
To simulate the severing experiments, the length of one flagellum is shortened at a particular time and the motors and tubulin within the severed part of the flagellum are lost.
For those simulations with no protein replenishment (Figure 3—videos 1, 2, 3 and Figure 4—video 1) we fix the total amount of tubulin $T$ and the total number of motors $M$. For those simulations in which we incorporate the replenishment of proteins in the basal pool (Figure 3—videos 4, 5, 6 and Figure 4—video 2), the target protein numbers ${\overline{T}}_{f}$ and ${\overline{M}}_{f}$ are fixed along with the timescale of replenishment ${\tau}_{r}$. At each time step, there is a possibility of adding or removing a protein to or from the basal pool. The probability of addition or removal is proportional to the difference between the target number and the number of proteins currently in the basal pool.
The videos were made using the following parameters: $D=1.7\phantom{\rule{thinmathspace}{0ex}}\mu {\mathrm{m}}^{2}/\mathrm{s}$, $k}_{\text{on}}=0.075\phantom{\rule{thinmathspace}{0ex}}{s}^{1$, $T=38\phantom{\rule{thinmathspace}{0ex}}\mu \mathrm{m}/\mathrm{f}\mathrm{l}\mathrm{a}\mathrm{g}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{u}\mathrm{m}$, $M=200/\mathrm{f}\mathrm{l}\mathrm{a}\mathrm{g}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{u}\mathrm{m}$, $\gamma =2.5\times {10}^{4}$, $d}_{0}=0.01\phantom{\rule{thinmathspace}{0ex}}\mu \mathrm{m}/\mathrm{s$, $d}_{1}=1.7\times {10}^{3}\phantom{\rule{thinmathspace}{0ex}}\mu {\mathrm{m}}^{2}/\mathrm{s$, $v=2.5\phantom{\rule{thinmathspace}{0ex}}\mu \mathrm{m}/\mathrm{s}$. In Figure 3—videos 4–6, the replenishment timescale is $\tau}_{r}=300\phantom{\rule{thinmathspace}{0ex}}\mathrm{s$.
Appendix 2
Parameter estimation
To estimate the model parameters, we fit to experimentallymeasured data. First, we extrapolate from Marshall et al. (2005) to estimate that the growth rate upon severing a flagellum is approximately 0.4 µm/min. This implies
We next estimate the product $\gamma {k}_{\text{on}}M$. To do so, we use the measured disassembly speed $d=0.5\phantom{\rule{thinmathspace}{0ex}}\mu \mathrm{m}/\mathrm{m}\mathrm{i}\mathrm{n}$ (Marshall and Rosenbaum, 2001) and an estimated initial tubulin pool of $T=2540\phantom{\rule{thinmathspace}{0ex}}\mu \mathrm{m}$. This yields $\gamma {k}_{\text{on}}M=2.3\times {10}^{2}$–$3.6\times {10}^{2}{\text{min}}^{1}$. The estimate $T$ for the tubulin pool of a single flagellum is based on the reported amount 76–94 μm for the total tubulin shared by two flagella (Marshall et al., 2005), allowing for some tubulin loss after severing. The estimate for $\gamma {k}_{\text{on}}M$ is not expected to be very precise, particularly given that the total tubulin pool size from Marshall et al. (2005) was itself obtained by fitting the parameters of a related model to data on mutants with extra flagella.
We subsequently estimate ${k}_{\text{on}}$ by fitting it to the steadystate length observed in the severing experiment. Returning to Equation 35, we may express ${k}_{\text{on}}$ in terms of quantities that have already been measured or estimated:
Plugging in the values for $\gamma kM$,$d$, and $T$ above as well as the measured values $v=150\phantom{\rule{thinmathspace}{0ex}}\mu \mathrm{m}/\mathrm{m}\mathrm{i}\mathrm{n}$ (Kozminski et al., 1993; Buisson et al., 2013), $D=102\phantom{\rule{thinmathspace}{0ex}}\mu {\mathrm{m}}^{2}/\mathrm{m}\mathrm{i}\mathrm{n}$ (Chien et al., 2017), and $L}_{ss}=10\phantom{\rule{thinmathspace}{0ex}}\mu \mathrm{m$ measured in Ludington et al. (2012), we obtain $k}_{\text{on}}=0.84.5\phantom{\rule{thinmathspace}{0ex}}\mathrm{m}\mathrm{i}{\mathrm{n}}^{1$. See the parameter values and definitions in Table 1.
Boundary conditions
Here we discuss potential boundary conditions besides the diffusive sink ${c}_{d}(0)=0$ described in Materials and methods. Repeating the previous calculation for a general prescribed concentration ${c}_{d}(0)={c}_{0}$ at the flagellar base yields
Therefore, to first order in $\mathrm{\Delta}L=L{L}_{ss}$ the effect of nonzero ${c}_{0}$ is equivalent to rescaling $M$ in Equation 4.
We also consider the scenario in which the basal pool concentration depends on the number of free molecular motors ${M}_{f}$ through ${c}_{d}(0)=A{M}_{f}/V$, where $A$ and $V$ are geometric parameters representing the flagellar cross sectional area $A$ and the basal pool volume $V$, respectively. Mass action kinetics of injection $J={k}_{\text{on}}{M}_{f}$ may be used to rewrite the basal concentration as ${c}_{d}(0)=AJ/({k}_{\text{on}}V)$. Substituting this expression into Equation 66 results in
Therefore, to first order in $\mathrm{\Delta}L$ the effect of the boundary condition in this case is equivalent to rescaling $v\to v/(1+(A/V)(v/{k}_{\text{on}}))$ in Equation 4.
Appendix 3
Concentrationdependent disassembly model
Separate pools
We test various modes of coupling within the concentrationdependent depolymerization model and find that only the case of fully shared biomolecule pools is consistent with data (Appendix 3—figure 1). Unlike the constant disassembly models we have considered, there is some degree of length recovery after severing when only one biomolecule is shared. However, there is still no length equalization after severing because of the asymmetical depletion caused by separate pools.
Biomolecules in excess
Flagellar growth in Chlamydomonas is limited by the supply of proteins. As discussed in Results, the importance of depletion effects is evidenced by the shortening of the long flagellum in severing experiments and by the cyclohexamide experiments of Rosenbaum et al. (1969) that blocked new protein synthesis and resulted in shorter flagella. Within the proposed active disassembly model this limitingpool mechanism may arise in three ways: (i) both tubulin and motors are limited, (ii) tubulin is limited and motors are in excess, or (iii) tubulin is in excess and motors are limited. In Figure 4 we considered case (i) given by Equations 20 and 21 in which both biomolecules are limited. Here for completeness we consider the other two cases as well. Case (ii) may immediately be ruled out: if the ratelimiting IFT protein were in excess, their injection rate would not depend on flagellar length as observed in experiments (Dentler, 2005). A limiting pool of IFT proteins is therefore necessary for agreement with the severing data. We further consider the following model in which tubulin is in excess:
where $T$ is constant since tubulin is in excess and the flux $J$ is given as before by
Simulations show that the model Equations 68 and 69 is consistent with severing data (Appendix 3—figure 2). Therefore, a limiting pool of IFT proteins is necessary for agreement with data, whereas tubulin may either be limited or in excess.
Linearization
Stability is established by linearizing about steadystate. We perform the linearization for the concentrationdependent disassembly model and discuss how the result generalizes to simultaneous length control with arbitrary flagellar number.
Let $\mathrm{\Delta}{L}_{1}$ and $\mathrm{\Delta}{L}_{2}$ be the deviations from steadystate such that ${L}_{1}={L}_{ss}+\mathrm{\Delta}{L}_{1}$ and ${L}_{2}={L}_{ss}+\mathrm{\Delta}{L}_{2}$. From (13), the flux $J$ is given by
Keeping only terms up to first order in $\mathrm{\Delta}{L}_{1}$ and $\mathrm{\Delta}{L}_{2}$ in the denominator,
so that the firstorder Taylor series expansion yields
Substituting this expression for the flux into the dynamical equations Equations 20 and 21 results in
where we have retained terms up to first order in $\mathrm{\Delta}{L}_{1}$ and $\mathrm{\Delta}{L}_{2}$. Defining
we may write this system in the matrix form
The matrix above is the sum of a rankone matrix and a diagonal perturbation. Roughly speaking, $a$ corresponds to the shared quantities whereas $b$ corresponds to the independent quantities. This system may be diagonalized in terms of the sum $\mathrm{\Sigma}=\mathrm{\Delta}{L}_{1}+\mathrm{\Delta}{L}_{2}$ and difference $\mathrm{\Gamma}=\mathrm{\Delta}{L}_{1}\mathrm{\Delta}{L}_{2}$ to yield
Note that the eigenvalues
are both negative, so that the steadystate is stable, since $b$ is clearly positive and $T>2{L}_{ss}$ implies the positivity of $a$. The fact that ${\lambda}_{\mathrm{\Sigma}}$ and ${\lambda}_{\mathrm{\Gamma}}$ are distinct is noteworthy as it provides a possible means to extract two independent parameters from experiment.
Arbitrary flagellar number
This rankone plus diagonal matrix structure also applies to the case of arbitrary flagellar number $N$ discussed in Results. Let $\mathrm{\Delta}{L}_{i}:={L}_{i}{L}_{ss}$ denote the deviation of the $i\text{th}$ flagellum from steadystate for $i=1,\mathrm{\dots},N$. The linearized equations satisfy
where $\mathrm{\Delta}\mathbf{\mathbf{L}}=(\mathrm{\Delta}{L}_{1},\mathrm{\dots},\mathrm{\Delta}{L}_{N})$ and the matrix $M$ is of the form
with $R={\mathrm{\U0001d7cf\U0001d7cf}}^{T}$ the rankone matrix satisfying ${R}_{ij}=1$ for all $i,j$ and $I$ the $N\times N$ identity matrix, for example for N = 3
$M$ is straightforward to diagonalize. The vector ${\mathbf{\mathbf{v}}}_{1}:=\mathrm{\U0001d7cf}={(1,\mathrm{\dots},1)}^{T}$ corresponding to the sum of all lengths is an eigenvector of $M$:
Further, for any vector $\mathbf{\mathbf{x}}=({x}_{1},\mathrm{\dots},{x}_{N})$ such that ${\sum}_{i=1}^{N}{x}_{i}=0$, we have $R\mathbf{\mathbf{x}}=0$ and
therefore it is an eigenvector with eigenvalue $\lambda =b$. Note that the pairwise differences
for $k=2,\mathrm{\dots},N$ form a convenient basis for the space of such vectors $\mathbf{\mathbf{x}}$ whose components sum to zero. In terms of the basis $\{{\mathbf{\mathbf{v}}}_{1},{\mathbf{\mathbf{v}}}_{2},\mathrm{\dots},{\mathbf{\mathbf{v}}}_{N}\}$ consisting of the sum and differences in lengths, the evolution equations diagonalize with eigenvalues
for the sum and differences, respectively, so that ${\lambda}_{\mathrm{\Sigma}}(N)$ is an eigenvalue of multiplicity 1 and ${\lambda}_{\mathrm{\Gamma}}(N)$ is an eigenvalue of multiplicity $N1$. Note that in addition to the explicit dependence of ${\lambda}_{\mathrm{\Sigma}}(N)$ and ${\lambda}_{\mathrm{\Gamma}}(N)$ on $N$ there is an implicit numberdependence through the steadystate length (and potentially the size of the pool $T$). Since ${\lambda}_{\mathrm{\Sigma}}(N)\ne {\lambda}_{\mathrm{\Gamma}}(N)$, the dynamics involve two distinct timescales ${\tau}_{\mathrm{\Sigma}}(N)={\lambda}_{\mathrm{\Sigma}}{(N)}^{1}$ and ${\tau}_{\mathrm{\Gamma}}(N)={\lambda}_{\mathrm{\Gamma}}{(N)}^{1}$ corresponding to these two eigenvalues.
Different depolymerizer and ratelimiting IFT protein
In the description of the concentrationdependent disassembly model in Results, for convenience we made the assumption that the depolymerizer is the ratelimiting IFT protein. Here, we consider the more general case that the depolymerizer is not the ratelimiting IFT protein, but rather a different protein that is carried to the flagellar tip by IFT and present in excess in the basal pool. The formula derived in the manuscript for the flux $J$ represents the flux of IFT particles, and in this case the flux of depolymerizer is $KJ$, where the capacity $K$ of depolymerizers per IFT particle is assumed constant. Assuming the depolymerizer diffuses back from the flagellar tip with diffusivity ${D}^{\prime}$, the concentration ${c}_{d}^{\prime}$ of diffusing depolymerizer proteins satisfies
so that the concentration at position $x$ along the flagellum satisfies ${c}_{d}^{\prime}(x)={c}_{0}^{\prime}+\frac{KJ}{{D}^{\prime}}x$ under the boundary condition ${c}_{d}^{\prime}(0)={c}_{0}^{\prime}$. (Primes are used to distinguish the parameters for the depolymerizer from those of the ratelimiting IFT protein.) In this case the dynamical equations for length become
Therefore Equations 20 and 21 derived in Results remain valid in this more general case upon making the identification ${d}_{0}\to {d}_{0}+{d}_{1}{c}_{0}^{\prime}$ and ${d}_{1}\to \frac{KD}{{D}^{\prime}}{d}_{1}$. We have verified these results through agentbased simulations (Appendix 3—figure 3a).
Exponential concentration gradient
To show that our model allows for nonlinear concentration gradients, we set up agent based simulations which would lead to an exponential concentration distribution, similar to Naoz et al. (2008).
In our simulations, after injection motors travel ballistically to the tip of the flagellum where they begin to diffuse back toward the base as discussed in Appendix 1. In addition motors may detach with rate ${k}_{d}$ to undergo diffusive motion and reattach with rate ${k}_{a}$ to switch from diffusive to ballistic motion. This leads to an exponential distribution with the concentration profile of diffusing motors being
where as before $J$ = injection rate = ${k}_{on}{M}_{f}/2$ and ${\lambda}_{\pm}$ is given by
The concentration profile of diffusing motors with a fit to the above equation is shown along with the length regulation of flagella in Appendix 3—figure 3b for rates ${k}_{a}=6.3\times {10}^{2}$ s^{−1} and ${k}_{d}=3.13\times {10}^{4}$ s^{−1}. The results are found to be qualitatively similar to the case of a linear concentration gradient, supporting our claim that concentrationdependent disassembly is able to control lengths independent of the precise form of the concentration gradient.
Processive depolymerizers
By taking the disassembly rate to depend on local depolymerizer concentration, we have implicitly assumed that the depolymerizer acts nonprocessively. However, this is not a fundamental restriction. We have used our agentbased model to explore a mechanism of processive depolymerization, in which depolymerizers diffusing within 1 micron of the flagellar tip bind at rate 9.4 × 10^{2} s^{−1} and depolymerize at a rate of 9.7 × 10^{4} µm/s. The depolymerization duration is drawn from an exponential distribution with prescribed mean. We have verified that this model achieves simultaneous length control over a range of mean depolymerization times. In Appendix 3—figure 3c we show the results of simulations using mean depolymerization times from 10 s (corresponding to 0.01 µm) up to 10 min (corresponding to 0.6 µm). For mean depolymerization times up to 100 s, we find the results to be qualitatively similar to the nonprocessive case, whereas for longer mean depolymerization times the steadystate lengths exhibit oscillations about the steadystate. The emergence of oscillations is not entirely surprising since processive depolymerization effectively introduces into the equations a time delay, which is known to give rise to oscillations in many contexts (Richard, 2003).
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Decision letter

Frank JülicherReviewing Editor; Max Planck Institute for the Physics of Complex Systems, Germany

Naama BarkaiSenior Editor; Weizmann Institute of Science, Israel
In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.
[Editors’ note: this article was originally rejected after discussions between the reviewers, but the authors were invited to resubmit after an appeal against the decision.]
Thank you for submitting your work entitled "Length regulation of multiple flagella that selfassemble from a shared pool of components" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Senior Editor. The reviewers have opted to remain anonymous.
Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.
The authors perform a theoretical study of the regulation flagellar length in Chlamydomonas based on intraflagellar transport of motors and tubulin subunits that govern assembly and disassembly. A key question is how the length of two flagella can be coordinated by exchange of components. This is an important question. The present paper extends an earlier model by Hendel, Thomson and Marshall. It presents simple analytic expressions for the length dynamics and steady state lengths in several scenarios of components that are shared or separate between cilia. After thorough discussion the reviewers concluded that this work is not a major advance as compared to the earlier work by Hendel et al. It provides an extension of that work which is of interest to specialists but does not represent a fundamental advance.
Reviewer #1:
The authors perform a theoretical study of the regulation flagellar length in Chlamydomonas based on intraflagellar transport of motors and tubulin subunits that govern assembly and disassembly. A key question is how the length of two flagella can be coordinated by exchange of components.
The paper is largely well written and interesting. First a reduced model is presented with several simplifying assumptions such as separation of timescales and simplified kinetic rules. In the context of this reduced model the coordination of two cilia is then discussed. It is shown that if a molecular component is not shared between two cilia then length can be coordinated. If all components are shared then coordination needs to be more subtle. The authors suggest that concentration dependent depolymerization could be responsible for length coordination.
This work is interesting but it also has shortcomings. Reading the Introduction, the paper makes a strong impression. However, when I worked through the main part of the paper weaknesses became apparent and the discussion seemed to be rather superficial. In the end it remains unclear what advance in our understanding the work actually achieves. In its present form I do not think that this work is suitable for publication in eLife.
Major points:
1) The main motivation of the paper is to provide a possible explanation of the cilia severing experiment shown in Figure 1B. This is an important and interesting problem. However, the manuscript fails to really advance this issue. The authors rule out the two independent flagella (Figure 3Bi) because they cannot account for the coordinated behavior of the experiment. It seems that the proposed model of coordinated flagella shown in Figure 4 does also not really capture the key feature of coupled flagella observed in experiments: the flagellum that is not severed shrinks to almost half its original length and then both flagella grow together to their final length. In fact, Figure 4 which is a key figure of the paper is not well presented. In a severing experiment the longer flagellum should start from the steady state length which both flagella reach at long times. Another problem with Figure 4 is that only stochastic simulations are shown. It would be better to show the true average which is obtained by solving the deterministic equations. Then it would be clear if the longer flagellum first reaches a minimum before it again increases its length to reach the steady state. The stochastic simulations can be misleading as the fluctuations conceal this important feature in the behavior or even give the impression of length minima but which arise only from the noise.
2) It is an interesting but not deep insight (given the simplifications of the model), that if all components are shared then only the total length is fixed but individual lengths are independent. As the authors show this feature is a result of the simplifications used. Taking more realistic aspects into account, such as the concentration dependent depolymerization, this degeneracy in the model is removed. However, there are most likely other possibilities that could provide such a lift of the degeneracy. The fact that one mechanism can lift the degeneracy is interesting but does not constitute a strong result given that the model fails to qualitatively account for the experimental data.
3) When discussing the cases where one component (M or T) is not shared but exists in two different pools, it is implicitly assumed that the separate pools (e.g. T_{1} and T_{2}) are equal. However, this is not stated clearly and it is not clear why they should be equal if they are completely independent.
Reviewer #2:
The authors have analyzed a class of models for length control of the two cilia of Chlamydomonas. The interesting result is that a naive model in which both tubulin and motor pools are shared between the two cilia doesn't work: only the summed ciliary length is determined and the individual lengths are undetermined. To get length control for two cilia, the authors add a lengthdependent depolymerization, which can be satisfied by a depolymerase whose concentration increases with length (which happens if anterograde transport of the depolymerase is advective but retrograde transport diffusive).
My main concern is: what is different/new compared to the Hendel et al. (2018) paper? Is this paper wrong, in the sense that their mechanism (which has no lengthdependent depolymerase) does not work, contrary to their claims? If this is the case, then then this point must be stressed. If the Hendel et al. paper is correct, then more justification of the novelty of the current work is necessary.
The following points need to be addressed in the manuscript
1) What is the concentration profile of the depolymerase? Please add this in a figure. I assume that it is a linear increasing concentration from the base (as in the Hendel paper) but I would like to see this.
2) Under what conditions can depolymerases regulate a single cilium? Is a limiting tubulin pool necessary? Is a limiting tubulin pool necessary for the case of two cilia? Is a limiting pool of IFT motors necessary? The general point I am raising is that lengthdependent depolymerization is a strong assumption and perhaps it is sufficient under very broad conditions. What are they? Then this point needs to be discussed.
3) The stochastic aspect of the work is irrelevant as it is not used and should be removed from the paper (perhaps put into another paper).
Reviewer #3:
The paper describes a very interesting study of the possible models that can account for the simultaneous length control in the flagella of a singlecell organism. The study is illuminating, and shows how simple models can help to shed light on the microscopic processes, simply from the analysis of largescale dynamics. I have a few comments:
1) How many MT are in each flagellum? How is this number controlled? Is this also a dynamic variable that selforganizes? The implicit assumption is that this is a constant (determined at the base, and therefore not dependent on length?), but should be explained.
2) The authors consider that the ballistic motion of the motors to the growing tip is uninterrupted, is motors and their cargo do not detach from each other or from the MT track until the tip. Is this known to be a good approximation of this system? It certainly simplifies the analysis, as they do not need to solve the spatial distribution of the density of motors, and cargo, along the flagella's length.
I would suggest discussing how this is different from models of the growth and steadystate of actinbased protrusions, where disassembly is at the base, and the length is a result of force balance (see Naoz et al., 2008; Orly et al., 2014).
3) The question about the return current of tipdirected motors is also an open issue for myosin motors along actinfilled protrusions. Interesting dynamics and traffic jams have been observed and modeled (see Yochelis et al., 2015; Pinkoviezky and Gov, 2014, 2017), I suggest contrasting with the situation in the flagella.
4) The noise in the reactions that control the growth at the tip should give rise to a term in (1) that has noise multiplied by J and T_{f}? Would this change the dynamics?
5) MTs undergo catastrophes and recoveries, and the data traces seem to show this. Does this type of dynamics matter for the model? This should be discussed.
[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]
Thank you for resubmitting your work entitled "Length regulation of multiple flagella that selfassemble from a shared pool of components" for further consideration at eLife. Your revised article has been favorably evaluated by Naama Barkai (Senior Editor), a Reviewing Editor, and two reviewers.
The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.
Summary:
The manuscript discusses the length regulation of the pair of flagellae of the green algae Chlamydomonas. The paper builds on earlier work of the Wallace Marshall group which proposed basic concepts of length regulation via tubulin transport along flagellae controlling assembly together with disassembly. Coupling of flagellae can be mediated by shaped molecular pools.
The present paper provides two important results:
i) Detailed theoretical analysis of the shared limitingpool mechanism, and showing that by itself this mechanism does not lead to length equilibration of the two flagella.
ii) A careful study of a length regulation by length dependent depolymerization, showing that length dependent depolymeriyation together with shared molecular pools can account for the experimental observations.
However, the paper also has some serious weaknesses. In particular:
1) The experimental basis for their proposal of length dependent depolymerization are much weaker than claimed. In fact, Piao et al., 2009 and 2013, do not give evidence for depolymerases at the flagellar tip in steady state.
2) The claim that length dependent depolymerization is the only mechanism to account for the data is an overstatement.
3) The discussion of previous work (Hendel et al.) that did find length regulation as seen in experiments without the need of length dependent depolymerization is discussed only superficially and with a somewhat negative attitude. This previous work should be taken more seriously and discussed more carefully. It does show that length regulation does not require length dependent depolymerization. However, this seems to be resulting from different details and assumptions in the model.
The authors should tone down their claims and more carefully relate their results to previous work. Then this paper could become a very valuable contribution that clarifies subtle but general aspects of length regulation and provides novel insights in the possible role of length dependent depolymerization.
The authors have to revise their manuscript carefully before it could be suitable for publication in eLife.
Essential revisions:
– The authors discuss a mechanism for length dependent depolymerization. However, several arguments are unclear. The authors begin with the assumption in the Introduction: "We will assume for now that the ratelimiting protein is kinesin2, which is the molecular motor responsible for transport toward the tip of the flagellum". But, then in subsection “Tubulin shared, motors shared and concentrationdependent disassembly” the authors write "although to this point we have assumed the ratelimiting protein is kinesin2, in fact the model is valid for any ratelimiting IFT protein. Therefore, in what follows we assume that the ratelimiting protein is a depolymerizer having the same motion as kinesin2, uninterrupted ballistic motion to the tip followed by diffusive motion to the base…"
The authors should clarify if there is a family of kinesins that possess both these properties, namely, anterograde transport of tubulin dimers in a directed manner along a microtubule as well as microtubule depolymerase activity. It seems no such motor is known.
– In support of their claim, the authors quote the experimental observations of Piao et al. Piao et al. clearly state in their paper that kinesin13 "was transported by IFT into flagella during flagellar shortening." Further elaborating on this mechanism, Wang et al. (2013) reported that "CrKin13 was barely detectable in the flagella of steady state cells, as shown previously (Piao et al., 2009). However, during rigorous phase of flagellar assembly at 15 and 30 min after deflagellation, CrKin13 was found to be enriched in the flagella and decreased to normal level in fully assembled flagella". Thus, unlike the mechanism proposed by Fai et al. in this paper, experiments indicate very little presence of kinesin13 depolymerases in the flagella under normal circumstances. Only amputation of one of these trigger a signal that leads to rapid entry of the kinesin13 into the uncut flagellum for its depolymerization that supplies tubulins for the initial regeneration of the amputated flagellum. Thus, the roles of kinesin2 and kinesin13 and their presence or absence in the flagellum should be treated separately and cannot be represented by an all encompassing single motor. The relation to experiments and the required properties of motors should be discussed more carefully (see also next point)).
– Piao et al. demonstrated the depolymerase activity of kinesin13 family (and not kinesin8 family) in the microtubule disassembly in cilia. Kinesin13 diffuses along microtubules to target either end and are not known to transport tubulin. Therefore, the claim of experimental support of the model seems to be a too strong statement.
– Subsection “Tubulin shared, motors shared and concentrationdependent disassembly”, the authors state "Unlike constant disassembly models in which a limitingpool mechanism is essential for length control,…" This appears to be an incorrect statement. Some constantdisassembly models, e.g., Timeofflight (TOF) model, which also assumes constant disassembly, does not assume "limiting pool". Instead TOF assumed "differential loading" of IFT particles, as indicated by the experiments of Wren et al.
Therefore, the statement "As we shall see, the constant disassembly models do not result in the rapid length equalization observed experimentally" is an overstatement and oversimplification.
– The key assumptions of Fai et al.'s model seem to be the following: (a) a concentration gradient of the depolymerases along the length of the flagellum and (b) the local depolymerization rate is proportional to the local concentration of the depolymerases. There appears to be an additional implicit assumption: the depolymerase is nonprocessive in its depolymerase activity (not to be confused with processivity in motility). Otherwise, depolymerases loaded on to the MT plusend at a location closer to the ciliary tip may continue to depolymerize even when the MT becomes shorter and its plusend reaches points where the depolymerase concentrations are quite different.
– The possibility of a concentration gradient of the motors arose naturally already in the paper of Hendel et al. (see page 667 of Hendel et al.). However, Hendel et al. did not associate this concentration gradient with that of depolymerases, which seems to be the appropriate picture.
– The authors present "testable predictions". However there are some problems. The experimental results already reported by Piao et al., 2009 and 2013, do not provide evidence for the presence of depolymerases at the tip in steady state. Also based on these experiments the existence of a concentration gradient of the depolymerases in the steadystate (that too, an increasing concentration towards the tip) seems to be unlikely.
The second testable prediction that the authors mention would not be a clear test for validation/refutation of their model. For all those models that depend on a dynamic balance of the rates of polymerization and depolymerization in the steadystate of the flagellum, lowering the entry flux of the IFT particles would lead to overall depolymerization although the local rate of depolymerization of the microtubules at the plus end would remain unchanged.
– The slight difference in the expressions for L_{ss} derived by Hendel et al. and that of Fai et al. arises from difference in the scenarios considered by the two. The two assumptions made by Hendel et al. were clearly stated in their paper. The first assumption of "a constant source of free motor protein at the tip" and the second assumption of "the approximation in which motors that have reached the base immediately transport back to the tip" are exactly the two conditions ("no tubulin depletion" and "instantaneous ballistic motion") that Fai et al. point out in reducing their result to that of Hendel et al. In this sense their derivation is only slightly improved compared to that of Hendel et al.
– I find the claims of the paper that Hendel’s model is somehow incorrect and that the present manuscript solves all open issues unconvincing. Hendel et al. show a scenario where length control works, probably based on different detailed assumptions. This should be more clearly discussed and not superficially mentioned as in the paragraph three of the Discussion.
– The authors discuss the case where molecular components stabilize their levels (Equations 18 and 19) and point out the problem that in steady state such a relaxation to fixed levels breaks length regulation. It remains unclear if that is also a problem for the proposed model based on length dependent depolymerization. Also, it would be important to know what happens when the motors alone are relaxing to a fixed level but the tubulin is limited and fixed. Would that be similar to the results of Hendel et al.?
– The authors seem to miss relevant references: Varga et al. (2009); Klein et al. (2005); Johann et al. (2012). These should be cited and their consequences for this paper should be discussed.
https://doi.org/10.7554/eLife.42599.sa1Author response
[Editors’ note: this article was originally rejected after discussions between the reviewers, but the authors were invited to resubmit after an appeal against the decision.]
The authors perform a theoretical study of the regulation flagellar length in Chlamydomonas based on intraflagellar transport of motors and tubulin subunits that govern assembly and disassembly. A key question is how the length of two flagella can be coordinated by exchange of components. This is an important question. The present paper extends an earlier model by Hendel, Thomson and Marshall. It presents simple analytic expressions for the length dynamics and steady state lengths in several scenarios of components that are shared or separate between cilia. After thorough discussion the reviewers concluded that this work is not a major advance as compared to the earlier work by Hendel et al. It provides an extension of that work which is of interest to specialists but does not represent a fundamental advance.
We appreciate the detailed and useful comments from the three reviewers. In light of these comments, we have performed full stochastic simulations to verify our analytical results and clarify their relation to the model described in Hendel et al. This has led to a new and surprising result that we are very excited about: of the five models discussed in our paper, only one leads to length control consistent with the available experimental data, and it is not the one of Hendel et al. Instead, it is the model in which length control is achieved by lengthdependent depolymerization. The essence of these new results are briefly explained below. We have also found supporting experimental evidence for this model in published papers on Chlamydomonas reinhardtii (Piao et al., 2009) and on Leishmania major (Blaineau et al., Current Biology 17, 778–782 (2007)), which both describe the role of a microtubule depolymerizing protein (kinesin 13) in the assembly and length regulation of flagella. With these new results in hand, we feel that our theoretical work significantly advances our understanding of flagellar length control. It challenges the existing paradigm of control by lengthdependent polymerization by switching focus on lengthdependent depolymerization. Finally, our model provides concrete predictions for new experiments.
We would like to emphasize that these results allow us to definitively address a major concern of the reviewers, which is how our manuscript advances the field beyond the work of Hendel et al. We have used new agentbased simulations to reproduce the results of Hendel et al. and confirmed that their model involves an additional control mechanism of the two motor pools associated with each flagellum; in their simulations of the”severing experiment” (in which one of the two flagella is cut), the amount of IFT lost upon cutting is added (in silico) to the basal pool. This assumption is required for agreement with the experiments. In effect, regeneration of the flagella to their original length after one has been severed is achieved in the model through this control mechanism that effectively monitors the total number of motors associated with each flagellum. There is no biological justification for this assumption, and it seems highly implausible as it would require the cell to count both the freely diffusing motors and those taking part in transport by IFTparticles, and to maintain this number at a fixed value. Our new results show that removing this additional control mechanism, while leading to length control of a single flagellum (as described by Hendel et al.), is incapable of regenerating equal lengths of both flagella after one is cut, in clear contradiction with experiment. This subtlety of the Hendel et al. model was missed by us as well, leading us to conclude previously that their model is consistent with experiments. We only became aware of the disparity upon careful and detailed stochastic simulations in which we attempted to replicate the work of Hendel et al. To our surprise, this left us with the conclusion that all available experimental evidence now clearly points in the direction of flagellar length being controlled by lengthdependent depolymerization (the last model discussed in our manuscript). As stated above, this is a paradigm shifting proposal in this field and we can offer concrete predictions for experiments that will test it (some of which have already been done in published papers mentioned above). The following is a list of the main changes, with references to relevant videos from full stochastic simulations included in the new submission
1) The Langevin equation (stochastic differential equation) formulation has been removed and replaced by a deterministic ordinary differential equation throughout the manuscript. We have included new stochastic simulations (see the new Appendix 1 and associated videos) that model the assembly process with more detail than the differential equation model and include stochastic effects.
2) Figure 2 describing the length dynamics of a single flagellum has been remade to emphasize the emergence of lengthdependent injection.
3) Figure 3 has been remade without assuming instantaneous recovery of the original number of motors in the flagellum (see also the new Figure 3—videos 1–3). These results rule out all four versions of the constant disassembly model.
4) We have considered a model of protein replenishment based on external control of basal body concentrations (Equations 18 and 19). Somewhat counterintuitively, this further destabilizes the constant disassembly models, as we show by an analytic argument included in our response and subsection “Controlling protein levels in the basal pool is incompatible with constant disassembly” of the revision and illustrate in the new Figure 3—videos 4–6.
5) Figure 4 has been remade and merged with the previous Figure 6. In Figure 4C we have added quantitative fits of the concentrationdependent disassembly model to severing data.
6) We have considered variants of the active disassembly model in which not all biomolecules are shared. Our results show that, in addition to active disassembly, all biomolecules must be shared to obtain agreement with the severing experiments (see the new Appendix 3—figure 1). We have also shown that, whereas a limiting pool of IFT proteins is required to capture the experimentallyobserved injection rates, tubulin may be in excess (new Appendix 3—figure 2).
Reviewer #1:
The authors perform a theoretical study of the regulation flagellar length in Chlamydomonas based on intraflagellar transport of motors and tubulin subunits that govern assembly and disassembly. A key question is how the length of two flagella can be coordinated by exchange of components.
The paper is largely well written and interesting. First a reduced model is presented with several simplifying assumptions such as separation of timescales and simplified kinetic rules. In the context of this reduced model the coordination of two cilia is then discussed. It is shown that if a molecular component is not shared between two cilia then length can be coordinated. If all components are shared then coordination needs to be more subtle. The authors suggest that concentration dependent depolymerization could be responsible for length coordination.
This work is interesting but it also has shortcomings. Reading the Introduction, the paper makes a strong impression. However, when I worked through the main part of the paper weaknesses became apparent and the discussion seemed to be rather superficial. In the end it remains unclear what advance in our understanding the work actually achieves. In its present form I do not think that this work is suitable for publication in eLife.
We thank the reviewer for the careful reading of the manuscript and helpful feedback. Inspired by the reviewers’ comments, we have made substantial changes that we believe address the shortcomings in our original submission and significantly improve this work.
As mentioned above, we have used new agentbased simulations to reproduce the results of Hendel et al. and confirmed that their model involves an additional control mechanism of the two motor pools associated with each flagellum; in their simulations of the”severing experiment” (in which one of the two flagella is cut), the amount of IFT motors lost upon cutting is added (in silico) to the basal pool. As we show in the Author response image 1 (to be compared with Figure 6A from Hendel et al.), this assumption is required for agreement with the experiments. In effect, regeneration of the flagella to their original length after one has been severed is achieved in the model through this control mechanism that effectively monitors the total number of motors associated with each flagellum. There is no biological justification for this assumption, and it seems highly implausible as it would require the cell to count both the freely diffusing motors and those taking part in transport by IFTparticles, and to maintain this number at a fixed value. Our new results show that removing this additional control mechanism, while leading to length control of a single flagellum (as described by Hendel et al.), is incapable of regenerating equal lengths of both flagella after one is cut, in clear contradiction with experiment.
The detailed stochastic simulations performed for the new submission have led us to realize a subtlety of the Hendel et al. model of severing experiments: upon correctly accounting not only for the tubulin, but also for the motors lost upon severing, all variants of the constant disassembly model become inconsistent with the data. Our work explains why lengthdependent disassembly, not lengthdependent assembly, is the essential ingredient for length control in Chlamydomonas.
In our model this lengthdependent disassembly is achieved by ballistictodiffusive IFT motion of a depolymerizer, which results in a concentration gradient in steadystate. We have also found supporting experimental evidence for this model in published papers on Chlamydomonas reinhardtii (Piao et al., 2009) and on Leishmania major (Blaineau et al., Current Biology 17, 778–782 (2007)), which both describe the role of a microtubule depolymerizing protein (kinesin 13) in the assembly and length regulation of flagella.
Our revised submission has three main results, as described in detail later on in the response. The first result is that models in which only one protein is separate do not capture the rapid length equalization observed experimentally. This is because of the asymmetry in protein levels after severing, an effect that was initially missed by us as well as Hendel et al., and it allows us to rule out all of the constant disassembly models on a casebycase basis via their shorttime behavior.
Whereas previously we focused on the shorttime dynamics and ignored replenishment of proteins, on longertimescales the importance in Chlamydomonas of replenishing pool levels was shown by experiments that used cyclohexamide to block new protein synthesis after severing and resulted in shorter flagella (Rosenbaum et al., 1969), an effect that others had included previously in theoretical models including Hendel et al. Our second result is that protein replenishment is fundamentally inconsistent with constant disassembly, as shown by a simple analytic argument. This allows us to rule out constant disassembly models on the basis of the longtime behavior over which protein replenishment takes place.
Our third main result is that the active disassembly model we propose is consistent with both the rapid length equalization and the protein replenishment observed in severing experiments; we show that the model agrees with data from Ludington et al. (2012) and Rosenbaum et al. (1969). With these new results in hand, we feel that our theoretical work significantly advances our understanding of flagellar length control. Finally, our model provides concrete predictions for new experiments which we can now propose.
Major points:
1) The main motivation of the paper is to provide a possible explanation of the cilia severing experiment shown in Figure 1B. This is an important and interesting problem. However, the manuscript fails to really advance this issue. The authors rule out the two independent flagella (Figure 3Bi) because they cannot account for the coordinated behavior of the experiment. It seems that the proposed model of coordinated flagella shown in Figure 4 does also not really capture the key feature of coupled flagella observed in experiments: the flagellum that is not severed shrinks to almost half its original length and then both flagella grow together to their final length. In fact, Figure 4 which is a key figure of the paper is not well presented. In a severing experiment the longer flagellum should start from the steady state length which both flagella reach at long times. Another problem with Figure 4 is that only stochastic simulations are shown. It would be better to show the true average which is obtained by solving the deterministic equations. Then it would be clear if the longer flagellum first reaches a minimum before it again increases its length to reach the steady state. The stochastic simulations can be misleading as the fluctuations conceal this important feature in the behavior or even give the impression of length minima but which arise only from the noise.
We have revised Figure 4 so that it now includes all relevant aspects of the severing experiment. We have redone all figures to show that the flagellar lengths are in steadystate prior to severing. In Figure 4B we focus on the shorttime dynamics without protein replenishment and illustrate the rapid length equalization exhibited by the model. In Figure 4C we add protein replenishment as previously mentioned and show that, when the model is fit to data, it is able to capture both the shorttime behavior and the slow recovery to the original steadystate.
We have also taken to heart the reviewer’s suggestion to solve the deterministic ODE rather than showing realizations of the stochastic differential equation (SDE). We have removed the SDE formulation throughout the manuscript, and now include results from the deterministic ODE as well as new agentbased stochastic simulations.
2) It is an interesting but not deep insight (given the simplifications of the model), that if all components are shared then only the total length is fixed but individual lengths are independent. As the authors show this feature is a result of the simplifications used. Taking more realistic aspects into account, such as the concentration dependent depolymerization, this degeneracy in the model is removed. However, there are most likely other possibilities that could provide such a lift of the degeneracy. The fact that one mechanism can lift the degeneracy is interesting but does not constitute a strong result given that the model fails to qualitatively account for the experimental data.
As shown in the new Figure 4C, we are now able to show that fitting the concentrationdependent disassembly model to data yields agreement with the primary qualitative features of severing experiments.
Although as the reviewer suggests that the degeneracy of the constant disassembly model could be lifted in multiple ways, we believe there are several compelling arguments for why concentrationdependent depolymerization is the leading candidate. First, there is experimental evidence in support of our model: it is known that in the absence of IFT, depolymerization is 50fold slower [1], and a particular IFT protein, kinesin13, has been shown to act as depolymerizers in both Chlamydomonas [2] and Leishmania [3]. Second, from a theoretical perspective this model fits well with all phases of the severing experiment, in particular the length equalization after severing.
After the first stage of rapid length equalization, protein replenishment is needed for the recovery of the flagellar lengths back to their original steadystate as demonstrated by experiments of Rosenbaum et al. that use cyclohexamide to block new protein synthesis after severing and resulted in shorter flagella [4]. Protein replenishment was included in the Hendel et al. model as well. Importantly, we have now realized that replenishing proteins based on protein levels in the basal body implies that length control must include nonconstant disassembly, in contrast to the prevailing model. This can be shown by the following simple argument: the final phase of severing experiments implies a replenishment of the pools at some relatively slow timescale τ_{r}. Assuming that the cell maintains target protein concentrations in the basal body, in steadystate these concentrations will be equal to their target levels. However, in the constant disassembly model the growth rate is determined solely by basal body concentrations, so that length drops out of the steadystate equations. That is, the constant disassembly is inconsistent not only with the first phase of rapid length equalization, but also with a plausible control mechanism describing the second phase of slow recovery.
More precisely, to include external control with a target tubulin level ${\overline{T}}_{f}$ and target motor number ${\overline{M}}_{f}$ in the basal body, we include the following equations:
In steadystate, ${T}_{f}={\overline{T}}_{f}$and ${M}_{f}={\overline{M}}_{f}$so that in steadystate the assembly term $\gamma {k}_{\text{on}}{\overline{M}}_{f}{\overline{T}}_{f}$ is constant, and the only way to obtain a balance point is through lengthdependent disassembly. Note that, although here we have used a linear restoring force for simplicity, the conclusion also holds for general nonlinear restoring forces having a stable steadystate at ${T}_{f}={\overline{T}}_{f}$ and $T}_{m}={\overline{M}}_{f$.
We have included this argument in the revision.
3) When discussing the cases where one component (M or T) is not shared but exists in two different pools, it is implicitly assumed that the separate pools (e.g. T_{1} and T_{2}) are equal. However, this is not stated clearly and it is not clear why they should be equal if they are completely independent.
We thank the reviewer for this important comment. Indeed, in the case of proteins with separate pools, after severing the amounts in each pool should be different. This point was neglected by us in our original submission and by Hendel et al. Upon properly accounting for this asymmetry in protein levels after severing, we find that the constant disassembly models do not capture the rapid length equalization after severing observed experimentally. This is shown in the new Figure 3, which focuses on the shorttime behavior and neglects protein replenishment. This has allowed us to rule out both cases in which one protein is shared and the other is separate, thereby excluding all of the constant disassembly models.
Reviewer #2:
The authors have analyzed a class of models for length control of the two cilia of Chlamydomonas. The interesting result is that a naive model in which both tubulin and motor pools are shared between the two cilia doesn't work: only the summed ciliary length is determined and the individual lengths are undetermined. To get length control for two cilia, the authors add a lengthdependent depolymerization, which can be satisfied by a depolymerase whose concentration increases with length (which happens if anterograde transport of the depolymerase is advective but retrograde transport diffusive).
My main concern is: what is different/new compared to the Hendel et al. (2018) paper? Is this paper wrong, in the sense that their mechanism (which has no lengthdependent depolymerase) does not work, contrary to their claims? If this is the case, then this point must be stressed. If the Hendel et al. paper is correct, then more justification of the novelty of the current work is necessary.
We thank the reviewer for this valuable feedback. We agree that in our original submission we did not effectively distinguish our results from those of earlier papers. We have performed new agentbased simulations to better place our model into the context of the Hendel et al. (2018) model. This has led to a new and surprising result: of the five models discussed in our paper, in fact only one leads to length control consistent with the available experimental data, and it is not the one of Hendel et al. Instead, it is the model in which length control is achieved by lengthdependent depolymerization (an idea supported by previously published papers on Chlamydomonas reinhardtii [2] and Leishmania major [3], which both describe the role of a microtubule depolymerizing protein (kinesin13) in the assembly and length regulation of flagella).
As mentioned above, our revised submission has three main results. First, models in which only one protein is separate do not capture the rapid length equalization observed experimentally, which allows us to rule out all of the constant disassembly models on a casebycase basis via their shorttime behavior.
To capture this absence of length equalization, one must properly account for the asymmetry in protein levels after severing, an effect which was initially missed by us as well as Hendel et al. We have used new agentbased simulations to reproduce the results of Hendel et al. and confirmed that their model involves a control mechanism that rapidly restores the motor pools after severing. In their simulations of the severing experiment, the amount of IFT lost upon cutting is returned immediately to the basal pool. This assumption is required for agreement with the experiments, yet we know of no biological justification for this assumption, which requires the cell to count the number of motors on the flagellum and to maintain this number at a fixed value. This subtlety of simulating the severing experiments, that not only tubulin but also motors must be depleted on severing, was initially missed by us and prevented us from seeing this additional control mechanism.
Our second main result is that protein replenishment is fundamentally inconsistent with constant disassembly, as shown by the simple analytic argument in our previous response. This allows us to rule out constant disassembly models on the basis of the longtime behavior over
which protein replenishment takes place.
It may seem plausible that adding an external control mechanism that replenishes protein levels would lead to length equalization, thus resolving the issue of unequal steadystate lengths after severing. Indeed the final phase of severing experiments implies a replenishment of the pools at some relatively slow timescale τ_{r}. However, adding such a control mechanism on free proteins levels in the basal body does not lead to length equalization. Instead, in this case the limitingpool mechanism completely fails to control lengths. This can be shown by the following simple argument: assuming that the cell maintains target protein concentrations in the basal body, in steadystate these concentrations will be equal to their target levels. However, in the constant disassembly model the growth rate is determined solely by basal body concentrations, so that length drops out of the steadystate equations. That is, the constant disassembly is inconsistent not only with the first phase of rapid length equalization, but also with a plausible control mechanism describing the second phase of slow recovery.
More precisely, to include external control with a target tubulin level $\overline{T}}_{f$ and target motor number in the basal body, we include the following equations:
In steadystate, $T}_{f}={\overline{T}}_{f$and ${M}_{f}={\overline{M}}_{f}$so that in steadystate the assembly term $\gamma {k}_{\text{on}}{\overline{M}}_{f}{\overline{T}}_{f}$ is constant, and the only way to obtain a balance point is through lengthdependent disassembly. Note that, although here we have used a linear restoring force for simplicity, the conclusion also holds for general nonlinear restoring forces having a stable steadystate at ${T}_{f}={\overline{T}}_{f}$ and $T}_{m}={\overline{M}}_{f$.
We have included this argument in the revision.
Our third main result is that the active disassembly model we propose is consistent with both the rapid length equalization and the protein replenishment observed in severing experiments; we show that the model agrees with data from Ludington et al. (2012) and Rosenbaum et al. (1969). These three results and the understanding acquired through our new agentbased simulations leads us to believe that all available experimental evidence points in the direction of flagellar length being controlled by lengthdependent depolymerization (the last model discussed in our original manuscript). With these new results in hand, we feel that our theoretical work significantly advances our understanding of flagellar length control by challenging the existing paradigm of control by lengthdependent polymerization and switching focus to lengthdependent depolymerization.
We have included this in the Discussion, and made changes throughout the manuscript to reflect this improved understanding.
The following points need to be addressed in the manuscript
1) What is the concentration profile of the depolymerase? Please add this in a figure. I assume that it is a linear increasing concentration from the base (as in the Hendel paper) but I would like to see this.
We have included above the concentration profile of the depolymerase resulting from our new agentbased simulations, which is in agreement to the theoretical prediction. The concentration profile is linear, confirming the reviewer’s comment and the quasi steadystate assumption. This figure has been included in the new Figure 2B(inset) of the revision.
2) Under what conditions can depolymerases regulate a single cilium? Is a limiting tubulin pool necessary? Is a limiting tubulin pool necessary for the case of two cilia? Is a limiting pool of IFT motors necessary? The general point I am raising is that lengthdependent depolymerization is a strong assumption and perhaps it is sufficient under very broad conditions. What are they? Then this point needs to be discussed.
As the reviewer points out, unlike the constant disassembly models, in which the limitingpool mechanism is essential, concentrationdependent disassembly yields length control under mild assumptions including the case that all biomolecules are in excess. However, in severing experiments on Chlamydomonas the shortening of the unsevered flagellum shows the importance of depletion effects, and limitingpools of biomolecules are necessary to capture this effect. This is also demonstrated by experiments of Rosenbaum et al. that use cyclohexamide to block new protein synthesis after severing and result in shorter flagella [4].
Within our model this limitingpool mechanism may arise in one of three ways: (i) both tubulin and motors are limited, (ii) tubulin is limited and motors are in excess, or (iii) tubulin is in excess and motors are limited. Previously we considered only case (i) in which both biomolecules were in excess. In the new Appendix 3 section “Biomolecules in excess” we show that we may rule out case
(ii) based on experimental data on lengthdependent injection rates. The new Appendix 3—figure 2 shows that not only the previouslyconsidered case (i) but also the case (iii) in which only motors are limited is consistent with the severing experiments. Therefore, to in response to the reviewer’s question, we find while in principle depolymerases are sufficient to regulate lengths of two flagella, a limiting pool of IFT proteins is necessary for agreement with data, whereas tubulin may either be limited or in excess.
In addition to studying whether proteins may be in excess in the active disassembly model, we have also investigated how proteins present in limiting amounts may be shared between basal pools. In the new Appendix 3—figure 1, we show that if both tubulin and IFT proteins are present in limiting amounts, the basal pools of both of these biomolecules must be shared in order to obtain the rapid length equalization observed in data.
3) The stochastic aspect of the work is irrelevant as it is not used and should be removed from the paper (perhaps put into another paper).
In the new version, we have emphasized the deterministic aspect of this work by removing the Langevin (SDE) formulation, which we plan to include in a more technical followup paper. We have replaced the individual simulated SDE trajectories by the solution of the deterministic ODE, together with the results of our new agentbased stochastic simulations, and removed the fluctuation analysis from the Appendix.
Reviewer #3:
The paper describes a very interesting study of the possible models that can account for the simultaneous length control in the flagella of a singlecell organism. The study is illuminating, and shows how simple models can help to shed light on the microscopic processes, simply from the analysis of largescale dynamics. I have a few comments:
We appreciate the reviewer’s feedback and enthusiastic response. Note that we have made significant changes in response to reviewers #1 and #2 as detailed above.
1) How many MT are in each flagellum? How is this number controlled? Is this also a dynamic variable that selforganizes? The implicit assumption is that this is a constant (determined at the base, and therefore not dependent on length?), but should be explained.
The axoneme (the microtubule structure of eukaryotic cilia and flagella) has 9 outer microtubule doublets surrounded by an inner microtubule central pair (often referred to as the 9+2 structure) [6], the detailed ultrastructure of which having been recently characterized by cryoEM microscopy [7, 8]. Although there are subtle variations in structure from the base to tip, given this highly regular structure it is reasonable to treat the crosssection as fixed and lengthindependent. We have included this clarification in the revised manuscript.
2) The authors consider that the ballistic motion of the motors to the growing tip is uninterrupted, is motors and their cargo do not detach from each other or from the MT track until the tip. Is this known to be a good approximation of this system? It certainly simplifies the analysis, as they do not need to solve the spatial distribution of the density of motors, and cargo, along the flagella's length.
I would suggest discussing how this is different from models of the growth and steadystate of actinbased protrusions, where disassembly is at the base, and the length is a result of force balance (see Naoz et al., 2008; Orly et al., 2014).
The ballistic motion of the motors kinesin2 and dynein responsible for IFT is uninterrupted and processive, as evidenced by kymographs containing the trajectories of IFT cargo [9, 10]. As a proof of concept, we assume within our model that the depolymerizing protein has the same motion as kinesin2 –uninterrupted ballistic motion to the tip followed by diffusive motion to the base – resulting in a linear concentration profile. However, the ballistictodiffusive assumption is not essential for the model; so long as the depolymerizer has a concentration gradient (e.g. an exponential profile such as the one described in the papers cited by the reviewer), the conclusion of simultaneous length control holds. This is indeed an important point as it highlights the robustness of the model and allows for candidate depolymerizers with different pattern of motion e.g. intermittent rather than uninterrupted ballistic motion from the base to tip. We have highlighted this point and cited the corresponding references in the revision.
3) The question about the return current of tipdirected motors is also an open issue for myosin motors along actinfilled protrusions. Interesting dynamics and traffic jams have been observed and modeled (see Yochelis et al., 2015; Pinkoviezky and Gov, 2014, 2017), I suggest contrasting with the situation in the flagella.
We thank the reviewer for pointing out these references. It is illuminating to see some of the phenomena that arise in analogous actinmyosin systems, and we have cited this previous work in the Discussion.
4) The noise in the reactions that control the growth at the tip should give rise to a term in (1) that has noise multiplied by J and T_{f}? Would this change the dynamics?
In our revised manuscript, we have replaced the SDE model with agentbased stochastic simulations that explicitly model the fluxes in motor and tubulin numbers and their associated fluctuations. The full simulations are in good agreement with the deterministic equations and show that the dynamics is not changed significantly by the effects of multiplicative noise.
5) MTs undergo catastrophes and recoveries, and the data traces seem to show this. Does this type of dynamics matter for the model? This should be discussed.
Early studies of axonemes showed that the microtubule doublets in the axoneme are highly stable [11]. More recent work, in which the researchers were able to purify axonemal tubulin, showed that although purified axonemal tubulin has slower dynamics and lower catastrophe frequency, it still undergoes dynamic instability [12]. It is possible that posttranslational modifications are responsible for the additional stability, but this is still an area of active research.
[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]
Summary:
[…]
However, the paper also has some serious weaknesses. In particular:
1) The experimental basis for their proposal of length dependent depolymerization are much weaker than claimed. In fact, Piao et al., 2009 and 2013, do not give evidence for depolymerases at the flagellar tip in steady state.
Recent experiments in Giardia (McInally et al., 2019), which has four pairs of cilia and an IFT machinery similar to Chlamydomonas, have shown that kinesin13 is involved in length regulation via its disassembly activity and is carried by IFT proteins. This observation made us anticipate its role in the disassembly of cilia in Chlamydomonas as well. Although the work of McInally was unpublished as of our previous submission, it is now available as a bioRxiv preprint and therefore we are able to cite it freely.
However, we acknowledge that we may have been too quick to suggest kinesin13 plays a similar role in Chlamydomonas. Given the Wang et al. (2013) reference pointed out by the reviewer, which reports only negligible amounts of kinesin13 in the flagellum at steadystate, any role for kinesin13 in length control is uncertain. Therefore, we have removed statements identifying kinesin13 as the depolymerizer throughout the manuscript and instead refer to a general and as of yet unknown depolymerizer. This is discussed in the following paragraph of the revision:
“Further experiments such as the single molecule turnaround experiments pioneered recently in C. elegans (Mijalkovic et al., 2018) are needed to establish the identity of the hypothesized depolymerizer. While recent experiments have shown kinesin13 to be involved in length control in Giardia (McInally et al., 2019), the observation that only negligible amounts of flagellar kinesin13 are present at steadystate (Wang et al., 2013) appears to preclude it from being the candidate depolymerizer of our model.”
2) The claim that length dependent depolymerization is the only mechanism to account for the data is an overstatement.
We thank the reviewer for this important comment. In the revision we have narrowed the scope of our claims by more clearly stating our assumptions and by delimiting the space of models under consideration.
As we now explain in the revision:
“Herein, we limit our theoretical exploration to the space of models defined by the following processes: IFT particle assembly and injection at the flagellar base, motion of IFT proteins along the flagellum, and tubulin polymerization and depolymerization at the flagellar tip (see schematic Figure 2A). We further assume that IFT particle injection satisfies firstorder chemical kinetics and allow for a control mechanism that regulates protein levels in the basal pool. Note that this model space does not include all possibilities. In particular, it does not include the timeofflight model (Wren et al., 2013), in which additional reactions affect protein state inside the flagellum (e.g. proteins enter in an activated state and deactivate at some rate).”
We have also refined our claim in the revision to say that within this model space, the models with constant disassembly break down whereas lengthdependent depolymerization allows for agreement with data:
“Within the model space outlined, our main results hold independent of these details. Notably, we find that lengthindependent disassembly of microtubules cannot account for the experimental results, whereas incorporating lengthdependent disassembly (e.g. through the ballistictodiffusive motion of a depolymerizing protein) leads to reasonable agreement with the experiments.”
3) The discussion of previous work (Hendel et al.) that did find length regulation as seen in experiments without the need of length dependent depolymerization is discussed only superficially and with a somewhat negative attitude. This previous work should be taken more seriously and discussed more carefully. It does show that length regulation does not require length dependent depolymerization. However, this seems to be resulting from different details and assumptions in the model.
The work of Hendel et al. and its predecessors including Marshall et al. (2001) and Marshall et al. (2005) serve as the intellectual foundation of our work. These models have inspired us to spend a considerable amount of time reproducing the results of Hendel et al. and understanding their assumptions, and our discussion and critiques of the model are in the spirit of building on this foundational work.
In the revision, we have elaborated on our discussion of Hendel et al. We have clarified that we have followed the simulation protocol described in their paper and are able to replicate their results using a constant disassembly model with shared tubulin and separate motors provided that the total number of motors are instantaneously replenished to their original amounts upon severing. However, whereas the Hendel et al. model does not yield length equalization in the case of no protein replenishment (see Figure 3Biii), severing experiments show that length equalization occurs even when protein synthesis is blocked using cyclohexamide (Rosenbaum et al., 1969). Note further that controlling motor number in the flagellum is not equivalent to controlling protein concentrations in the basal pool; when we modify the Hendel et al. model by replacing the control on flagellar motor number by a control on motor concentration in the flagellar base, the model breaks down as described in the Results section titled “Controlling protein levels in the basal pool is incompatible with our constant disassembly models”.
The following explanation is included in the revision:
“We remark on the differences between our model and (Hendel et al., 2018), in which simultaneous length control was obtained using a balance point model with constant disassembly. […] As shown in Results, when basal pool concentrations are controlled according to (18)–(19), there is a breakdown of length control for the constant disassembly models.”
We further explain:
“The model equations (57)–(58) [corresponding to shared tubulin pools and separate motor pools with constant disassembly] have a similar form to existing models (Marshall et al., 2005; Hendel et al., 2018), in which the assembly rates involve a factor of T − L_{1} − L_{2} and either a 1/L_{i} or 1/L^{2}_{i} dependence in the denominator, for i = 1, 2 as discussed earlier in the context of a single growing flagellum. Although the equations are similar, the absence of length equalization in our model (Figure 3Biii) contrasts with the length equalization achieved in Hendel et al. by an additional control mechanism that instantaneously replenishes the number of motors on the flagellum after severing. As noted in the Discussion, the importance of protein replenishment for the model appears to be inconsistent with experimental results (Rosenbaum et al., 1969), which show that length equalization occurs even in the absence of new protein synthesis.”
The authors should tone down their claims and more carefully relate their results to previous work. Then this paper could become a very valuable contribution that clarifies subtle but general aspects of length regulation and provides novel insights in the possible role of length dependent depolymerization.
The authors have to revise their manuscript carefully before it could be suitable for publication in eLife.
Essential revisions:
– The authors discuss a mechanism for length dependent depolymerization. However, several arguments are unclear. The authors begin with the assumption in the Introduction: "We will assume for now that the ratelimiting protein is kinesin2, which is the molecular motor responsible for transport toward the tip of the flagellum". But, then in subsection “Tubulin shared, motors shared and concentrationdependent disassembly” the authors write "although to this point we have assumed the ratelimiting protein is kinesin2, in fact the model is valid for any ratelimiting IFT protein. Therefore, in what follows we assume that the ratelimiting protein is a depolymerizer having the same motion as kinesin2, uninterrupted ballistic motion to the tip followed by diffusive motion to the base…"
The authors should clarify if there is a family of kinesins that possess both these properties, namely, anterograde transport of tubulin dimers in a directed manner along a microtubule as well as microtubule depolymerase activity. It seems no such motor is known.
We thank the reviewer for pointing out this issue and will now clarify the mechanism of depolymerizer transport we have in mind. We do not assert that a family of kinesins exists that achieves both anterograde transport of tubulin and microtubule depolymerization. Indeed, we do not know of a kinesin possessing both of these properties. Rather, the mechanism we consider to be most likely is that the depolymerizer is a nonmotile protein, which is transported ballistically to the flagellar tip as IFT cargo and diffuses back to the flagellar base. In this scenario, the ballistic speed in the anterograde direction is determined by the speed at which kinesin2 transports IFT particles. (Another possibility, discussed below in response to the reviewer comments but not the main focus of this work, is that the depolymerizer itself is motile). This clarification has been added to the revision, where we state:
“We will assume that the depolymerizer has the same motion as kinesin2 – uninterrupted ballistic motion to the tip followed by diffusive motion to the base – resulting in a linear concentration profile. This would be the case for any nonmotile protein that is transported ballistically to the flagellar tip as IFT cargo and diffuses back to the flagellar base.”
Kinesin13 is an example of such a nonmotile depolymerizer, and it is known to be transported into the flagellum via IFT (Piao, 2009). Also, it has recently been demonstrated to be involved in length control in Giardia (McInally et al., 2019). However, as mentioned above, the evidence available for Chlamydomonas does not indicate that kinesin13 is involved in length control. Therefore, although our model suggests that such a nonmotile, depolymerizing IFT protein could solve the length control problem, further investigation is needed to determine if such a protein is responsible for length control in Chlamydomonas. These qualifications have been added to the revised manuscript, where we state:
“Although our results suggest that having a depolymerizer – one which is ballistically transported to the tip and then diffuses back – provides an appealing model for simultaneous length control, such a depolymeriser has yet to be identified in Chlamydomonas.”
We have also clarified our discussion of the ratelimiting protein for IFT and its relationship to the depolymerizer. Previously, we assumed that the ratelimiting protein for IFT was the depolymerizer. However, this assumption does not cause any loss of generality; as we now explain, the same formulas are obtained with rescaled parameters in the more realistic scenario that the ratelimiting protein and the depolymerizer are different. As stated in the revision:
“In the description of the concentrationdependent disassembly model in Results, for convenience we made the assumption that the depolymerizer is the ratelimiting IFT protein. […] Therefore the equations of the manuscript remain valid in this more general case upon making the identification
${d}_{0}\to {d}_{0}+{d}_{1}{c}_{0}^{\prime}$ and ${d}_{1}\to \frac{KD}{{D}^{\prime}}{d}_{1}$.”
We have also verified this result in agentbased simulations that include different populations of depolymerizer and ratelimiting IFT protein, with depolymerizers present in excess in the basal pool, carried to the flagellar tip by IFT, and diffusing back. These simulations result in simultaneous length control, as we now show in the revision in Appendix 3—figure 3A.
– In support of their claim, the authors quote the experimental observations of Piao et al. Piao et al. clearly state in their paper that kinesin13 "was transported by IFT into flagella during flagellar shortening." Further elaborating on this mechanism, Wang et al. (2013) reported that "CrKin13 was barely detectable in the flagella of steady state cells, as shown previously (Piao et al., 2009). However, during rigorous phase of flagellar assembly at 15 and 30 min after deflagellation, CrKin13 was found to be enriched in the flagella and decreased to normal level in fully assembled flagella". Thus, unlike the mechanism proposed by Fai et al. in this paper, experiments indicate very little presence of kinesin13 depolymerases in the flagella under normal circumstances. Only amputation of one of these trigger a signal that leads to rapid entry of the kinesin13 into the uncut flagellum for its depolymerization that supplies tubulins for the initial regeneration of the amputated flagellum. Thus, the roles of kinesin2 and kinesin13 and their presence or absence in the flagellum should be treated separately and cannot be represented by an all encompassing single motor. The relation to experiments and the required properties of motors should be discussed more carefully (see also next point)).
As mentioned above, recently published experimental data identified kinesin13 as a depolymerizer involved in flagellar length control in Giardia, whose four pairs of flagella have different steadystate lengths (McInally et al., 2019). When CRISPRimediated knockdown was used to deplete kinesin13 levels by approximately 60%, the flagellar lengths of each of these pairs was found to decrease by 5–20%. This led us to speculate on a similar role for kinesin13 in Chlamydomonas.
However, we acknowledge that the evidence from Piao et al. (2009) and Wang et al. (2013) appears to preclude kinesin13 from being involved in length control in Chlamydomonas. Therefore, as mentioned above, we have removed the previous statements identifying kinesin13 as the depolymerizer and refer instead to a general and as of yet unidentified depolymerizer.
We have also disentangled the roles of kinesin2 and the depolymerizer as discussed in detail in the response to the previous comment.
– Piao et al. demonstrated the depolymerase activity of kinesin13 family (and not kinesin8 family) in the microtubule disassembly in cilia. Kinesin13 diffuses along microtubules to target either end and are not known to transport tubulin. Therefore, the claim of experimental support of the model seems to be a too strong statement.
As mentioned above, we have removed the claims of experimental support for any particular depolymerizing protein and generalized our model to the case of a nonmotile depolymerizer different from the ratelimiting IFT protein and carried by IFT alongside tubulin.
– Subsection “Tubulin shared, motors shared and concentrationdependent disassembly”, the authors state "Unlike constant disassembly models in which a limitingpool mechanism is essential for length control,…" This appears to be an incorrect statement. Some constantdisassembly models, e.g., Timeofflight (TOF) model, which also assumes constant disassembly, does not assume "limiting pool". Instead TOF assumed "differential loading" of IFT particles, as indicated by the experiments of Wren et al.
We thank the reviewer for this comment and have corrected our claims. As mentioned above, we have better delimited the model space under consideration and acknowledged that mechanisms such as timeofflight fall outside the space of models we consider.
Therefore, the statement "As we shall see, the constant disassembly models do not result in the rapid length equalization observed experimentally" is an overstatement and oversimplification.
We have qualified this claim by restricting it to the model space we have explored. We have edited this statement, which now reads:
“As we shall see, the constant disassembly models within our model space do not result in the rapid length equalization observed experimentally.”
– The key assumptions of Fai et al.'s model seem to be the following: (a) a concentration gradient of the depolymerases along the length of the flagellum and (b) the local depolymerization rate is proportional to the local concentration of the depolymerases. There appears to be an additional implicit assumption: the depolymerase is nonprocessive in its depolymerase activity (not to be confused with processivity in motility). Otherwise, depolymerases loaded on to the MT plusend at a location closer to the ciliary tip may continue to depolymerize even when the MT becomes shorter and its plusend reaches points where the depolymerase concentrations are quite different.
The reviewer is correct that the assumption that depolymerase activity depends on local concentration excludes processive depolymerizers that could remove more than a few tubulin subunits from the flagellum before falling off into a deactivated state. To investigate whether this is a fundamental restriction, we have used our agentbased model to explore a mechanism of processive depolymerization, in which depolymerizers within 1 micron of the flagellar tip may bind and begin depolymerizing at a fixed rate. The depolymerization duration is randomly drawn from an exponential distribution with prescribed mean. As we now state in the revision and show in the Appendix 3—figure 3C:
“In Appendix 3—figure 3C we show the results of simulations using mean depolymerization times from 10 s (corresponding to 0.01 µm) up to 10 min (corresponding to 0.6 µm). For mean depolymerization times up to 100 s, we find the results to be qualitatively similar to the nonprocessive case, whereas for longer mean depolymerization times the steadystate lengths exhibit oscillations about the steadystate. The emergence of oscillations is not entirely surprising since processive depolymerization effectively introduces into the equations a time delay, which is known to give rise to oscillations in many contexts (Richard, 2003).”
Further, the model is not fundamentally restricted to a proportional dependence of depolymerase activity on concentration. If instead the local depolymerization rate were a nonlinear function of concentration, a Taylor series expansion may be performed as in (Klein et al., 2005) to obtain the corresponding linearized system discussed in the Appendix. We have clarified these points in the revision, where we say:
“Although here we have taken depolymerase activity to depend linearly on concentration, the model generalizes to the nonlinear case in a straightforward manner. For a local depolymerization rate that is an arbitrary function of concentration, a Taylor series expansion may be performed as in (Klein et al., 2005) to obtain the corresponding linearized system discussed in Appendix 3.”
– The possibility of a concentration gradient of the motors arose naturally already in the paper of Hendel et al. (see page 667 of Hendel et al.). However, Hendel et al., did not associate this concentration gradient with that of depolymerases, which seems to be the appropriate picture.
We thank the reviewer for this comment. To make this point clear, we have included the following in the revision:
“How proteins can develop and maintain such concentration profiles is therefore one of the key questions raised by the model. Indeed, concentration gradients (unassociated with depolymerizing activity) were observed already in (Hendel et al., 2018) as the result of ballistictodiffusive motion.”
Note that the concentrationdependent disassembly is not limited to the case of a linear concentration distribution, as we now explain below and demonstrate in the new Appendix 3—figure 3B:
“Although here as proof of principle this concentration gradient is achieved by the mechanism of ballistictodiffusive motion, our main results are independent of the detailed form of this concentration gradient and how it is generated. As shown in the new Appendix 3—figure 3B, our model allows for depolymerizers with nonlinear concentration profiles and different patterns of motion, e.g. exponential concentration distributions such as those recently observed in Giardia (McInally et al., 2019) and those generated by motile proteins that bind and unbind to cytoskeletal filaments, as theorized in the context of actinmyosin systems (Naoz et al., 2008; Orly et al., 2014; Pinkoviezky and Gov, 2014;, Pinkoviesky and Gov, 2017; Yochelis and Gov, 2017).”
We have specified the form of this exponential dependence in the following section added to the Appendix: “To show that our model allows for nonlinear concentration gradients, we set up an agent based simulations which would lead to an exponential concentration distribution, similar to (Naoz et al., 2008). […] The results are found to be qualitatively similar to the case of a linear concentration gradient, supporting our claim that concentrationdependent disassembly is able to control lengths independent of the precise form of the concentration gradient.”
– The authors present "testable predictions". However there are some problems. The experimental results already reported by Piao et al., 2009 and 2013, do not provide evidence for the presence of depolymerases at the tip in steady state. Also based on these experiments the existence of a concentration gradient of the depolymerases in the steadystate (that too, an increasing concentration towards the tip) seems to be unlikely.
The second testable prediction that the authors mention would not be a clear test for validation/refutation of their model. For all those models that depend on a dynamic balance of the rates of polymerization and depolymerization in the steadystate of the flagellum, lowering the entry flux of the IFT particles would lead to overall depolymerization although the local rate of depolymerization of the microtubules at the plus end would remain unchanged.
As mentioned above, we have replaced the references to kinesin13 by a more general and as of yet unidentified depolymerizer. We describe experiments that may be helpful for searching for likely candidates. In the revision we now state:
“In addition to our claim that the depolymerization rate is nonconstant and dependent on length, our model leads to testable predictions that may be useful in identifying candidate depolymerizers. For example, according to our model the depolymerizer active in length control is not uniformly distributed along the flagellum; its concentration increases toward the flagellar tip. This could be tested experimentally by fluorescently labeling candidate depolymerizers and studying their concentration profiles along the flagellum as recently done to characterize the concentration profile of kinesin13 in Giardia (McInally et al., 2019).”
Further, we have clarified that:
“Our model predicts that the tubulin and depolymerizer pools must both be shared for the concentrationdependent disassembly model to capture the rapid length equalization observed (Appendix 3). This illustrates the dramatic consequences in behavior that can occur when biomolecules are shared between compartments, and highlights the importance of knowing which proteins are exchanged between the basal pools in the context of flagellar length control. In particular, having a protein that is not exchanged can provide simultaneous length control by a limitingpool mechanism, but it introduces asymmetries that contradict the length equalization observed in severing experiments.”
– The slight difference in the expressions for L_{ss} derived by Hendel et al. and that of Fai et al. arises from difference in the scenarios considered by the two. The two assumptions made by Hendel et al. were clearly stated in their paper. The first assumption of "a constant source of free motor protein at the tip" and the second assumption of "the approximation in which motors that have reached the base immediately transport back to the tip" are exactly the two conditions ("no tubulin depletion" and "instantaneous ballistic motion") that Fai et al. point in reducing their result to that of Hendel et al. In this sense their derivation is only slightly improved compared to that of Hendel et al.
In fact, there are subtle differences between our expressions and those of Hendel et al., e.g. the constant term in the denominator that prevents the flux from becoming singular at L = 0 even in the instantaneous ballistic motion limit v → ∞. However, we agree that these differences are not the main results of our work. As discussed above, the key difference between Hendel et al. and our constant disassembly model with shared tubulin and separate motor pools is that whereas they effectively control the number of motors loaded on the flagellum, we only allow for control on basal protein levels (which we show is incompatible with their model).
We agree that the previous wording was confusing, as it overly emphasized the subtle differences in these formulas. For clarification, we have now added the following sentence after the discussion of the different formulas obtained by us and Hendel et al.:
“Note however that the essential difference between our model and Hendel et al. (2018) lies in their effective control mechanism on the number of motors loaded on the flagella, which is not captured by any differences in these formulas (see Discussion).”
– I find the claims of the paper that Hendel’s model is somehow incorrect and that the present manuscript solves all open issues unconvincing. Hendel et al. show a scenario where length control works, probably based on different detailed assumptions. This should be more clearly discussed and not superficially mentioned as in the paragraph three of the Discussion.
As mentioned above, we have more clearly delineated why the control mechanism used by Hendel et al. to maintain a constant number of motors loaded on the flagellum falls outside our scope of models, which only allows for control mechanisms on protein levels in basal pools. Moreover, we have shown that seemingly analogous models that would fall within our model space, e.g. those that control basal protein concentrations, or those in which all biomolecules are shared, do not achieve simultaneous length control under the constant disassembly assumption.
– The authors discuss the case where molecular components stabilize their levels (Equations 18 and 19) and point out the problem that in steady state such a relaxation to fixed levels breaks length regulation. It remains unclear if that is also a problem for the proposed model based on length dependent depolymerization. Also, it would be important to know what happens when the motors alone are relaxing to a fixed level but the tubulin is limited and fixed. Would that be similar to the results of Hendel et al.?
Unlike the constant disassembly models we have considered, the depolymerizer model leads to a unique steadystate length in the presence of protein replenishment. This is demonstrated in Figure 4C and in Figure 4—video 2, as we now state in the revision:
“Another feature of the concentrationdependent disassembly model is that it allows for an external control mechanism on protein levels in the basal pool, unlike the constant disassembly models we have considered. As shown in Figure 4C and Figure 4—video 2, upon including protein replenishment via eq. (18)–(19) on a timescale of τ_{r} = 10 mins, the recovery of the flagella back to their original lengths is in reasonable agreement with experimental data.”
Note that in the absence of protein replenishment, the depolymerizer model yields rapid length equalization (Figure 4B) whereas the constant disassembly model comparable to Hendel et al. does not (Figure 3Biii).
The case of motors replenishing to a fixed level but tubulin limited and fixed does not reduce to the model of Hendel et al.: in their simulations, Hendel et al. replenish tubulin to restore the flagellar lengths back to their initial values.
– The authors seem to miss relevant references: Varga et al. (2009); Klein et al. (2005); Johann et al. (2012). These should be cited and their consequences for this paper should be discussed.
We thank the reviewers for pointing out these references. We have discussed their relevance to the paper as follows in the revision:
“Although here we have taken depolymerase activity to depend linearly on concentration, the model generalizes to the nonlinear case in a straightforward manner. […] The aggregation of motile depolymerizing proteins has also been demonstrated in previous theoretical studies of microtubule length control (Klein et al., 2005; Johann et al., 2012; Reese et al., 2014); note however that these previous works differed from our model of flagellar IFT in that they considered isolated microtubules surrounded by a constant concentration bath and/or significant steric interactions between motile proteins.”
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https://doi.org/10.7554/eLife.42599.sa2Article and author information
Author details
Funding
National Science Foundation (CAREER 1752024)
 Ariel Amir
National Science Foundation (DMS1502851)
 Thomas G Fai
National Science Foundation (DMR1610737)
 Jane Kondev
National Science Foundation (MRSEC1420382)
 Jane Kondev
National Science Foundation (PHY1806818)
 Prathitha Kar
Alfred P. Sloan Foundation
 Ariel Amir
Kavli Foundation
 Ariel Amir
Simons Foundation
 Lishibanya Mohapatra
 Jane Kondev
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Acknowledgements
We acknowledge useful discussions with Wallace Marshall, William Ludington, Shashank Shekhar, and Shane Mcinally. We thank Jie Lin and Ethan Levien for reading a draft of this manuscript and offering helpful feedback. We acknowledge support from National Science Foundation grants DMS1502851 (TGF), grant PHY1806818 (PK), grant DMR1610737 (JK), MRSEC1420382 (JK), the Simons Foundation (JK, LM), the A P Sloan Foundation (AA), NSF CAREER award 1752024 (AA), and the Kavli Institute (AA).
Senior Editor
 Naama Barkai, Weizmann Institute of Science, Israel
Reviewing Editor
 Frank Jülicher, Max Planck Institute for the Physics of Complex Systems, Germany
Publication history
 Received: October 5, 2018
 Accepted: October 8, 2019
 Accepted Manuscript published: October 9, 2019 (version 1)
 Version of Record published: November 19, 2019 (version 2)
 Version of Record updated: December 4, 2019 (version 3)
Copyright
© 2019, Fai et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
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