Testing the ion-current model for flagellar length sensing and IFT regulation
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This paper is valuable and of interest to scientists studying primary cilia/flagellar formation and regulation. It addresses how ciliary/flagellar length is controlled and whether calcium negatively regulates Intraflagellar transport (IFT) injection. The study convincingly demonstrates that calcium influx correlates with flagellar length, but calcium does not appear to work as a negative regulator of IFT injection, which challenges a previous model. The models and methods are generally sound, but some conclusions would be strengthened by additional experimental support.
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Abstract
Eukaryotic cilia and flagella are microtubule-based organelles whose relatively simple shape makes them ideal for investigating the fundamental question of organelle size regulation. Most of the flagellar materials are transported from the cell body via an active transport process called intraflagellar transport (IFT). The rate of IFT entry into flagella, known as IFT injection, has been shown to negatively correlate with flagellar length. However, it remains unknown how the cell measures the length of its flagella and controls IFT injection. One of the most-discussed theoretical models for length sensing to control IFT is the ion-current model, which posits that there is a uniform distribution of Ca 2+ channels along the flagellum and that the Ca 2+ current from the flagellum into the cell body increases linearly with flagellar length. In this model, the cell uses the Ca 2+ current to negatively regulate IFT injection. The recent discovery that IFT entry into flagella is regulated by the phosphorylation of kinesin through a calcium-dependent protein kinase has provided further impetus for the ion-current model. To test this model, we measured and manipulated the levels of Ca 2+ inside of Chlamydomonas flagella and quantified IFT injection. Although the concentration of Ca 2+ inside of flagella was weakly correlated with the length of flagella, we found that IFT injection was reduced in calcium-deficient flagella, rather than increased as the model predicted, and that variation in IFT injection was uncorrelated with the occurrence of flagellar Ca 2+ spikes. Thus, Ca 2+ does not appear to function as a negative regulator of IFT injection, hence it cannot form the basis of a stable length control system.
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Author Response
Reviewer #1 (Public Review):
Ciliary length control is a basic question in cell biology and is fascinating. Regulation of IFT via calcium is a simple model that can explain length control. In this model, ciliary elongation associates with an increase in intraciliary calcium level that leads to calcium increase at the ciliary base. Calcium increase acts to reduce IFT injection and thus ciliary assembly rate. The longer the cilia, the more increase of calcium level and the more reduction of IFT injection and thus the ciliary assembly rate. When the cilia approach the genetic defined length, the gradual reducing assembly rate eventually balances the constitutive disassembly activity. Cilia then stop elongation and a final length is achieved. This work tested this model by manipulating the calcium level in cilia by using …
Author Response
Reviewer #1 (Public Review):
Ciliary length control is a basic question in cell biology and is fascinating. Regulation of IFT via calcium is a simple model that can explain length control. In this model, ciliary elongation associates with an increase in intraciliary calcium level that leads to calcium increase at the ciliary base. Calcium increase acts to reduce IFT injection and thus ciliary assembly rate. The longer the cilia, the more increase of calcium level and the more reduction of IFT injection and thus the ciliary assembly rate. When the cilia approach the genetic defined length, the gradual reducing assembly rate eventually balances the constitutive disassembly activity. Cilia then stop elongation and a final length is achieved. This work tested this model by manipulating the calcium level in cilia by using an ion channel mutant and treatment of the cells with EGTA. In addition, IFT injection was measured before and after calcium ciliary influx. Based on the outcome of these and other experiments, it was concluded that there is no correlation between changes in calcium level and IFT injection, thus challenging the previous model. This work is well written and the experiments appear to be properly executed. It nicely showed an increase of intraciliary calcium during cilia elongation, and beautifully showed that ciliary calcium influx depends on extracellular calcium. However, I felt the current data are inadequate to support the author's conclusion.
We thank the reviewer for the positive assessment of the interest in our work, and we have performed additional experiments to address the reviewers concerns as discussed below.
The authors showed that ciliary calcium increases along with ciliary elongation, which correlates with reduction of IFT injection. Thus, this result would support that calcium increase reduces IFT injection. To test whether reducing calcium influx would alter the IFT injection, the authors used an ion channel mutant cav2. Indeed, ciliary calcium level in the mutant cilia appears to be lower compared to the control in average. After measuring ciliary calcium level and IFT injection during ciliary elongation with mathematical analysis, it was concluded that reducing ciliary calcium level did not lead to increased IFT injection, which is distinct from the control cells. Thus, the authors concluded that calcium does not act as a negative regulator of IFT injection. However, if one examines the calcium flux in Figure 3B and IFT injection in Figure 4B of cilia less than 6 micron, one may draw a different conclusion. For the mutant cilia, the calcium influx is higher than that in control cilia and IFT injection is reduced compared to the control. Thus, this analysis is the opposite of the authors' conclusion, and is supporting the previous model. There is a rapid change in ciliary assembly rate at the early stages of ciliary assembly (see Figure 1C), thus, the changes in calcium influx and IFT injection in the earlier assembly stage would be more appropriate to assess the relationship between intraciliary calcium level and IFT injection.
We thank the reviewer for raising this issue, which led us to examine the data more carefully. In looking at the numbers of cells with flagella in each length range, we became concerned that the apparently low calcium influx in shorter flagella in control cells compared to ppr2 or EGTA treatment might actually due to bias from technical issues: it is relatively difficult to image shorter flagella in our TIRF imaging setup, because shorter flagella have less flagellar surface area to attach the coverslip. The more motile the flagella are, the more likely are the cells to detach when their flagella are short, because the bending force of the flagella is strong enough to pull them away from their small area of adhesion. This effect is much stronger in control cells than in either the ppr2 mutants or EGTA treated cells, whose flagella are less motile. This led to a reduced number of cells examined with flagella shorter than 6 um (17 versus 34 for control and ppr2 cells, respectively). To overcome the difficulties and biased result, we observed more flagella in control cells. The new data has now been integrated with our previous data and shown in Figure 3. The new result shows that calcium influx in control cells is in fact higher than in the ppr2 mutant cells. So, our result is remains consistent with our conclusion, and we believe that it is not useful to analyze the shorter flagella separately.
The authors used EGTA treatment to support their conclusion. However, EGTA treatment may induce a global calcium change of the cell, the outcome may not reflect actual regulation of IFT injection by ciliary calcium influx. For example, as reported elsewhere, the change of cAMP level in the cell body and cilia has a different impact on ciliary length and hedgehog regulation. The slower assembly of cilia in EGTA treated cells may be caused by many other factors instead of sole regulation by IFT.
It is certainly possible that EGTA is affecting some process inside the cell that then indirectly affects IFT. Our experiments cannot rule this out. The fact that similar effects are seen with the ppr2 mutant argues against this idea, but again cannot rule it out. We have added the following caveat to the discussion:
"Other calcium dependent processes in the cytoplasm might also potentially address IFT, and our results cannot rule out this possibility. However, we note that the ppr2 mutant also fails to show the effect on IFT or regeneration predicted by the ion current model."
The authors only examined the impact of reducing ciliary calcium influx. To further support the authors' conclusion, it is recommended that the authors should examine IFT injection in a condition where ciliary calcium level is increased. Using calcium ionophore may not be a good choice as it may change the global calcium level. One approach to consider is using mutants of a calcium pump present in cilia.
We thank the reviewers for this suggestion. The calcium current model would predict that if a calcium pump mutant failed to export calcium, the increased calcium building up inside the flagellum should lead to decreased IFT entry and a shorter flagellar length. We found at least two calcium pumps in the published Chlamydomonas flagella proteome (Pazour et al., 2005) and ordered several mutant strains from Chlamydomonas Library Project (CLiP) which are annotated as affecting these pumps. We measured the flagellar length of these potential calcium pump mutant strains, but none showed a statistically significant difference in length relative to control cells. We have now included this data as Figure S4. Because no length change was observed, we did not perform the extremely time consuming process of constructing strains that contain these mutations along with DRC4-GCaMP and KAP-GFP.
As an alternative strategy to get at this reviewer's suggestion, we measured DRC4-GCaMP and KAP-GFP intensity in 1 mM CaCl2 treated flagella and found that CaCl2 treatment increases both the flagellar calcium level (Figure 3, see below) and IFT injection (Figure 4). This increase in IFT injection is the opposite of what the calcium current model predicts.
Based on these results, we think the calcium pump experiment is not necessary because of the following reasons. 1. These calcium pump mutants might not increase the flagellar calcium level. 2. Even if the flagellar calcium was increased in these mutants, it does not affect the flagellar length and thus our conclusions would still hold. 3. These mutant strains might still have functional calcium pumps since the existing data on calcium pumps in flagella is likely to be incomplete. 4. The CaCl2 experiment clearly increased the flagellar calcium level inside flagella, directly addressing the point that the reviewer is getting at.
The conclusion on line 272-273 may need more evidence. The authors showed that addition of 1 mM CaCl2 does not change ciliary assembly, and used this as one of the evidences to argue against the ion-current model. The addition of calcium extracellularly may not alter intracellular/intraciliary calcium level given that cells have robust systems to control calcium homeostasis. To support the authors' conclusion, one should measure the changes of calcium level in the cell/cilia or revise their conclusion.
We have now performed these measurements and have included the data in Figure 3D.
The authors showed nicely the changes in IFT properties before, during and after ciliary calcium influx and found that the intensity and frequency of IFT do not have a correlation with calcium influx though calcium influx restarts paused IFT trains for retrograde transport as previously reported (Collingride 2013). The authors again concluded that this is supporting their conclusions in that there is no correlation between IFT injection and calcium influx. However, I am not sure whether the short pulses of calcium influx at one time point would change the calcium level in the whole cilia in a significant way that would alter IFT injection at the ciliary base.
We agree that individual pulses might not have an effect on the average level of IFT injection. We were specifically trying to see if, having previously ruled out the predicted correlation at the level of average rates, there might still be a trace of the correlation for individual events.
Reviewer #2 (Public Review):
The authors use a genetically encoded calcium indicator to measure Ca in flagella to establish that Ca influx correlates with flagellar length. (Despite this correlation, there is so much noise that it is dubious that Ca level can regulate the flagella's length.) Then, they show that reduced Ca decreases the rate of IFT trains entering flagella, which ruins the ion-current model of regulating flagella's length. (Ca can still be one of the factors that sets the target length.) Ca does not seem to change the disassembly rate either. There are also no correlations between Ca influx spikes and IFT injection events. Curiously, these spikes broke pauses of retrograde IFT trains, but that still did not affect IFTs entering dynamics.
Some other possibilities like Ca regulating unloading rates are discussed and convincingly rejected.
The study ends with an interesting Discussion, which talks about other possible models, and concludes that the only model not easily rejected so far is the mechanism relying on diffusion time for kinesins from flagella to the cell body being greater in longer flagella.
The paper is well written, very thorough, contains significant results.
We thank the reviewer for this strong positive assessment.
Reviewer #3 (Public Review):
This work by Ishikawa et. al is focused on testing the hypothesis first proposed by Rosenbaum that Ca2+ levels in the primary cilia act as an internal regulator of cilia length by negatively regulating intraflagellar transport (IFT) injection and/or microtubule assembly. The authors first built a mathematical model for Ca2+ based regulation of cilia length through the activity of a Ca2+ dependent kinase. They then tested this model in the growing cilia of Chlamydomonas cells expressing an axonemal localized GCaMP. Ca2+ levels were manipulated genetically with a calcium channel deficient mutant line and with the addition of EGTA. While increases in Ca2+ levels do correlate with cilia length as expected by the model they found that IFT injection was positively correlated with IFT injection and increased axonemal stability which contradicts its potential as a mechanism for the cell to internally regulate cilia length.
Overall the conclusions of the paper are supported by their data. They greatly benefit from first establishing their model in a clear form and then experimentally interrogating the model from multiple angles in order to test its viability. The importance of cilia length to our understanding of human health has only become greater in recent history and the authors are making a significant contribution to our understanding of ciliary length regulation.
We thank the reviewer for this positive assessment, including of the relevance of the model. We have attempted to address all suggestions.
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eLife assessment
This paper is valuable and of interest to scientists studying primary cilia/flagellar formation and regulation. It addresses how ciliary/flagellar length is controlled and whether calcium negatively regulates Intraflagellar transport (IFT) injection. The study convincingly demonstrates that calcium influx correlates with flagellar length, but calcium does not appear to work as a negative regulator of IFT injection, which challenges a previous model. The models and methods are generally sound, but some conclusions would be strengthened by additional experimental support.
-
Reviewer #1 (Public Review):
Ciliary length control is a basic question in cell biology and is fascinating. Regulation of IFT via calcium is a simple model that can explain length control. In this model, ciliary elongation associates with an increase in intraciliary calcium level that leads to calcium increase at the ciliary base. Calcium increase acts to reduce IFT injection and thus ciliary assembly rate. The longer the cilia, the more increase of calcium level and the more reduction of IFT injection and thus the ciliary assembly rate. When the cilia approach the genetic defined length, the gradual reducing assembly rate eventually balances the constitutive disassembly activity. Cilia then stop elongation and a final length is achieved. This work tested this model by manipulating the calcium level in cilia by using an ion channel …
Reviewer #1 (Public Review):
Ciliary length control is a basic question in cell biology and is fascinating. Regulation of IFT via calcium is a simple model that can explain length control. In this model, ciliary elongation associates with an increase in intraciliary calcium level that leads to calcium increase at the ciliary base. Calcium increase acts to reduce IFT injection and thus ciliary assembly rate. The longer the cilia, the more increase of calcium level and the more reduction of IFT injection and thus the ciliary assembly rate. When the cilia approach the genetic defined length, the gradual reducing assembly rate eventually balances the constitutive disassembly activity. Cilia then stop elongation and a final length is achieved. This work tested this model by manipulating the calcium level in cilia by using an ion channel mutant and treatment of the cells with EGTA. In addition, IFT injection was measured before and after calcium ciliary influx. Based on the outcome of these and other experiments, it was concluded that there is no correlation between changes in calcium level and IFT injection, thus challenging the previous model. This work is well written and the experiments appear to be properly executed. It nicely showed an increase of intraciliary calcium during cilia elongation, and beautifully showed that ciliary calcium influx depends on extracellular calcium. However, I felt the current data are inadequate to support the author's conclusion.
The authors showed that ciliary calcium increases along with ciliary elongation, which correlates with reduction of IFT injection. Thus, this result would support that calcium increase reduces IFT injection. To test whether reducing calcium influx would alter the IFT injection, the authors used an ion channel mutant cav2. Indeed, ciliary calcium level in the mutant cilia appears to be lower compared to the control in average. After measuring ciliary calcium level and IFT injection during ciliary elongation with mathematical analysis, it was concluded that reducing ciliary calcium level did not lead to increased IFT injection, which is distinct from the control cells. Thus, the authors concluded that calcium does not act as a negative regulator of IFT injection. However, if one examines the calcium flux in Figure 3B and IFT injection in Figure 4B of cilia less than 6 micron, one may draw a different conclusion. For the mutant cilia, the calcium influx is higher than that in control cilia and IFT injection is reduced compared to the control. Thus, this analysis is the opposite of the authors' conclusion, and is supporting the previous model. There is a rapid change in ciliary assembly rate at the early stages of ciliary assembly (see Figure 1C), thus, the changes in calcium influx and IFT injection in the earlier assembly stage would be more appropriate to assess the relationship between intraciliary calcium level and IFT injection.
The authors used EGTA treatment to support their conclusion. However, EGTA treatment may induce a global calcium change of the cell, the outcome may not reflect actual regulation of IFT injection by ciliary calcium influx. For example, as reported elsewhere, the change of cAMP level in the cell body and cilia has a different impact on ciliary length and hedgehog regulation. The slower assembly of cilia in EGTA treated cells may be caused by many other factors instead of sole regulation by IFT.
The authors only examined the impact of reducing ciliary calcium influx. To further support the authors' conclusion, it is recommended that the authors should examine IFT injection in a condition where ciliary calcium level is increased. Using calcium ionophore may not be a good choice as it may change the global calcium level. One approach to consider is using mutants of a calcium pump present in cilia.
The conclusion on line 272-273 may need more evidence. The authors showed that addition of 1 mM CaCl2 does not change ciliary assembly, and used this as one of the evidences to argue against the ion-current model. The addition of calcium extracellularly may not alter intracellular/intraciliary calcium level given that cells have robust systems to control calcium homeostasis. To support the authors' conclusion, one should measure the changes of calcium level in the cell/cilia or revise their conclusion.
The authors showed nicely the changes in IFT properties before, during and after ciliary calcium influx and found that the intensity and frequency of IFT do not have a correlation with calcium influx though calcium influx restarts paused IFT trains for retrograde transport as previously reported (Collingride 2013). The authors again concluded that this is supporting their conclusions in that there is no correlation between IFT injection and calcium influx. However, I am not sure whether the short pulses of calcium influx at one time point would change the calcium level in the whole cilia in a significant way that would alter IFT injection at the ciliary base.
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Reviewer #2 (Public Review):
The authors use a genetically encoded calcium indicator to measure Ca in flagella to establish that Ca influx correlates with flagellar length. (Despite this correlation, there is so much noise that it is dubious that Ca level can regulate the flagella's length.) Then, they show that reduced Ca decreases the rate of IFT trains entering flagella, which ruins the ion-current model of regulating flagella's length. (Ca can still be one of the factors that sets the target length.) Ca does not seem to change the disassembly rate either. There are also no correlations between Ca influx spikes and IFT injection events. Curiously, these spikes broke pauses of retrograde IFT trains, but that still did not affect IFTs entering dynamics.
Some other possibilities like Ca regulating unloading rates are discussed and …
Reviewer #2 (Public Review):
The authors use a genetically encoded calcium indicator to measure Ca in flagella to establish that Ca influx correlates with flagellar length. (Despite this correlation, there is so much noise that it is dubious that Ca level can regulate the flagella's length.) Then, they show that reduced Ca decreases the rate of IFT trains entering flagella, which ruins the ion-current model of regulating flagella's length. (Ca can still be one of the factors that sets the target length.) Ca does not seem to change the disassembly rate either. There are also no correlations between Ca influx spikes and IFT injection events. Curiously, these spikes broke pauses of retrograde IFT trains, but that still did not affect IFTs entering dynamics.
Some other possibilities like Ca regulating unloading rates are discussed and convincingly rejected.
The study ends with an interesting Discussion, which talks about other possible models, and concludes that the only model not easily rejected so far is the mechanism relying on diffusion time for kinesins from flagella to the cell body being greater in longer flagella.
The paper is well written, very thorough, contains significant results.
-
Reviewer #3 (Public Review):
This work by Ishikawa et. al is focused on testing the hypothesis first proposed by Rosenbaum that Ca2+ levels in the primary cilia act as an internal regulator of cilia length by negatively regulating intraflagellar transport (IFT) injection and/or microtubule assembly. The authors first built a mathematical model for Ca2+ based regulation of cilia length through the activity of a Ca2+ dependent kinase. They then tested this model in the growing cilia of Chlamydomonas cells expressing an axonemal localized GCaMP. Ca2+ levels were manipulated genetically with a calcium channel deficient mutant line and with the addition of EGTA. While increases in Ca2+ levels do correlate with cilia length as expected by the model they found that IFT injection was positively correlated with IFT injection and increased …
Reviewer #3 (Public Review):
This work by Ishikawa et. al is focused on testing the hypothesis first proposed by Rosenbaum that Ca2+ levels in the primary cilia act as an internal regulator of cilia length by negatively regulating intraflagellar transport (IFT) injection and/or microtubule assembly. The authors first built a mathematical model for Ca2+ based regulation of cilia length through the activity of a Ca2+ dependent kinase. They then tested this model in the growing cilia of Chlamydomonas cells expressing an axonemal localized GCaMP. Ca2+ levels were manipulated genetically with a calcium channel deficient mutant line and with the addition of EGTA. While increases in Ca2+ levels do correlate with cilia length as expected by the model they found that IFT injection was positively correlated with IFT injection and increased axonemal stability which contradicts its potential as a mechanism for the cell to internally regulate cilia length.
Overall the conclusions of the paper are supported by their data. They greatly benefit from first establishing their model in a clear form and then experimentally interrogating the model from multiple angles in order to test its viability. The importance of cilia length to our understanding of human health has only become greater in recent history and the authors are making a significant contribution to our understanding of ciliary length regulation.
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