Kinesin-4 KIF21B limits microtubule growth to allow rapid centrosome polarization in T cells
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Summary: This is a very interesting study addressing the question of microtubule cytoskeleton reorganization in the immunological synapse. Specifically, the work demonstrates the contribution of KIF21B for the control of the T cell microtubule (MT) network required for T cell polarization during immunological synapse formation. The authors use a variety of microscopy techniques, including expansion microscopy, controlled perturbations of the cell, and computer simulations to generate their results. The authors show that knockout of KIF21B results in longer MTs that result in an inability to polarise the MT network by a mechanism consistent with dynein motor function at the immunological synapse to capture long MTs and center the MT aster at the synapse. They use the Jurkat cell line, which is a classical model for this step in immune synapse function and fully appropriate. They show that KIF21B-GFP can rescue the knockout phenotype and then use this as a way to follow KIF12B dynamics in the Jurkat cells. KIF21B works by inducing pausing and catastrophe, thus, more MTs are shorter when present. They also rescue the defect in the KIF21B KOs with 0.5 nM vinblastine, that directly increases catastrophes, shortens the MTs and restores MT network polarization to the synapse. As a functional surrogate they investigate lysosome positioning at the synapse, which is one of the proposed functions of this cytoskeletal polarization. The use of expansion microscopy in this system is relatively new and clearly very powerful. The modelling component adds to the story and supports the sliding model proposed by Poenie and colleagues in 2006, but cannot say that there is no component of end capture and shrinkage as proposed by Hammer and colleagues more recently. Experiments and modelling are performed to a high standard and the results advance the field.
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Abstract
When a T cell and an antigen-presenting cell form an immunological synapse, rapid dynein-driven translocation of the centrosome towards the contact site leads to reorganization of microtubules and associated organelles. Currently, little is known about how the regulation of microtubule dynamics contributes to this process. Here, we show that the knockout of KIF21B, a kinesin-4 linked to autoimmune disorders, causes microtubule overgrowth and perturbs centrosome translocation. KIF21B restricts microtubule length by inducing microtubule pausing typically followed by catastrophe. Catastrophe induction with vinblastine prevented microtubule overgrowth and was sufficient to rescue centrosome polarization in KIF21B-knockout cells. Biophysical simulations showed that a relatively small number of KIF21B molecules can restrict microtubule length and promote an imbalance of dynein-mediated pulling forces that allows the centrosome to translocate past the nucleus. We conclude that proper control of microtubule length is important for allowing rapid remodeling of the cytoskeleton and efficient T cell polarization.
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Author Response
Author Response refers to a revised version of the manuscript, Version 2, which was posted on December 17, 2020 (https://doi.org/10.1101/2020.08.28.271643).
Summary: This is a very interesting study addressing the question of microtubule cytoskeleton reorganization in the immunological synapse. Specifically, the work demonstrates the contribution of KIF21B for the control of the T cell microtubule (MT) network required for T cell polarization during immunological synapse formation. The authors use a variety of microscopy techniques, including expansion microscopy, controlled perturbations of the cell, and computer simulations to generate their results. The authors show that knockout of KIF21B results in longer MTs that result in an inability to polarise the MT network by a mechanism consistent with dynein motor function …
Author Response
Author Response refers to a revised version of the manuscript, Version 2, which was posted on December 17, 2020 (https://doi.org/10.1101/2020.08.28.271643).
Summary: This is a very interesting study addressing the question of microtubule cytoskeleton reorganization in the immunological synapse. Specifically, the work demonstrates the contribution of KIF21B for the control of the T cell microtubule (MT) network required for T cell polarization during immunological synapse formation. The authors use a variety of microscopy techniques, including expansion microscopy, controlled perturbations of the cell, and computer simulations to generate their results. The authors show that knockout of KIF21B results in longer MTs that result in an inability to polarise the MT network by a mechanism consistent with dynein motor function at the immunological synapse to capture long MTs and center the MT aster at the synapse. They use the Jurkat cell line, which is a classical model for this step in immune synapse function and fully appropriate. They show that KIF21B-GFP can rescue the knockout phenotype and then use this as a way to follow KIF12B dynamics in the Jurkat cells. KIF21B works by inducing pausing and catastrophe, thus, more MTs are shorter when present. They also rescue the defect in the KIF21B KOs with 0.5 nM vinblastine, that directly increases catastrophes, shortens the MTs and restores MT network polarization to the synapse. As a functional surrogate they investigate lysosome positioning at the synapse, which is one of the proposed functions of this cytoskeletal polarization. The use of expansion microscopy in this system is relatively new and clearly very powerful. The modelling component adds to the story and supports the sliding model proposed by Poenie and colleagues in 2006, but cannot say that there is no component of end capture and shrinkage as proposed by Hammer and colleagues more recently. Experiments and modelling are performed to a high standard and the results advance the field.
We thank the reviewers for their thoughtful and constructive suggestions, and for the positive feedback.
Reviewer #1:
This is an excellent study of centrosome polarization in the process of establishing immunological synapse and the effect of kinesin-4 on this process. The authors use a variety of microscopy techniques and controlled perturbations of the cell to obtain beautiful images that clearly suggest that kinesin-4, by increasing frequency of pauses and subsequent MT catastrophes, limits MT length, which assists dynein pulling in polarizing the centrosome. They complement the experiments with modeling based on Cytosim; the model supports the conclusions from the data, and suggests some interesting ideas.
I am not an expert in experimental techniques, though I understand what's been done, and in my limited opinion, the results are first-rate. The paper is well written and accurate. Modeling, which I know intimately, is done very well.. I have just a few minor comments:
- I was not quite clear what does the modeling say about the centrosome sometimes being in apical position, and sometimes half-way between apical and basal positions.
The model predicts the centrosome to be either in an apical or a basal position, while in the experimental data from the KIF21B knockout cells, it can be polarized halfway. Our results indicate that in the knockout cells, the MT network is under a constant force pointed towards the synapse. This force can lead to major deformations of the nucleus and the centrosome can indent the nucleus. This indentation allows the centrosome to be located at a position half-way between an apical and a basal position. In our simulations, we assume that the nucleus is relatively stiff and cannot change size or shape. Therefore, we only find centrosomes at the apical or basal side. To clarify this point, we added a text to the 6th paragraph of the Discussion:
“Our simulations suggest that when centrosome translocation is impaired, the MT network is experiencing balanced forces. As a consequence, we predict that in these situations one would observe major deformations of the nucleus because it is trapped in a contracting cage of MTs spanning between the centrosome and the synapse. These deformations could also allow the centrosome to be located half-way between an apical and a basal position of the cell (Figure 4H). In our simulations, we assume a relatively stiff nucleus and therefore we only find the centrosome in an apical or basal position. It could be also possible that nuclear deformations push MTs towards the synapse, where they form dense peripheral MT bundles to accommodate the least curvature (Figure 2A and B).”
- I understand that 2d modeling cannot address this issue explicitly, but can the authors speculate about the apparent ring of MTs along the periphery of the synapse in the non-polarized case?
The MTs in the non-polarized case of some of the panels in Figure 2 and S2B are densely located along the periphery of the synapse. This could indicate that dynein-mediated force generation actively binds these MTs to the synapse plane through multiple motors. Another option could be that these systems are force-balanced, and thus the nucleus is experiencing a downward force. The deformable nucleus would then push all surrounding MTs down into the synapse plane as well, creating this phenomenon of MT alignment along the synapse plane. From our current data, we cannot distinguish the two processes. However, we added a text on the deformability of the nucleus to the 6th paragraph of the Discussion (page 19 of the revised paper):
“Our simulations suggest that when centrosome translocation is impaired, the MT network is experiencing balanced forces. As a consequence, we predict that in these situations one would observe major deformations of the nucleus because it is trapped in a contracting cage of MTs spanning between the centrosome and the synapse. These deformations could also allow the centrosome to be located half-way between an apical and a basal position of the cell (Figure 4H). In our simulations, we assume a relatively stiff nucleus and therefore we only find the centrosome in an apical or basal position. It could be also possible that nuclear deformations push MTs towards the synapse, where they form dense peripheral MT bundles to accommodate the least curvature (Figure 2A and B)."
- My perhaps most significant comment: the model nicely integrates and explains the data, but is it predictive? A detailed model like that clearly can generate some nontrivial prediction that could be experimentally tested.
As recognized by the reviewer, the main focus of our model was to “integrate and explain the data”. Nonetheless, we can draw at least two nontrivial predictions from the model. A strong prediction with important consequences is the length regulation of MTs by only a small number of KIF21B molecules. This length regulation mechanism could be tested in a reconstituted in vitro system in which the dependence on the number of KIF21B molecules can be systematically changed, or by exact quantification of KIF21B units through fluorescent labeling. This prediction could also potentially be tested in vivo, by the rescue of KIF21B knockout with KIF21B-GFP at different expression levels. However, these experimental validations of the small number of involved KIF21B molecules are very laborious and beyond the scope of this study. The second prediction is related to the KIF21B knockout system. In such a system the centrosome is not repositioned to the synapse. Our simulations suggest that in this case, the MT network is under constant force, but not able to rearrange. Therefore, we predict strong deformations of the nucleus by the MT network. However, we did not directly investigate such deformations in our simulations in which the nucleus is a rather stiff object. To emphasize the predictions from our model, we added the following text in the 4th paragraph of the Discussion (see above).
- "Interestingly, in our simulations, a small number of KIF21B motors was sufficient to prevent the overgrowth of the MT network." - this is a bit counter-intuitive: if the motor number is less than MT number, how would this work? Or, by a "small number of KIF21B motors" you mean still greater than ~ 100?
We agree with the referee that at first sight, it may seem counterintuitive that 10 KIF21B motors can regulate 100 MTs. Key is to realize that length regulation by KIF21B is a very dynamic process. The motor binds to a MT, induces its shrinkage, detaches, and is ready to bind to a different MT. If this happens in about 10s, 10 motors can induce shrinkage of 100 MTs in about 100s. A single motor molecule can thus initiate shrinkage of several different MTs within a short time. To clarify this point, we added a text as explained above in the answer to the second major concern raised by the reviewers.
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Reviewer #2:
This is a fascinating study demonstrating the role of KIF21B in control of T cell microtubule network required for T cell polarization during immunological synapse formation. The authors show that knockout of KIF21B results in longer microtubules that result in an inability to move the polarise the MT network by a mechanism consistent with dynein motor function at the immunological synapse to capture long MT and center the MT aster at the synapse. They use the Jurkat cell line, which is a classical model for this step in immune synapse function and fully appropriate. They show that KIF21B-GFP can rescue the knockout phenotype and then use this as a way to follow KIF12B dynamics in the Jurkat cells. KIF21B works by binding to the + end and inducing pausing and catastrophe, thus, more MT that are shorter when present. They …
Reviewer #2:
This is a fascinating study demonstrating the role of KIF21B in control of T cell microtubule network required for T cell polarization during immunological synapse formation. The authors show that knockout of KIF21B results in longer microtubules that result in an inability to move the polarise the MT network by a mechanism consistent with dynein motor function at the immunological synapse to capture long MT and center the MT aster at the synapse. They use the Jurkat cell line, which is a classical model for this step in immune synapse function and fully appropriate. They show that KIF21B-GFP can rescue the knockout phenotype and then use this as a way to follow KIF12B dynamics in the Jurkat cells. KIF21B works by binding to the + end and inducing pausing and catastrophe, thus, more MT that are shorter when present. They also rescue the defect in the KIF21B Kos with 0.5 nM vinblastine, that directly increases catastrophes, shortens the MT and restores MT network polarization to the synapse. As a functional surrogate they investigate lysosome positioning at the synapse, which is one of the proposed functions of this cytoskeletal polarization. The use of expansion microscopy in this system is relatively new and clearly very powerful. The modelling component adds to the story and supports the sliding model proposed by Poenie and colleagues in 2006, but cannot say that there is no component of end capture and shrinkage as proposed by Hammer and colleagues more recently.
-
Reviewer #1:
This is an excellent study of centrosome polarization in the process of establishing immunological synapse and the effect of kinesin-4 on this process. The authors use a variety of microscopy techniques and controlled perturbations of the cell to obtain beautiful images that clearly suggest that kinesin-4, by increasing frequency of pauses and subsequent MT catastrophes, limits MT length, which assists dynein pulling in polarizing the centrosome. They complement the experiments with modeling based on Cytosim; the model supports the conclusions from the data, and suggests some interesting ideas.
I am not an expert in experimental techniques, though I understand what's been done, and in my limited opinion, the results are first-rate. The paper is well written and accurate. Modeling, which I know intimately, is done very …
Reviewer #1:
This is an excellent study of centrosome polarization in the process of establishing immunological synapse and the effect of kinesin-4 on this process. The authors use a variety of microscopy techniques and controlled perturbations of the cell to obtain beautiful images that clearly suggest that kinesin-4, by increasing frequency of pauses and subsequent MT catastrophes, limits MT length, which assists dynein pulling in polarizing the centrosome. They complement the experiments with modeling based on Cytosim; the model supports the conclusions from the data, and suggests some interesting ideas.
I am not an expert in experimental techniques, though I understand what's been done, and in my limited opinion, the results are first-rate. The paper is well written and accurate. Modeling, which I know intimately, is done very well.. I have just a few minor comments:
I was not quite clear what does the modeling say about the centrosome sometimes being in apical position, and sometimes half-way between apical and basal positions.
I understand that 2d modeling cannot address this issue explicitly, but can the authors speculate about the apparent ring of MTs along the periphery of the synapse in the non-polarized case?
My perhaps most significant comment: the model nicely integrates and explains the data, but is it predictive? A detailed model like that clearly can generate some nontrivial prediction that could be experimentally tested.
"Interestingly, in our simulations, a small number of KIF21B motors was sufficient to prevent the overgrowth of the MT network." - this is a bit counter-intuitive: if the motor number is less than MT number, how would this work? Or, by a "small number of KIF21B motors" you mean still greater than ~ 100?
-
Summary: This is a very interesting study addressing the question of microtubule cytoskeleton reorganization in the immunological synapse. Specifically, the work demonstrates the contribution of KIF21B for the control of the T cell microtubule (MT) network required for T cell polarization during immunological synapse formation. The authors use a variety of microscopy techniques, including expansion microscopy, controlled perturbations of the cell, and computer simulations to generate their results. The authors show that knockout of KIF21B results in longer MTs that result in an inability to polarise the MT network by a mechanism consistent with dynein motor function at the immunological synapse to capture long MTs and center the MT aster at the synapse. They use the Jurkat cell line, which is a classical model for this step in immune …
Summary: This is a very interesting study addressing the question of microtubule cytoskeleton reorganization in the immunological synapse. Specifically, the work demonstrates the contribution of KIF21B for the control of the T cell microtubule (MT) network required for T cell polarization during immunological synapse formation. The authors use a variety of microscopy techniques, including expansion microscopy, controlled perturbations of the cell, and computer simulations to generate their results. The authors show that knockout of KIF21B results in longer MTs that result in an inability to polarise the MT network by a mechanism consistent with dynein motor function at the immunological synapse to capture long MTs and center the MT aster at the synapse. They use the Jurkat cell line, which is a classical model for this step in immune synapse function and fully appropriate. They show that KIF21B-GFP can rescue the knockout phenotype and then use this as a way to follow KIF12B dynamics in the Jurkat cells. KIF21B works by inducing pausing and catastrophe, thus, more MTs are shorter when present. They also rescue the defect in the KIF21B KOs with 0.5 nM vinblastine, that directly increases catastrophes, shortens the MTs and restores MT network polarization to the synapse. As a functional surrogate they investigate lysosome positioning at the synapse, which is one of the proposed functions of this cytoskeletal polarization. The use of expansion microscopy in this system is relatively new and clearly very powerful. The modelling component adds to the story and supports the sliding model proposed by Poenie and colleagues in 2006, but cannot say that there is no component of end capture and shrinkage as proposed by Hammer and colleagues more recently. Experiments and modelling are performed to a high standard and the results advance the field.
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