Glucose-stimulated KIF5B-driven microtubule sliding organizes microtubule networks in pancreatic β cells

Reviewer #1 (Public review):

This study investigates the role of microtubules (MT) in regulating insulin secretion from pancreatic islet beta cells. This is of great importance considering that controlled secretion of insulin is essential to prevent diabetes. Previously, it has been shown that KIF5B plays an essential role in insulin secretion by transporting insulin granules to the plasma membrane. High glucose activates KIF5B to increase insulin secretion resulting in cellular uptake of glucose. In order to prevent hypoglycemia, insulin secretion needs to be tightly controlled. Notably, it is known that KIF5B plays a role in MT sliding. This is important, as the authors described previously that beta cells establish a peripheral sub-membrane MT array, which is critical for withdrawal of excessive insulin granules from the secretion sites. At high glucose, the sub-membrane MT array is destabilized to allow for robust insulin secretion. Here the authors aim to answer the question how the peripheral array is formed. Based on the previously published data the authors hypothesize that KIF5B organizes the sub-membrane MT array via microtubule sliding.

General comment:
This manuscript provides data that indicate that KIF5B, like in many other cells, mediates MT sliding in beta cells to establish a non-radial sub-membrane MT array. This study is based mainly on in vitro assays and one cell line. To demonstrate the importance of KIF5B in vivo/under physiological conditions, the MT pattern and directionality in beta cells within whole isolated pancreatic islets from KIF5B KO mice was analyzed in comparison to their WT littermates. While the presented effects appear often rather small, it is important to note that small changes in MT configuration can have strong effects. However, the authors provide no link to insulin secretion and glucose uptake. Finally, it remains unclear whether a KIF5B-dependent mechanism regulating microtubule sliding plays a major role in controlling insulin secretion.

Specific comments:
(1) It is difficult to appreciate that there is a "peripheral sub-membrane microtubule array" as it is not well defined in the manuscript. This reviewer assumes that this is in the respective field clear. Yet, while it is appreciated that there is an increased amount of MTs close to the cytoplasmic membrane, the densities appear very variable along the membrane. Please provide a clear description in the Introduction what is meant with "peripheral sub-membrane microtubule array".
(2) The authors described a "consistent presence of a significant peripheral array in the C57BL/6J control mice, while the KO counterparts exhibited a partial loss of this peripheral bundle. Specifically, the measured tubulin intensity at the cell periphery was significantly reduced in the KO mice compared to their wild-type counterparts". In vitro "control cells had convoluted non-radial MTs with a prominent sub-membrane array, typical for β cells (Fig. 2A), KIF5B-depleted cells featured extra-dense MTs in the cell center and sparse receding MTs at the periphery (Fig. 2B,C)". Please comment/discuss why in vivo there are no "extra-dense MTs in the cell center".
(3) Authors should include in the Discussion a paragraph discussing the fact that small changes in MT configuration can have strong effects.

Reviewer #2 (Public review):

This elegant study provides significant and impactful insights into the factors contributing to the distinct arrangement of sub-membrane microtubules within mouse β-cells of the pancreas. The authors propose that in these cells, the motor protein KIF5B plays a crucial role in sliding existing microtubules toward the cell periphery and aligning them with one another along the plasma membrane. Furthermore, similar to other physiological features of β-cells, high glucose levels enhance this microtubule sliding process. A precise arrangement of microtubules beneath the cell membrane in β-cells is vital for the regulated secretion of pancreatic enzymes and hormones; thus, KIF5B has a significant role in pancreatic activity in both healthy conditions and diseases. The authors support their model by demonstrating that the levels of KIF5B mRNA in MIN6 cells are higher than those of other known kinesins. They show that microtubule sliding becomes less efficient when KIF5B is genetically silenced using two different short hairpin RNAs (shRNAs). Additionally, silencing of KIF5A in the same cells results in a general reorganization of microtubules throughout the cell. Specifically, while control cells exhibit a convoluted and non-radial arrangement of microtubules near the cell membrane, KIF5B-depleted cells display a sparse and less dense sub-membrane array of microtubules. Based on these findings, the authors conclude that the loss of KIF5B strongly affects the localization of microtubules to the cell periphery. Using a dominant-negative approach, the authors also demonstrate that KIF5B facilitates the sliding of microtubules by binding to cargo microtubules through the kinesin-1 tail binding domain. They present evidence suggesting that KIF5B-mediated microtubule sliding is glucose-dependent, similar to the activity levels of kinesin-1, which increase in the presence of glucose. Lastly, they show that this is glucose-dependent.

Strengths:

This study unveils a previously unexplained mechanism that regulates the specific rearrangement of microtubules beneath the cell membrane in pancreatic β-cells. The findings have significant implications because the precise regulation of the microtubule array at the secretion zone plays a critical role in controlling pancreatic function in both healthy and diseased states. The provided data supports the authors' conclusions well, and the study demonstrates the use of state-of-the-art methodologies, including quantification techniques and elegant dominant-negative experiments.

Weaknesses: None

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public Review):

This study investigates the role of microtubules in regulating insulin secretion from pancreatic islet beta cells. This is of great importance considering that controlled secretion of insulin is essential to prevent diabetes. Previously, it has been shown that KIF5B plays an essential role in insulin secretion by transporting insulin granules to the plasma membrane. High glucose activates KIF5B to increase insulin secretion resulting in the cellular uptake of glucose. In order to prevent hypoglycemia, insulin secretion needs to be tightly controlled. Notably, it is known that KIF5B plays a role in microtubule sliding. This is important, as the authors described previously that beta cells establish a peripheral sub-membrane microtubule array, which is critical for the withdrawal of excessive insulin granules from the secretion sites. At high glucose, the sub-membrane microtubule array is destabilized to allow for robust insulin secretion. Here the authors aim to answer the question of how the peripheral array is formed. Based on the previously published data the authors hypothesize that KIF5B organizes the sub-membrane microtubule array via microtubule sliding.

General comment:

This manuscript provides data that indicate that KIF5B, like in many other cells, mediates microtubule sliding in beta cells. This study is limited to in vitro assays and one cell line. Furthermore, the authors provide no link to insulin secretion and glucose uptake and the overall effects described are moderate. Finally, the overall effect of microtubule sliding upon glucose stimulation is surprisingly low considering the tight regulation of insulin secretion. Moreover, the authors state "the amount of MT polymer on every glucose stimulation changes only slightly, often undetectable…. In fact, we observe a prominent effect of peripheral MT loss only after a long-term kinesin depletion (three-four days)". This challenges the view that a KIF5Bdependent mechanism regulating microtubule sliding plays a major role in controlling insulin secretion.

(1) Our initial study was indeed done in a cell line, which is a normal approach to addressing molecular mechanisms of a phenomenon in a challenging cell model: primary pancreatic beta cells are prone to rapidly dedifferentiate outside of the organism and are hard to genetically modify. To address this reviewer’s comment, in the revised manuscript we now confirm the phenotype in beta cells within intact pancreatic islets from a KIF5B KO mouse model (New Figure 2 – Supplemental Figure 1).

(2) We agree that testing the effect of microtubule sliding on insulin secretion is an important question. Unfortunately, the experimental design needed to accomplish this task is not straighDorward. Importantly, besides microtubule sliding, KIF5B is heavily engaged in insulin granule transport, and GSIS deficiency upon KIF5B inactivation is well documented (e.g. Varadi et al 2002). In this study, we choose not to repeat this GSIS assay because of ample existing data. However, this reported GSIS deficiency could result from a combination of lack of insulin granule delivery to the periphery (previous data) and from the depletion of insulin granules from the periphery due to the loss of the submembrane MT bundle (this study and Bracey et al 2020). In order to exclusively test the role of MT sliding in secretion, a significant investment in mutant tool development would be needed. Ideally, a new mutant mouse model where insulin granule transport is allowed by MT sliding in blocked must be developed to specifically address this question. To conclude, answering this question will be the subject for another, follow-up study.

(3) We respecDully disagree with the reviewer’s opinion that the effect of MT sliding in beta cells is moderate. As MT networks go, even a slight change in MT configuration often has dramatic consequences. For example, in mitotic spindles, a tiny overgrowth of microtubule ends during metaphase, which causes them to attach to both kinetochores rather than just one, is very significant for the efficiency of chromosome segregation, causing aneuploidy and cancer. The changes in beta-cell MT networks that we are reporting are much stronger: the effect on the peripheral MT network accumulated over three days of KIF5B depletion is dramatic (Fig 2 B, C). Short-term gross MT network configurations after a single glucose stimulation are harder to detect, but MTs at the cell periphery are, in fact, destabilized and fragmented, as we and others have previously reported (Ho et al 2020, Mueller et al 2021). Preventing this MT rearrangement completely blocks GSIS (Zhu et al 2015, Ho et al 2020).

One of the most fascinating features of insulin secretion regulation is that the amount of generated insulin granules significantly exceeds the normal physiological needs for insulin secretion (~100 times more than needed). At the same time, even slightly facilitated glucose depletion can be devastating. Accordingly, the excessive insulin content of a beta cell resulted in the development of multiple levels of control, preventing excessive secretion. Our previous data suggest that the peripheral MT array provides one of those mechanisms. This study indicates that microtubule sliding is necessary to form the proper peripheral network in the long term. Short-term glucose-induced changes in the peripheral MT array likely need to be subtle to prevent over-secretion. Thus, we are not surprised that a dramatic effect of sliding inhibition is only detectable by our approaches after the changes in the MT network accumulate over time. In the revised paper, we now discuss the potential impact of peripheral MT sliding on positive and negative regulation of secretion and add a schematic model illustrating these processes.

Specific comments:

(1) Notably, the authors have previously reported that high glucose-induced remodeling of microtubule networks facilitates robust glucose-stimulated insulin secretion. This remodeling involves the disassembly of old microtubules and the nucleation of new microtubules. Using real-time imaging of photoconverted microtubules, they report that high levels of glucose induce rapid microtubule disassembly preferentially in the periphery of individual β-cells, and this process is mediated by the phosphorylation of microtubule-associated protein tau. Here, they state that the sub-membrane microtubule array is destabilized via microtubule sliding. What is the relevance of the different processes?

In this comment, the summary of our previous conclusions is correct, but the conclusion of this current study is re-stated incorrectly. Indeed, we have previously shown that in high glucose, MTs are destabilized at the cell periphery and nucleated in the cell interior. However, this current paper does not state that “the sub-membrane microtubule array is destabilized via microtubule sliding”. To answer this reviewer’s question, our data support a model where, during glucose stimulation, MT sliding within the peripheral bundle might move fragments of MTs severed by other mechanisms. Importantly, we propose that MT sliding restores the partially destabilized peripheral bundle by delivery of MTs that are nucleated at the cell interior and incorporating them into that bundle. In our overall model, three processes (destabilization, nucleation, and sliding to restore the bundle) are coordinated to maintain beta cell fitness on each GSIS cycle.

(2) On one hand the authors describe how KIF5B depletion prevents sliding and the transport of microtubules to the plasma membrane to form the sub-membrane microtubule array. This indicates KIF5B is required to form this structure. On the other hand, they describe that at high glucose concentration, KIF5B promotes microtubule sliding to destabilize the sub-membrane microtubule array to allow robust insulin secretion. This appears contradictory.

We never intended to make an impression that MT sliding destabilized the sub-membrane bundle. Apologies if there was a reason in our wording that caused this misunderstanding of our model. We propose that while the bundle is destabilized downstream of glucose signaling (e.g. due to tau phosphorylation, please see Ho et al Diabetes 2020), MT sliding remodels the bundle and thereafter rebuilds it to prevent over-secretion. In the revised manuscript, we have doublechecked the whole text to make sure that such misunderstanding is avoided.

(3) Previously, it has been shown that KIF5B induces tubulin incorporation along the microtubule shaft in a concentration-dependent manner. Moreover, running KIF5B increases microtubule rescue frequency and unlimited growth of microtubules. Notably, KIF5B regulates microtubule network mass and organization in cells (PMID: 34883065). Consequently, it appears possible that the here observed phenomena of changes in the microtubule network might be due to alterations in these processes.

We thank the reviewer for proposing this alternative explanation to the observed change in microtubule networks after KIF5B depletion. We have now directly tested this possibility. Namely, we have re-expressed the kinesin-1 motor domain in MIN6 cells depleted of KIF5B. This motor domain construct by itself is not capable of driving microtubule sliding because it lacks the tail domain. At the same time, it is known to move very efficiently at microtubules and should provide the effects as reported in the article cited by the reviewer. We found that the reexpression of the kinesin motor domain does not rescue microtubule network defects in beta cells (see new Figure 2 – Supplemental Figure 2). Thus, we conclude that the effects of kinesin depletion on the microtubule network in beta cells are due to the lack of microtubule sliding, as reported here.

(4) The authors provide data that indicate that microtubule sliding is enhanced upon glucose stimulation. They conclude that these data indicate that microtubule sliding is an integral part of glucose-triggered microtubule remodeling. Yet, the authors fail to provide any evidence that this process plays a role in insulin secretion or glucose uptake.

We would like to point out that we do not “fail” but rather choose not to overload our study by repeating insulin secretion assays in KIF5B-inactivated cells because this would not have been very informative. It has been found previously that kinesin-1 inactivation or knockout significantly attenuates insulin secretion because kinesin-1 is actively transporting insulin granules and kinesin-1 activity is enhanced under high glucose conditions (e.g. Varadi et al 2002, Cui et al., 2011, Donelan et al, 2002). That said, our current finding is very much in line with these previous data. When kinesin is depleted, two things would be happening at the same time: in the absence of sub-membrane microtubule bundle pre-existing insulin granules would be over-secreted, and new insulin would not be delivered to the periphery, both decreasing GSIS. Unfortunately, we do not have tools yet that would allow us to dissect which part of the insulin secretion defect is due to prior over-secretion (the consequence of deficient MT sliding) and which part is due to the lack of new granule delivery. We plan to develop such tools in the future and elaborate on them in a follow-up study. Here, our goal is to understand microtubule organization principles in beta cells, and we choose not to extend the scope of the current study to metabolic assays.

(5) The authors speculate that the sub-membrane microtubule array prevents the over-secretion of insulin. Would one not expect in this case a change in the distribution of insulin granules at the plasma membrane when this array is affected? Or after glucose stimulation? Notably, it has been reported that "the defects of β-cell function in KIF5B mutant mice were not coupled with observable changes in islet morphology, islet cell composition, or β-cell size" and "the subcellular localization of insulin vesicles was found to not be affected significantly by the decreased Kif5b level. The cytoplasm of both wild-type and mutant β-cells was filled with insulin vesicles. Insulin vesicle numbers per square μm were determined by counting all insulin vesicles in randomly photographed β-cells. More insulin granules were found in Kif5b knockout β-cells compared with control cells. This phenomenon is consistent with the observation that insulin secretion by β-cells is affected" whereby "Insulin vesicles (arrowheads) were distributed evenly in both mutant and control cells" (PMID: 20870970).

Quantitative analyses in the study cited by the reviewer do not include assays that would be relevant to our study. Particularly, in that study neither the amount of insulin granules at the cell periphery nor the ratio between the number of granules at the periphery and the beta cell interior has been analyzed. In addition, in our preliminary observations not shown here, insulin content in beta cells in KIF5B KO mice is highly heterogeneous, with a subpopulation of cells severely depleted of insulin. This opens a new avenue of investigation into beta cell heterogeneity, which is out of the scope of this current study. Thus, we chose to restrict this current study to microtubule organization data.

(6) Does the sub-membrane microtubule array exist in primary beta cells (in vitro and/or in vivo) and how it is affected in KIF5B knockout mice?

Yes, it does exist. In fact, we have first reported it in mouse islets (Bracey et al 2020, Ho et al 2020). Now, we report that the sub-membrane bundle is defective, and microtubules are misaligned in KIF5B KO mice (new Figure 2 – Supplemental Figure 1).

Reviewer #2 (Public Review):

In this article, Bracey et al. provide insights into the factors contributing to the distinct arrangement observed in sub-membrane microtubules (MTs) within mouse β-cells of the pancreas. Specifically, they propose that in clonal mouse pancreatic β-cells (MIN6), the motor protein KIF5B plays a role in sliding existing MTs towards the cell periphery and aligning them with each other along the plasma membrane. Furthermore, similar to other physiological features of β-cells, this process of MTs sliding is enhanced by a high glucose stimulus. Because a precise alignment of MTs beneath the cell membrane in β-cells is crucial for the regulated secretion of pancreatic enzymes and hormones, KIF5B assumes a significant role in pancreatic activity, both in healthy conditions and during diseases.

The authors provide evidence in support of their model by demonstrating that the levels of KIF5B mRNA in MIN6 cells are higher compared to other known KIFs. They further show that when KIF5B is genetically silenced using two different shRNAs, the MT sliding becomes less efficient. Additionally, silencing of KIF5A in the same cells leads to a general reorganization of MTs throughout the cell. Specifically, while control cells exhibit a convoluted and non-radial arrangement of MTs near the cell membrane, KIF5B-depleted cells display a sparse and less dense sub-membrane array of MTs. Based on these findings, the Authors conclude that the loss of KIF5B strongly affects the localization of MTs to the periphery of the cell. Using a dominant-negative approach, the authors also demonstrate that KIF5B facilitates the sliding of MTs by binding to cargo MTs through the kinesin-1 tail binding domain. Additionally, they present evidence suggesting that KIF5B-mediated MT sliding is dependent on glucose, similar to the activity levels of kinesin-1, which increase in the presence of glucose. Notably, when the glucose concentrations in the culturing media of MIN6 cells are reduced from 20 mM to 5 mM, a significant decrease in MT sliding is observed.

Strengths:

This study unveils a previously unexplained mechanism that regulates the specific rearrangement of MTs beneath the cell membrane in pancreatic β-cells. The findings of this research have implications and are of significant interest because the precise regulation of the MT array at the secretion zone plays a critical role in controlling pancreatic function in both healthy and diseased states. In general, the author's conclusions are substantiated by the provided data, and the study demonstrates the utilization of state-of-the-art methodologies including quantification techniques, and elegant dominant-negative experiments.

Weaknesses:

A few relatively minor issues are present and related to data interpretation and the conclusions drawn in the study. Namely, some inconsistencies between what appears to be the overall and sub-membrane MT array in scramble vs. KIF5B-depleted cells, the lack of details about the sub-cellular localization of KIF5B in these cells and the physiological significance of the effect of glucose levels in beta-cells of the pancreas.

We thank the reviewer for this insighDul review. In the revised version, we provided re-worded and extended interpretations and conclusions to prevent any issues or misunderstandings. We trust that while some noted apparent inconsistencies may reflect the intrinsic heterogeneity of the beta cell population, all data presented here indicate the same trend in phenotypes. In the revised version, we have provided additional cell views and, in places, alternative representative images and videos, to clear out any apparent inconsistencies. We also would like to point out that we in fact reported KIF5B localization: not surprisingly, KIF5B predominantly localized to insulin granules and the punctate staining fills the whole cytoplasm (Figure 2A, bottom panel). However, as pointed out in detail in our response to reviewer 1, we choose to leave out an extensive study of the physiological and metabolic consequences of the reported microtubule network dynamics to a follow-up study.

Reviewer #3 (Public Review):

Prior work from the Kaverina lab and others had determined that beta-cells build a microtubule network that differs from the canonical radial organization typical in most mammalian cell types and that this organization facilitates the regulated secretion of insulin-containing secretory granules (IGs). In this manuscript, the authors tested the hypothesis that kinesin-driven microtubule sliding is an underlying mechanism that establishes a sub-membranous microtubule array that regulates IG secretion. They employed knock-down and dominant-negative strategies to convincingly show microtubule sliding does, in fact, drive the assembly of the sub-membranous microtubule band. They also used live cell imaging assays to demonstrate that kinesin-mediated microtubule sliding in beta-cells is triggered by extracellular high glucose. Overall, this is an interesting and important study that relates microtubule dynamics to an important physiological process. The experiments were rigorous and well-controlled.

We truly appreciate this reviewer’ opinion.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

Figures:

(1) Figure 1:

a) Why can one not see here, and in most following images, the peripheral sub-membrane microtubule array? One can also not see an accumulation of microtubules in the cell interior.

Microtubule pattern in beta cells is variable, and the sub-membrane array is seen in the whole population to a variable extent (see directionality histogram in Figure 2E for statistics). In fact, an array of peripheral MTs parallel to the cell border is present in the example shown in Figure 1 and in all following control images. To make it clearer, we now show the pre-bleach images in Figure 1 D-F at a lower magnification, so that the differences in MT density at the cell periphery and cell center are more clearly seen: MTs lack at the periphery in KF5B-depleted but not the control cells.

b) 5 min appears to be a long time and enough time to polymerize a significant number of new microtubules.

We interpret this comment as the reviewer’s concern that in FRAP assays, fluorescently-labeled MTs moving into the bleached area might be newly polymerizing MTs rather than preexisting MT relocated into that area. However, this is not the case because newly polymerized MTs contain predominantly quenched “dark” tubulin molecules and only a small percent of fluorescent tubulin. These dim MTs are not included in MT sliding assay analysis, where a threshold for bright MTs is introduced. Now, we added more details for the quantification of these data to Materials and Methods section.

c) The overall effects appear minor. It is unclear how Fig. 1-Suppl-Fig.1, where no significant difference is shown, is translated into Figure 1 J and K showing a significant difference.

With all due respect, we do not agree that the effect is minor. Please see our response to the Public Review where we discuss the major consequences of MT defects in detail.

To answer this specific comment, we show that there are significant differences in the number of rapidly moving MTs (5-sec displacement over 0.3 µm) and in the amount of stationary MTs (5sec displacement is below 0.15 µm). There is no significant difference in the amount of slightly displaced MTs (displacements between 0.15 and 0.3 µm; the central part of the histogram). This might indicate that these slight displacements do not depend on kinesin-1 motor but rather are caused by experimental noise, pushing by moving organelles, and/or myosin-dependent forces in the cell. In the revised manuscript, we have this quantification more clearly detailed in Methods and included in Figure legends.

d) The authors utilize single molecule tracking to further strengthen their conclusion that KIF5B promotes microtubule sliding. The observed effects are weaker than the data obtained from photobleaching experiments. The videos clearly show that there is still significant movement also in KIF5B-depleted cells. If K560RigorE236A binds irreversibly to a microtubule and this microtubule is growing (not only by the addition of tubulin dimers to the plus end; see PMID: 34883065) wouldn't that also result in movement of the tagged K560RigorE236A? As KIF5B is also required in the transport of insulin granules, it should also label "interior microtubules". And in Video 2 it appears that pretty much all "labeled" microtubules are moving.

K560RigorE236A forms fiducial marks along the whole MTs lattice, as previously shown in (Tanenbaum et al., 2014). When it is bound to MT lattice, K560RigorE236A moves with the whole MT if it is being relocated. The mechanism described in (PMID: 34883065) appears to be absent or minor in beta cells (see Figure 2- Supplemental Figure 2), thus, even if this mechanism would displace already polymerized MTs, this is not happening in this cell type.

The reviewer is correct, K560RigorE236A does mark all MTs throughout a beta cell. All MTs are moving slightly in a living cell because they are pushed around by moving organelles, actin contractility, etc. MTs may also be slid by other MT-dependent motors (dynein against the membrane and such). So, it is not surprising that the MT network is “breezing,” and kinesindependent sliding is only a part of MT movement. What we show here is that the KIF5Bdependent MT sliding is responsible for a relatively “long-distance” relocation of MTs manifested in long, directional displacement of fiducial marks. This does not exclude other movements. This makes extraction of kinesin-dependent MT movements somewhat challenging, of course, that is why we needed to do those extensive analyses.

e) Figure 1 G to K is misleading, at least in the context of the provided videos. There are several microtubules that move extensively in shRNA#2-treated cells and overall there appears more movement in this cell as in the control cell. Figure 1I is clearly not representative of the movement shown in Video 2.

We apologize if our selection of representative movies/figures for this experiment was imperfect. Indeed, in all depleted cells, SunTag puncta still move to a certain extent, either due to incomplete depletion or to alternative intracellular forces dislocating microtubules. However, there is a clear difference in the fraction of persistently moving puncta (please see Figure 1K and histogram in Figure 1 - Supplemental Figure 1B). Unfortunately, when the number of SunTag puncta per a cell is variable, it sometimes prevents a good visual perception of the actual distribution of moving versus stationary microtubules. We now show an alternative representative movie for the Figure 1I and the corresponding Video 2, with a goal to compare cells with more consistent numbers of Sun-Tag puncta.

(2) Figure 2A.

a) This is the only image that clearly shows the existence of a sub-membrane microtubule array and the concentration of microtubules in the cell interior. The differences are unclear between the experimental setups including the length of cultivation and knockdown of KIF5B or expression of mutants.

We now provide a more detailed description of each image acquisition and processing in Materials and Methods. In brief, while the morphology of MT patterns is intrinsically variable in beta cells, all control cells have populated peripheral MTs that exhibit a more parallel configuration as compared to depletions and mutants.

b) The authors state "While control cells had convoluted non-radial MTs with a prominent sub-membrane array, typical for beta cells (Fig. 2A), KIF5B-depleted cells featured extra-dense MTs in the cell center and sparse reseeding MTs at the periphery (Fig. 2B, C)". Could that not be explained with the observation that "Kinesin-1 controls microtubule length" (PMID: 34883065)?

Thank you for this interesting alternative idea. It does not appear to be the case for beta cells.

Please see Figure 2-Supplemental Figure 2 and our response to Public Review Comment #3.

Also, our apologies for the typo in the original manuscript: this is “receding” nor “reseeding”.

(3) Figure 3:

a) This is an elegant way to determine whether KIF5B is involved in microtubule sliding independent of the fact that the effect appears very small.

Thank you!

b) The assay depends on ectopic expression of a dominant negative mutant. It appears important to show that KIFDNwt is high enough expressed to indeed block the binding of endogenous KIF5B. The authors need to provide a control for this. Furthermore, authors need to provide evidence that other functions of KIF5B are not impaired such as transport of insulin granules and tubulin incorporation or microtubule stability and length.

Expression of cargo-binding motor domains routinely causes a dominant-negative effect of their cargo transport. This exact construct has been used for the purpose of dominant-negative action previously (Ravindran et al., 2017). It does prevent the membrane cargo binding of KIF5B (Ravindran et al., 2017), thus the transport of insulin granules is also impaired in overexpression cells. Confirming this fact would not influence our study conclusions, so we chose not to repeat these assays for the sake of time.

c) N-numbers should be similar. The data for KIFDNmut are difficult to interpret with possibly 2 experiments showing little to no displacement and 3 showing displacement.

In the revised manuscript, additional data have been added to increase N-numbers.

(4) Figure 4 and supplements: The morphology of the KIFDNwt cells is greatly affected and this makes it difficult to say whether the effect on microtubules at the cell periphery is a direct or indirect effect.

Yes, these cells often have less spread appearance, obscuring visual perception of MT distribution. We have now replaced the image of KIFDNwt cell (Figure 4, Supplemental Figure 1 A) to a more visually representative example.

Things to do:

(1) Notably, the authors have previously reported that high glucose-induced remodeling of microtubule networks facilitates robust glucose-stimulated insulin secretion. This remodeling involves the disassembly of old microtubules and the nucleation of new microtubules. Here, they state that the sub-membrane microtubule array is destabilized via microtubule sliding. What is the relevance of the different processes? Please discuss these in the manuscript.

Thank you, we have now extended our discussion of these points and our prior findings. We have also added a schematic model figure for clarity (Figure 7).

(2) 5 min appears to be a long time and enough time to polymerize a significant number of new microtubules. Do the authors have any information about the speed of MT formation in MIN6 cells? Can the authors repeat this experiment by preventing MT polymerization? Or repeat the experiment with EB1/EB3 reporter to visualize microtubule growth in the same experimental setting?

While some MT polymerization will happen in this timeframe, newly polymerized MTs contain predominantly quenched “dark” tubulin molecules and only a small percent of fluorescent tubulin. These dim MTs are not included in MT sliding assay analysis, where a threshold for bright MTs is introduced. We apologize for initially omitting certain details from the FRAP assay analysis. Now these details have been added.

Are the microtubules shown on the cell surface (TIRF microscopy) or do we see here all microtubules?

Please see Materials and Methods for microscopy methods and image processing for each figure. Specifically, FRAP assays show a maximum intensity projection of spinning disk confocal stacks over 2.4µm in height (approximately the ventral half of a cell).

(3) Previously, it has been shown that KIF5B induces tubulin incorporation along the microtubule shaft in a concentration-dependent manner. Moreover, running KIF5B increases microtubule rescue frequency and unlimited growth of microtubules. Notably, KIF5B regulates microtubule network mass and organization in cells (PMID: 34883065). Consequently, it appears possible that the here observed phenomena of changes in the microtubule network might be due to alterations in these processes. Authors need to exclude these possibilities and discuss them.

Thank you for this interesting alternative idea. It does not appear to be the case for beta cells. Please see Figure 2-Supplemental Figure 2 and our response to Public Review Comment #3.

(4) It is important that the authors describe in the text and possibly in the figure legends the differences between the experimental set-ups including the length of cultivation and knock down of KIF5B or expression of mutants.

Thank you, please see these details in the text (Materials and Methods section).

(5) Figure 5: Does KIF5B depletion rescue the kinesore-induced defects

Thank you for suggesting this control. We have now conducted corresponding experiments. The answer is yes, it does. Kinesore does not induce detectable changes in MT patterns in KIF5Bdepleted cells (new Figure 5-Supplemental Figure 2).

(6) Can the authors block kinesin-1 resulting in microtubule accumulation in the cell center and then release the block, and best inhibiting microtubule formation, to see whether the microtubules accumulated in the cell center will be transported to the periphery?

This proposed experiment would have been a nice illustration to the study, however it has proven to be too challenging. Unfortunately we have to leave it for the future studies. However, the experiments already included in the paper are sufficient to prove our conclusions.

Minor comments:

(1) The English needs to be improved. Oaen it is unclear what the authors try to convey. The manuscript is difficult to read and contains several overstatements.

The revised manuscript has been through several rounds of proof-reading for clarity.

(2) It is important to describe in more detail in the introduction what is known about KIF5B in beta cells. Previously, it has been demonstrated that silencing, or inactivation by a dominant negative form of KIF5B, blocks the sustained phase of glucose-stimulated insulin secretion (PMID: 9112396, PMID: 12356920, PMID: 20870970).

Yes, this is of course very important and have been cited in the original manuscript. Now, we have expanded the discussion on the matter.

(3) Figure 1B and Fig. 1 Suppl Fig.1: Please provide band sizes and provide information on the size of KIF5B.

We have replaced Fig. 1B and Suppl Fig 1A with quantitative analysis of KIF5B depletion, not found in new Fig. 1B and Suppl Fig. 1A-C.

(4) It is important to state the used glucose concentrations in Figure 1D (based on the methods section it is probably 25 mM glucose) and all subsequent experiments. Is this correct and comparable to Figure 6A or B? For the non-specialized reader, more information should be provided on why initial glucose starvation is performed.

Cell culture models of pancreatic beta cells are routinely maintained at glucose levels that at considered “high”, or stimulatory for secretion. This is needed to prevent the loss of cells’ capacity to respond to glucose stimulation over generations. In order to test GSIS, cells need to be equilibrated at low (fasting, standardly 2.8mM) glucose levels for several hours, so that they are capable of secreting insulin upon glucose addition. 25mM glucose is normally used to stimulate GSIS in cell culture models of beta cells, like MIN6. This is a higher concentration as compared to what is needed to stimulate primary beta cells in islets.

Reviewer #2 (Recommendations For The Authors):

I have the following specific questions that pertain to data interpretation and the conclusions drawn.

(1) The morphology of the overall MT array before the bleach treatment in both control cells and KIF5B-KD cells depicted in Figure 1D-F and Figure 2A-C appears to be distinct. In Figure 1, it seems that the absence of KIF5B results in a general augmentation of MT mass, whereas the arrangement presented in Figure 2 indicates the contrary. Even in the sub-membrane areas, this phenomenon appears to hold true. However, the images used in this study, which depict entire cells or a significant portion of cells, may not be ideal for visualizing the sub-membrane regions.

It would be beneficial if the author could offer some explanations for this apparent inconsistency.

While beta cell population is intrinsically heterogeneous, all data presented here indicate the same trend in phenotypes. Possibly, some apparent inconsistency between figure 1 and 2 appeared because in the original manuscript we did not show the pre-bleach whole-cell overview in Figure 1. In the revised version, we now show the whole cells for pre-bleach so that MT organization at the cell periphery can be assessed. Please note that in the control cell, MTs are more or less equally distributed over the cell, while in KIF5B depletions the cell periphery is significantly less populated than the cell center. Furthermore, we did not detect MT mass augmentation or increase in KIF5B depletions. One possible explanation for such reviewer’s impression from Figure 2 is that Figure 2 F-H shows thresholded images where threshold was adjusted to highlight peripheral MTs in each cell. Please note that this is not the same threshold for each cell (see Figure 2 - Supplemental Figure 2 and 3). Thus, KIF5B-depleted cells that have fewer MTs at the periphery appear brighter in these thresholded images. For the true comparison of MT intensity, please see Figure 2 A-C (grayscale image, not the threshold).

(2) It would be helpful if the author could provide a visual representation or comment on the sub-cellular localization of KIF5B in MIN6 cells. Is it predominantly localized in the submembrane region, or is it more evenly distributed throughout the cytoplasm?

Please see Fig 2A, lower panel. KIF5B is seen across the cell as a punctate staining, in agreement with previous findings that it mostly localize at IGs.

(3) The alteration in microtubule (MT) organization and sliding in the absence of KIF5B seems to initiate in proximity to the apparent microtubule organizing center (MTOC) depicted in Figure 2A, and then "simply" extends towards the sub-membrane region. Although the authors acknowledge it, it would be advantageous for the readers to have a clearer indication that the sub-membrane microtubule (MT) reorganization in the absence of KIF5B is a result of a broader MT reorganization rather than a specific occurrence restricted to the sub-membrane regions.

Thank you for this comment. We now extend our discussion to clearer state our conclusions and interpretations of this point. We also have added a schematic Figure 7 as an illustration.

(4) Regarding the "glucose experiments," it is common to add 20-25 mM glucose to culture media, but physiological concentrations of glucose typically hover around 5 mM. Therefore, it is somewhat unclear what the implications are when investigating the impact of KIF5B depletion on MT sliding at 2.8 mM of glucose. It would be helpful if the authors could provide some commentary on this matter, particularly in relation to physiological and pathological conditions.

2.8 mM glucose is a standard low glucose condition used to model glucose deprivation/fasting. For functional primary beta cells within pancreatic islets, GSIS can be triggered by glucose stimulation as low as 8-12 mM glucose. However, for glucose stimulation of cultured beta cells such as MIN6 used in this paper, 20-25 mM glucose is standardly used because these cell lines have a higher threshold of stimulation compared to primary beta cells and whole islets.

(5) In supplementary Figure 1A, it would be helpful if the lanes in the WB were marked indicating what is what. In my observation, it appears that Supplementary Figure 1A, particularly lanes #2, 3, and 4, display the GAPDH protein (MW 36 kDa) (or is it alpha-tubulin, as mentioned in the Material and Methods section and indicated in lane #409?) relative to Figure 1A. I am curious about KIF5B (MW 108 kDa). Is it represented by the upper band? Did the author probe the same membrane simultaneously with two different primary antibodies? This should be clarified, and the author should indicate the molecular weight of the ladder.

Indeed, in the original WB two antibodies have been used together, due to a challenge in collecting a sufficient number of shRNA-expressing beta cells. It caused a confusion and improper interpretation of the loading control. We thank the reviewer for catching this. We have now replaced old Fig. 1B and Suppl. Fig. 1A with quantitative analysis of KIF5B depletion based on single-cell immunofluorescent staining. It is now found in new Fig. 1B and Suppl Fig. 1A-C.

Reviewer #3 (Recommendations For The Authors):

In all of the figures that present microtubule orientations (e.g. Figure 2E) the error bars obscure the vertical bins making them difficult to read or interpret. If they were rendered at a larger scale, it would be easier to read and interpret these results.

Thank you pointing this out. We now show these histograms with a different format of error bars and without outliers that obscure the view. A variant with outliers is now shown in the supplement.

Some of the callouts to the videos in the paper are inaccurate. Perhaps the authors reordered sections of the paper but failed to correctly renumber the video citations?

Thank you for this comment, we have corrected all callouts now.