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

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 KIF5B-dependent mechanism regulating microtubule sliding plays a major role in controlling insulin secretion.

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?

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.

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.

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.

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).

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?

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.

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.