Author response:
The following is the authors’ response to the previous reviews
Reviewer #1 (Public review):
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".
A definition has been added to the Introduction.
(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 nonradial 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".
We now add a discussion of this point, which we believe could be a manifestation of 3D shape of a beta cell in tissue and/or compensatory mechanisms in organisms.
(3) Authors should include in the Discussion a paragraph discussing the fact that small changes in MT configuration can have strong effects.
A paragraph added to the discussion.
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
(1) Figure 1: Even though the reviewer appreciates that minor changes of MT configuration have severe effects, still the overall effects appear minor (40 vs. <50% or 35% vs. around 28%). Notably, there are no statistically significant differences in the different groups in Fig. 1Suppl-Fig.1 D. This reviewer is not sure if the combination of many not significantly different data points can result in significant changes and this should be checked by a statistician. Authors should include in the Discussion a paragraph discussing the fact that small changes in MT configuration can have strong effects.
We have now added the requested paragraph to the discussion. Indeed, the differences are small, and the significance is only detected in a data set with a large sample size in Fig. 1J,K (combined data sets with smaller sizes from Fig. 1-Suppl-Fig.1 D), consistent with the fact that a larger sample size generally provides more power to detect an effect.
(2) Unfortunately, the authors cannot block kinesin-1 resulting in microtubule accumulation in the cell center and then release the block (best inhibiting microtubule formation), to show that the MTs accumulated in the cell center will be transported to the periphery.
This is indeed the case at the moment, yes.
Minor comments:
- Abstract: β-cells vs. β cells (and throughout the manuscript)
- Page 4: "MTOC, the Golgi, (Trogden et al. 2019), and"
- Page 5: "β-cell specific"
- MT-sliding vs. MT sliding
- Kinesin 1 vs. kinesin-1
- Page 6, line 1: "β cells. actively"
- Page 7: "a microtubule probe", should be "MT"
- Page 9: "1μm" vs. "1 μm"
- Page 10: "demonstrate a dramatic effect" recommended is: "demonstrate a marked effect"
- Page 13, line 1: dramatically vs. markedly
- Page 13, line 5: "50μm" vs. "50 μm" (in general, there should be a space between number and unit?)
- "37 degrees C" vs. "37{degree sign}C"
- Animal protocol number?
- "Mice were euthanized by isoflurane inhalation"? What concentration? How long? More details are needed (no cervical dislocation?).
- Antibodies: more identifiers are needed.
- Antibody information in Key reagents and under 5. Reagents and antibodies do not fit (1:500 and 1:1000).
Thank you, we corrected all relevant information now.
