Organization and dynamics of the cortical complexes controlling insulin secretion in β-cells

ABSTRACT Insulin secretion in pancreatic β-cells is regulated by cortical complexes that are enriched at the sites of adhesion to extracellular matrix facing the vasculature. Many components of these complexes, including bassoon, RIM, ELKS and liprins, are shared with neuronal synapses. Here, we show that insulin secretion sites also contain the non-neuronal proteins LL5β (also known as PHLDB2) and KANK1, which, in migrating cells, organize exocytotic machinery in the vicinity of integrin-based adhesions. Depletion of LL5β or focal adhesion disassembly triggered by myosin II inhibition perturbed the clustering of secretory complexes and attenuated the first wave of insulin release. Although previous analyses in vitro and in neurons have suggested that secretory machinery might assemble through liquid–liquid phase separation, analysis of endogenously labeled ELKS in pancreatic islets indicated that its dynamics is inconsistent with such a scenario. Instead, fluorescence recovery after photobleaching and single-molecule imaging showed that ELKS turnover is driven by binding and unbinding to low-mobility scaffolds. Both the scaffold movements and ELKS exchange were stimulated by glucose treatment. Our findings help to explain how integrin-based adhesions control spatial organization of glucose-stimulated insulin release.


Original submission
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Reviewer 1
Advance summary and potential significance to field This paper presents some new and interesting findings regarding the role that integrin-based adhesions play in controlling the spatial organization of glucose-stimulated insulin release. It also addresses the nature of ELKS puncta using photobleaching and single molecule imaging. That data argues that ELKS turnover is driven by its binding and unbinding to low-mobility scaffolds.

Comments for the author
Overall I liked this study. I think the topic is quite interesting, and the paper presents some new and interesting findings regarding the role that integrin-based adhesions play in controlling the spatial organization of glucose-stimulated insulin release. I found most of the data convincing. That said, I must confess that while I followed the arguments based on photobleaching and singlemolecule imaging that ELKS localization is driven by binding and unbinding to low-mobility scaffolds rather than by liquid-liquid phase separation, I might not be the best person to vet those results! One significant experimental concern relates to the LL5B knockdown data. Regarding this, the text reads "we depleted LL5β in INS-1E cells using two different siRNAs and observed a 30-40% reduction in the LL5β signal on Western blots (Fig. S2A,B). Immunofluorescence cell staining showed that LL5β-positive puncta were almost completely lost in ~30-40% of the cells ( Fig. 2A,B)." To me these results raise a couple of questions and issues. If all the cells had on average ~30-40% less LL5B, they why did only ~30-40% of cells exhibit a near complete loss of LL5B puncta? Or is it that ~30-40% of the cells had near complete knockdown of LL5B, and it was these cells that exhibited a near complete loss of LL5B? If it's the latter, the authors should show on an individual cell basis that the loss of puncta correlates with the loss of LL5B protein (by IF). My uncertainty about all this made it hard to be sure about the subsequent data on insulin secretion using the knockdown cells. Also, given the modest degree of knockdown, it seems a rescue experiment would be needed to be sure the effects seen are really due to LL5B knockdown.
Minor concerns: On page 7, the text reads "We therefore co-stained INS-1E cells for insulin and RIM, and analyzed the cells with dispersed RIM puncta, as we observed that such cells were strongly depleted of LL5β ( Fig. 2A)." Isn't the data on dispersal of RIM puncta shown in Figure 2D? On page 10, the authors state "These small clusters were distributed non-homogeneously, often showing local enrichment areas. Such areas often localized around focal adhesions at the base of stress fibers (Fig. 4D), supporting the findings described above." Can they provide some quantification of this data like they did for other, similar experiments? On page 13, the authors say in the first sentence in the Discussion "controlling the very rapid Ca2+regulated neurotransmitter secretion". I think they might need a reference here since they did not study this in their paper. Can the authors add statistical analysis for Figure 2D? In Figure 3A and elsewhere (text, figure legends), please specify the phosphorylation of FAK. It is pretty hard to see any differences in Figure 3F. Perhaps a graph like in Figure S3D would be more helpful.

Reviewer 2
Advance summary and potential significance to field Noordstra and van den Berge et al. present a thorough data sets to support the conclusions that have made in this manuscript. The experiments are presented with the proper controls. I particularly found the evidence that LLPS is not a major mechanism convincing.

Comments for the author
The only suggestion that I have at this time that will make this manuscript harder is for the authors to repeat the original over-expression experiments to test if ELKS forms liquid-like droplets. If it is from over expression, the authors should be able to separate exogenously expressing beta cells into low and high expressing groups to show if more ELKS makes liquid droplet-like structures appear. If this is the case, then much of the experiments in the LLPS may need to be re-examined.

Author response to reviewers' comments
We thank the reviewers for their supportive feedback. We have revised the manuscript in light of their comments, and textual changes are indicated in blue in the Revised manuscript.

Reviewer 1 Comments for the Author:
Overall, I liked this study. I think the topic is quite interesting, and the paper presents some new and interesting findings regarding the role that integrin-based adhesions play in controlling the spatial organization of glucose-stimulated insulin release. I found most of the data convincing. That said, I must confess that while I followed the arguments based on photobleaching and single-molecule imaging that ELKS localization is driven by binding and unbinding to low-mobility scaffolds rather than by liquid-liquid phase separation, I might not be the best person to vet those results! One significant experimental concern relates to the LL5B knockdown data. Regarding this, the text reads "we depleted LL5β in INS-1E cells using two different siRNAs and observed a 30-40% reduction in the LL5β signal on Western blots (Fig. S2A,B). Immunofluorescence cell staining showed that LL5β-positive puncta were almost completely lost in ~30-40% of the cells (Fig. 2A,B).

" To me, these results raise a couple of questions and issues. If all the cells had on average ~30-40% less LL5B, they why did only ~30-40% of cells exhibit a near complete loss of LL5B puncta? Or is it that ~30-40% of the cells had near complete knockdown of LL5B, and it was these cells that exhibited a near complete loss of LL5B? If it's the latter, the authors should show on an individual cell basis that the loss of puncta correlates with the loss of LL5B protein (by IF)
. My uncertainty about all this made it hard to be sure about the subsequent data on insulin secretion using the knockdown cells. Also, given the modest degree of knockdown, it seems a rescue experiment would be needed to be sure the effects seen are really due to LL5B knockdown.
Reply: We confirm that the latter statement of the reviewer is correct. ~30-40% of the cells had a near complete knockdown of LL5β. As a result, these cells exhibit a near complete loss of LL5β puncta. To clarify this point, and as the reviewer suggests, we included an immunofluorescence staining of LL5β and E-cadherin in LL5β knockdown cells ( Fig S2A). As E-cadherin has been shown to mediate homophilic cell adhesion between B-cells (Carvell et al., Cell Physiol Biochem. 2007;20(5):617-26), it can be used to define cell borders, which allows for analysis on an individual cell basis. This experiment clearly shows that some cells exhibit a near complete loss of LL5β puncta (highlighted by *), whereas others still express LL5β. In contrast, all cells express LL5β when transfected with control siRNA's.
We agree with the reviewer that rescue experiments would strengthen our observations. Unfortunately, after many attempts with different transfection protocols, we were not able to transfect INS-1E cells with DNA. We believe, however, that the strong correlation between phenotypes and knockdown with 2 independent siRNA's provides a solid and reproducible base for our findings.

Minor concerns:
On page 7, the text reads "We therefore co-stained INS-1E cells for insulin and RIM, and analyzed the cells with dispersed RIM puncta, as we observed that such cells were strongly depleted of LL5β ( Fig. 2A)." Isn't the data on dispersal of RIM puncta shown in Figure 2D?
Reply: We thank the reviewer for the correction. The dispersed RIM puncta, shown in figure 2A, is indeed quantified in figure 2D. We now refer to both figure 2A and 2D.
On page 10, the authors state "These small clusters were distributed non-homogeneously, often showing local enrichment areas. Such areas often localized around focal adhesions at the base of stress fibers (Fig. 4D), supporting the findings described above." Can they provide some quantification of this data like they did for other, similar experiments?
Reply: We thank the reviewer for the excellent suggestion and quantified GFP-ELKS distribution relative to focal adhesions as we have done in figure (S3D). The new data is shown in Figure S4I.
On page 13, the authors say in the first sentence in the Discussion "controlling the very rapid Ca2+-regulated neurotransmitter secretion". I think they might need a reference here since they did not study this in their paper.
Reply: We now added the correct references to the discussion.
Can the authors add statistical analysis for Figure 2D?
Reply: We added error bars representing SEMs to figure 2D (and 3I, as this is a similar quantification). To directly compare the groups, we did statistical analysis on weighted averages of the distance between nearest puncta and added additional bar graphs (Fig 2E, 3J) correlated to the distribution graphs.
In Figure 3A and elsewhere (text, figure legends), please specify the phosphorylation of FAK.
Reply: In the methods we mentioned that the antibody recognizes FAK phosphorylation on Tyr397. We now also added this to the text and figure legends.
It is pretty hard to see any differences in Figure 3F. Perhaps a graph like in Figure S3D would be more helpful.
Reply: To improve readability of the graph, and as suggested by the reviewer, we changed Fig 3F to a bar graph.

Reviewer 2 Advance Summary and Potential Significance to Field:
Noordstra and van den Berg et al. present a thorough data sets to support the conclusions that have made in this manuscript. The experiments are presented with the proper controls. I particularly found the evidence that LLPS is not a major mechanism convincing.