Functional hierarchy among different Rab27 effectors involved in secretory granule exocytosis
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eLife assessment
This is a well-done study to understand how Rab27 and its effectors regulate insulin secretion. The present work examines the relative hierarchy of exophilin-8 and melanophilin using single vs double knockouts and rescue experiments to show that melanophilin functions downstream of and potentially redundantly from Exo8. Imaging and protein co-localization studies were done in a rigorous way. The data are solid, and some additional data will make the work fully compelling. Overall this is an important study that sheds new light on the regulation of insulin granule exocytosis.
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Abstract
The Rab27 effectors are known to play versatile roles in regulated exocytosis. In pancreatic beta cells, exophilin-8 anchors granules in the peripheral actin cortex, whereas granuphilin and melanophilin mediate granule fusion with and without stable docking to the plasma membrane, respectively. However, it is unknown whether these coexisting effectors function in parallel or in sequence to support the whole insulin secretory process. Here, we investigate their functional relationships by comparing the exocytic phenotypes in mouse beta cells simultaneously lacking two effectors with those lacking just one of them. Analyses of prefusion profiles by total internal reflection fluorescence microscopy suggest that melanophilin exclusively functions downstream of exophilin-8 to mobilize granules for fusion from the actin network to the plasma membrane after stimulation. The two effectors are physically linked via the exocyst complex. Downregulation of the exocyst component affects granule exocytosis only in the presence of exophilin-8. The exocyst and exophilin-8 also promote fusion of granules residing beneath the plasma membrane prior to stimulation, although they differentially act on freely diffusible granules and those stably docked to the plasma membrane by granuphilin, respectively. This is the first study to diagram the multiple intracellular pathways of granule exocytosis and the functional hierarchy among different Rab27 effectors within the same cell.
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eLife assessment
This is a well-done study to understand how Rab27 and its effectors regulate insulin secretion. The present work examines the relative hierarchy of exophilin-8 and melanophilin using single vs double knockouts and rescue experiments to show that melanophilin functions downstream of and potentially redundantly from Exo8. Imaging and protein co-localization studies were done in a rigorous way. The data are solid, and some additional data will make the work fully compelling. Overall this is an important study that sheds new light on the regulation of insulin granule exocytosis.
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Reviewer #1 (Public Review):
Rab27 is a major regulator of insulin granule exocytosis from beta cells, and it acts via (at least) three distinct effector proteins; Granuphilin, Melanophilin and Exophilin-8. Although the role of each of these three Rab-effectors in the regulation of insulin secretion is fairly well-established from studies of KO mice, the functional hierarchy between the effectors remains largely unknown. This study by Zhao et al addresses this question by investigating how simultaneous loss of two these effectors influence insulin granule exocytosis and also provide an explanation for their differential regulation of this process. They propose that Exophilin-8 acts upstream of Melanophilin, which in turn is involved in crash-fusion of granules with the plasma membrane, and that the interaction between these two …
Reviewer #1 (Public Review):
Rab27 is a major regulator of insulin granule exocytosis from beta cells, and it acts via (at least) three distinct effector proteins; Granuphilin, Melanophilin and Exophilin-8. Although the role of each of these three Rab-effectors in the regulation of insulin secretion is fairly well-established from studies of KO mice, the functional hierarchy between the effectors remains largely unknown. This study by Zhao et al addresses this question by investigating how simultaneous loss of two these effectors influence insulin granule exocytosis and also provide an explanation for their differential regulation of this process. They propose that Exophilin-8 acts upstream of Melanophilin, which in turn is involved in crash-fusion of granules with the plasma membrane, and that the interaction between these two effectors require the exocyst complex. This mode of exocytosis is relatively rare and only accounts for around 20% of all fusion events. The majority of fusion events instead involves exocytosis of granules stably docked at the plasma membrane. The authors propose that this mode of exocytosis also depends on Exophilin-8, now acting by removal of a Granuphilin-mediated exocytic clamp.
Technically, this is a superb study where the authors use primary mouse islets isolated from both single and double KO mice and perform both bulk secretion assays and single-cell granule imaging to elucidate the role of Rab27 effectors in glucose-stimulated insulin secretion. Unfortunately, while visualization of granule dynamics is performed in living cells, visualization of the Rab27 effectors and the ecoxyst components is restricted to static immunofluorescence imaging. It is therefore difficult to reconcile granule dynamics with effector action. While the results are clearly presented and largely consistent with previous work, I feel that many of the conclusions are based on over-interpretation of data and that important control experiments are missing. The authors are able to confirm their and others' previous observations that each of the three Rab27 effectors have distinct functions during insulin secretion. A connection between insulin granule exocytosis and the Exocyst complex has also been established in previous studies. The most intriguing finding in this study is that the Exocyst complex function in cooperation with Rab27 and its effectors, thus connecting these two pathways, and that there appears to be a functional hierarchy amongst the Rab27 effectors where Exophilin-8 act upstream of the other two. What remains unclear to me is how this entire process is regulated and how it relates to prevailing models of insulin granule pools and modes of exocytosis.
Understanding the mechanism that regulate insulin secretion is imperative for understanding how this process fails in certain types of diabetes. This study reinforces the concept that the secretion of insulin granules is a very heterogenous process that involves multiple pools of granules and modes of exocytosis and provides important new information on how cross-talk between these pathways help to shape the secretory response and give it robustness.
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Reviewer #2 (Public Review):
Insulin exocytosis is a tightly orchestrated process that involves proteins acting in complexes near the plasma membrane. The authors have contributed much of the field's knowledge on how exophilin anchors insulin granules in cortical actin and works with other effectors to prepare granules for exocytosis. Here they find that, while both exophilin and melanophilin localize to the exocyst, functionally they are not equivalent. TIRF imaging of monolayer dispersed beta cells, although a non-physiologic model to study islet cell secretion (which requires homotypic and heterotypic cell coupling), is nonetheless an established method that the authors have used with expert proficiency. The imaging and quantitative methods here should be broadly applicable to those studying secretory events at cellular resolution, …
Reviewer #2 (Public Review):
Insulin exocytosis is a tightly orchestrated process that involves proteins acting in complexes near the plasma membrane. The authors have contributed much of the field's knowledge on how exophilin anchors insulin granules in cortical actin and works with other effectors to prepare granules for exocytosis. Here they find that, while both exophilin and melanophilin localize to the exocyst, functionally they are not equivalent. TIRF imaging of monolayer dispersed beta cells, although a non-physiologic model to study islet cell secretion (which requires homotypic and heterotypic cell coupling), is nonetheless an established method that the authors have used with expert proficiency. The imaging and quantitative methods here should be broadly applicable to those studying secretory events at cellular resolution, and practical details e.g. the need for double transfection in RNAi experiments, are helpful and appreciated.
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Reviewer #3 (Public Review):
In this manuscript Zhao et al investigated how multiple Rab27 effectors work to regulate insulin secretion by murine pancreatic b-cells. They do this by comparing the phenotypes of b-cells/islets lacking effectors doubly or singly. Their main findings/contributions are that:
Mlph works downstream of Myrip/exophilin-8 to mobilise granules for fusion from the actin network to the plasma membrane after stimulation.
Mlph and exophilin-8 interact via the exocyst
Down-regulation of exocyst affects exocytosis in cells expressing exophilin-8
Exophilin-8 promotes fusion of granules docked by granuphilin at the membrane
Exophilin-8 not required for Grph related granule docking at the plasma membrane
A model for how the three effectors coordinate ISG secretion. According to this model there are 2 insulin secretion …
Reviewer #3 (Public Review):
In this manuscript Zhao et al investigated how multiple Rab27 effectors work to regulate insulin secretion by murine pancreatic b-cells. They do this by comparing the phenotypes of b-cells/islets lacking effectors doubly or singly. Their main findings/contributions are that:
Mlph works downstream of Myrip/exophilin-8 to mobilise granules for fusion from the actin network to the plasma membrane after stimulation.
Mlph and exophilin-8 interact via the exocyst
Down-regulation of exocyst affects exocytosis in cells expressing exophilin-8
Exophilin-8 promotes fusion of granules docked by granuphilin at the membrane
Exophilin-8 not required for Grph related granule docking at the plasma membrane
A model for how the three effectors coordinate ISG secretion. According to this model there are 2 insulin secretion pathways in b-cells; a) where Exo8 acts upstream of Mlph and with actin/Myosin Va/VIIa, exocyst and syntaxin 4 to move dock granules in actin and promote exocytosis, and b) where Exo8 works in an antagonistic manner with Grph promoting secretion of granules docked at the membrane by Grph.
This is an interesting/important question and the authors make important contributions (above). In general experiments are well designed and controlled but there are some questions that remain open that could have been included to make the study a more comprehensive analysis of Rab27 effectors in insulin secretion.
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