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. HoweverIn mammalian cells, the exocyst complex is assembled after separate formation of the subcomplex 1  and the subcomplex 2  . BecauseSEC10 knockdown markedly decreased GSIS in WT cells, but induced no further decrease of GSIS in Exo8KO cells, which was already decreased by half compared with WT cells . These fAs shown in However, the above findings were unexpexted because exophilin-8 deficiency markedly affects the resident-type exocytosis in the presence of granuphilin . We prevBecause the exocyst is also involved in the resident-type exocytosis , we nextDrosophila with mutation in SEC5 displays a defect in neurite outgrowth but not in neurotransmitter secretion  or after staying beneath the plasma membrane for a while (the visitor-type exocytosis) . The exoLeaden (C57J/L) mice with nonfunctional mutation of the gene encoding melanophilin (Mlph) (IMSR_JAX:000668), and were backcrossed with C57BL/6N mice 10 times to generate MlphKO mice. Exo8KO mice in the genetic background of C57BL/6N mice were described previously  or female . By crossing these F1 mice, GE8DKO mice, as well as the WT, GrphKO, and Exo8KO mice, were generated in the F2 generation and used for experiments. Although the resultant F2 mice have a mixture of C57BL/6N and C3H/He genomes, we expected that significant phenotypic changes due to the loss of Rab27 effectors may be preserved despite any influences due to randomly distributed differences in the genome. In fact, we found similar differences in exocytic profiles between WT and Exo8KO cells both in the C57BL/6N background . Only male mice and their tissues and cells were phenotypically characterized in this study. n (Mlph)  were pureviously . ME8DKO eviously . The malckground  and in tckground . Furtherckground , consistckground . Pancreackground . Brieflyhttp://n2t.net/addgene:53755-53762; RRID:Addgene_53755-53762; Mouse cDNAs of granuphilin , exophilCVCL_0431) were originally provided by Dr. Jun-ichi Miyazaki  were purchased from Invitrogen (Cat# R70507). Guinea pig anti-insulin serum was a gift from H. Kobayashi (Gunma University). Rabbit polyclonal anti-exophilin-8 (αExo8N) and anti-granuphilin (αGrp-N) antibodies are described previously . Immunoblotting and immunoprecipitation were performed as described previously . Green and red fluorescence of beads was observed by confocal laser scanning microscopy. Acquisition of images was performed under fixed conditions. For immunofluorescence procedures, monolayer primary beta cells seeded at 3–5 × 104 cells on a poly-L-lysine-coated glass base dish were cultured overnight in RPMI-1640 medium (11.1 mmol/L glucose) supplemented with 10% fetal calf serum and were fixed by 4% paraformaldehyde for 30 min at room temperature. The cells were washed by phosphate buffered saline (PBS) and permeabilized by PBS containing 0.1% Triton X-100 and 50 mmol/L NH4Cl for 30 min. After blocking with 1% bovine serum albumin in PBS for 30 min, the cells were immunostained and observed under confocal microscopy with a ×100 oil immersion objective lens (1.49 NA). Quantification of immunoblot signals was performed using ImageQuant TL software (Cytiva). Quantification of colocalization between two proteins was performed using NIS Element Viewer software (Nikon).MIN6 cells (RRID:eviously . mCherryeviously . The soueviously . VIP asseviously . BrieflyTIRF microscopy was performed as described previously . Briefly7 cells in ten 15 cm dishes) were infected with adenovirus encoding FLAG, FLAG-melanophilin, or MEF-exophilin-8. The anti-FLAG immunoprecipitates were subjected to gel electrophoresis and visualized by Oriole fluorescent gel staining (Bio-Rad). Specific bands were excised and digested in gels with trypsin, and the resulting peptide mixtures were analyzed by a LC-MS/MS system, as described previously (Mus musculus (mouse) proteome data set (UP000000589) at the Uniplot using Protein pilot software (SCIEX).MIN6 cells (2 × 10eviously . All MS/5 cells/280 μL were transfected with 100 nmol/L siRNA using Lipofectamine RNAiMAX reagent (Invitrogen). After plating on a 24-well plate or glass base dish for 48 hr, the cells were transfected with the same siRNA for the second time. After another 48 hr, the cells were subjected to immunoblotting analyses, immunofluorescent staining, insulin secretion assays, or TIRF microscopy after infection with adenovirus encoding insulin-EGFP.The On-Target plus Set of four siRNA against mouse Exoc5  and mouse Exoc4 , and the On-Target plus non-targeting pool siRNA were purchased from Horizon Discovery Ltd. Mouse pancreatic islet cells suspended in 1 × 10t test or a one-way ANOVA with a Tukey multiple-comparison using GraphPad Prism software.All quantitative data were assessed as the mean ± SEM. p-Values were calculated using Student’s All data generated or analyzed during this study are included in the manuscript and supporting files. All noncommercially available resources generated and/or analyzed during this study are available from the corresponding author upon reasonable request. This study examines how Rab27 and its different effectors regulate insulin secretory granule exocytosis. Using single and double knockouts and rescue experiments, the work presents convincing data to characterize the relative hierarchy of the Rab27 effectors, their action with the exocyst, and their roles in distinct types of exocytosis. Overall, this is a valuable study that sheds new light on the regulation of distinct forms of secretory granule exocytosis. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Anna Akhmanova as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Jing Hughes (Reviewer #2); Alistair Hume (Reviewer #3).Thank you for submitting your article "Functional hierarchy among different Rab27 effectors involved in secretory granule exocytosis" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1) Metabolic phenotyping of the DKO mouse needs to be performed, ideally including dynamic measurements of insulin secretion (Reviewer 2)2) Additional data are require to strengthen the connection between the exocyst and the Rab27 effectors by additional loss of function experiments combined with live cell imaging of the components. It would also be important to clarify how melanophilin expression is affected by Exo8 KO (Reviewer 1).3) The manuscript should use more precise language, noting the limitations of the interpretation and providing alternate explanations, particularly for the possibility that another pathway(s) for insulin secretion exists in these preparations (Reviewer 3).Reviewer #1 (Recommendations for the authors):1) The authors show both biochemically and by granule imaging that GSIS is reduced from β cells of exophilin-8 and melanophilin KO mice. However, information about granule density at the plasma membrane and insulin content in the cells is lacking. It is therefore difficult to know whether the secretory defect can also be explained by a relative insulin deficiency.2) A problem with the study is that the authors follow the dynamic process of insulin secretion by live cell TIRF microscopy of glucose-stimulated cells, but then look at the localization of the Rab27 effectors by immunofluorescence staining of fixed cells, where the culture conditions at the time-point of fixation are unknown (e.g. glucose concentration). This makes it difficult to directly compare the results obtained by these two methods. The paper would be much strengthened from live cell imaging of Exophilin-8, melanophilin, granuphilin  and components of the exocyst complex. Such experiments will allow the authors to draw much more accurate conclusions about the identity of different granule subpopulation and how they relate to the different Rab27 effectors. This becomes particularly important for the exocyst components, where the authors data indicate that these would be present on a majority of granules that undergo exocytosis.3) The authors show that melanophilin levels are reduced by 50% in exophilin-8 KO β cells, and suggest based on this that Exo8 may be required for stabilization of Melanophilin. This however, complicates the interpretation of the data in this model. How can the authors know that part of the exophilin-8 KO phenotype is not due to the simultaneous reduction in melanophilin expression? Although the authors show that the KO phenotype can be rescued by re-expression of exo-8, they don't show what happens with melanophilin expression under these conditions. It would be essential to show this, and also to test to what extent melanophilin re-expression affect the phenotype of the Exo-8 KO cells.4) In figure 1 – supplement 1 the authors show a strikingly high degree of colocalization between exo-8 and Mlph. However, based on functional data and on the model shown in Figure 7, these two proteins would only be expected to colocalize on a small subset of granules that immediately fuse with the plasma membrane. What is the reason for this discrepancy?5) The authors demonstrate, through biochemical assays, interactions between the exocyst complex and both Exo-8 and Mlph. Based on results from IP-experiments, the authors conclude that Exo-8 interacts with subcomplex I via Sec8 and that Mlph interacts with subcomplex 2 via Exo70. These are interesting results that corroborates earlier studies indicating a link between insulin granule mobilization/secretion and the exocyst complex.However, it is very difficult to envisage how this interaction plays into the regulation of granule mobilization and exocytosis. In particular, it is unclear how the interaction with the exocyst complex affects the localization of the Rab27 effectors and what its role is in Grph-dependent exocytosis. The evidence for the Rab27 effectors associating with the exocyst complex would be strengthened by showing the loss of effectors from Rab27 positive granules following knockdown of Sec8 (for Exo-8) and Exo70 (for Mlph) and showing that the interaction between Exo-8 and Mlph is lost following KD by IP. It would also be relevant to determine to what extent Grph-dependent exocytosis also require the Exocyst complex . To show colocalization with Sec6 as is done now  is not appropriate since it is unclear what granule pool is labelled by this protein and whether its localization is affected by Sec10 KD. In fact, it is unclear to me why the authors chose to reduce the expression of Sec10 and not Sec8/Exo70, which might have allowed the authors to more specifically dissect the Exo-8/Mlph pathways.6) The components of the exocyst complex can have functions independent of the exocyst. Since the authors only reduce the expression of Sec10, it may be difficult to draw conclusions about the entire complex. More extensive loss of function studies would be required or at a minimum this possibility should be discussed.7) The authors state that exophilin-8 and melanophilin were re-expressed at the endogenous level , which is also what the Western blot shows. However, re-expression of exophilin-8 results in a much larger proportion of cells with peripheral granule accumulation . A likely explanation for this is that not all cells are transduced with the adenovirus. It would therefore be better to not say at the endogenous level but instead just talk about re-expression.8) Could the authors explain the rational for the choice of glucose concentrations when stimulating the β cells. 2.8 mM is sub-physiological and 25 mM is supra-physiological. More reasonable would have been to increase the glucose concentration from 5 mM to 10 mM.9) It is shown that exo-8 is not involved in Grph-mediated exocytosis of docked granules but still somehow involved in exocytosis of docked granules. How can Exo-8 deficiency primarily influence the release of granules that are already docked at the plasma membrane? Based on the proposed mechanism, Exo-8 would be expected to primarily influence exocytosis of granules that are mobilized from the cytosol along actin filaments.10) It would be appropriate to also include Grph KO and WT cells in the experiments presented in Figure 6, so that one can compare the degree of rescue obtained.11) How do the authors propose that actin is involved? Is there an actin-independent (granuphilin) and actin-dependent (melanophilin) form of exocytosis in β cells?12) Quantify the degree of colocalization between Sec6 and insulin in Figure 3 —figure supplement 1.13) Cropped images in figure 1A and 1B are not correctly cropped.14) The small decrease in Pearson correlation coefficient reported in Figure 3C should not be artificially inflated by y-axis cropping.15) The authors several times talk about "immediate" binding. Could they please clarify what they mean by this. Is it direct binding?16) The authors state that GSIS from the GE8DKO islets exhibit an intermediate level between GrphKO and Exo8KO, but this is not what is shown in Figure 5A .17) In Figure 5C, Exo-8 KO is without effect on insulin secretion visualized by granule imaging . This is different from other experiments and complicates interpretations also from the GE8DKO cells. Can the authors please comment on the reason for this.18) How can the authors know that they are recording exocytic events from β cells and not from other islet cell types?Reviewer #2 (Recommendations for the authors):Suggestions for additional experiments or clarifications:– While Exo8 and melanophilin loss-of-function mouse models have been previously characterized , the metabolic phenotype of double knockout ME8DKO mice is not presented here. It would be informative to include some standard in vivo metabolic testing such as IPGTT, body morphometrics, fasting glucose and diabetes incidence on chow or challenge diets, to see if these mice phenocopy Exo8KO and what does the loss of this pathway mean for diabetes propensity.– Dynamic insulin secretion as the authors have previously performed  would complement the static secretion data in Figure 1C by showing whether the secretory defect lies in 1st or 2nd phase, and how this might correspond to the release of different insulin pools – resident, passenger, or visitor.– The observation that melanophilin-only knockouts (MlphKO) have a milder phenotype than Exo8KO or ME8DKO suggests that there may be effectors other than melanophilin downstream of Exo8. Now that we have double KO data, what are the authors' conclusion regarding role of melanophilin, is it redundant and therefore expendable for Exo8 or Rab27 function? Under what physiologic conditions might this melanophilin-mediated, passenger-type exocytosis pathway be important?Reviewer #3 (Recommendations for the authors):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 that1. Mlph works downstream of Myrip/exophilin-8 to mobilise granules for fusion from the actin network to the plasma membrane after stimulation.2. Mlph and exophilin-8 interact via the exocyst3. Down-regulation of exocyst affects exocytosis in cells expressing exophilin-84. Exophilin-8 promotes fusion of granules docked by granuphilin at the membrane5. Exophilin-8 not required for Grph related granule docking at the plasma membraneA 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.In support of 1 they show that:insulin granules are docked at the cortex (a prelude to fusion) in WT and MlphKO, but not Exo8KO and double ME8DKO b-cells .In ME8DKO cells they show that expression of Exo8 but not Mlph restores cortical distribution of granules .Using TIRFM analysis of insulin secretion in pancreatic b-cells that fusion of non-resident granules (passenger and visitor types) is reduced in MlphKO cells compared with controls in response to stimulation .In support of 2 they show that:over-expressed/tagged and endogenous Exo8 and mlph can be co-precipitated in MIN6 cells .exocyst components Sec6 and Sec10 co-IP with Mlph and Exo8 .Depletion of sec10 reduces Mlph association with sec6 positive insulin granules .However, it would be important to show this directly e.g., by showing the results of Mlph/Exo8 IP in exocyst depleted cells to confirm that Mlph/Exo8 interaction is via the exocyst.In support of 3 they show that:using ELISA assays that glucose dependent insulin secretion is reduced in wild-type, but not Exo8KO, cells when Sec10 is KD .In support of 4 they show that:insulin secretion in double granuphilin (Grph)/Exo8 DKO b-cells is reduced compared with single Grph KO cells . Related to this they show that granule number in the TIRF field is affected GrphKO and GE8DKO but not Exo8KO. This seems surprising given that in Figure 1 the authors show that the number of docked granules is increased in Exo8KO cells.In support of 5they use TIRFM to show that granules are docked at the membrane by Exo8KO but not GrphKO or GE8DKO .Overall, their data support the model shown in Figure 6.This is an interesting study but leaves open some important questions regarding insulin secretion and the role of Rab27a and its effectors in this process.The finding that GE8DKO islets release more insulin than wild-type  is striking and suggests that another pathway(s) for insulin secretion exists in these preparations. Can the authors discuss the possible molecular mechanism of this pathway in their manuscript/include this in their model to present a more complete picture of the mechanisms of granule exocytosis?Related to this while I understand that it may be out of the remit of this project to characterise Grph/Exo8 independent pathways from scratch it would nevertheless be highly relevant to this study on Rab27a effectors for the authors to investigate the possibility that Rab27 and Mlph are involved in this pathway. For example, by generating triple Mlph, Exo8 and Grph8 KO islets or depleting in Mlph in GE8DKO islets and examining the effect of this on exocytosis and how this relates to exocytosis in the absence of Rab27a.Regarding the possible different pathways have the authors noticed any differences in the kinetic profile of fusion events as monitored by TIRFM this might provide interesting insight into the relationship and mechanism between the proposed different pathways. Essential revisions:1) Metabolic phenotyping of the DKO mouse needs to be performed, ideally including dynamic measurements of insulin secretion (Reviewer 2)We have performed the above experiments and presented the data in Figure 2B, 2E, and Figure 2—figure supplement 1.2) Additional data are require to strengthen the connection between the exocyst and the Rab27 effectors by additional loss of function experiments combined with live cell imaging of the components. It would also be important to clarify how melanophilin expression is affected by Exo8 KO (Reviewer 1).We have performed additional loss of function experiments combined with live cell imaging of the components and presented the data in Figure 7 and Figure 7—figure supplement 1. We found differential roles in the resident type exocytosis between exophilin-8 and the exocyst. Please see our responses to Reviewer 1’s comments 2) and 5). Furthermore, we clarify the effect of melanophilin expression in Exo8KO cells by performing experiments in heterozygous MlphKO cells . Please see our response to Reviewer 1’s comment 3).3) The manuscript should use more precise language, noting the limitations of the interpretation and providing alternate explanations, particularly for the possibility that another pathway(s) for insulin secretion exists in these preparations (Reviewer 3).We have asked a professional external editor in the US to correct English and reflected the changes in a revised manuscript. Furthermore, we have added description about the existence of another pathway in the last part of Discussion.Reviewer #1 (Recommendations for the authors):1) The authors show both biochemically and by granule imaging that GSIS is reduced from β cells of exophilin-8 and melanophilin KO mice. However, information about granule density at the plasma membrane and insulin content in the cells is lacking. It is therefore difficult to know whether the secretory defect can also be explained by a relative insulin deficiency.We have added the data of insulin content in Figure 2—figure supplement 1B and presented the data of granule density in Figure 2C.2) A problem with the study is that the authors follow the dynamic process of insulin secretion by live cell TIRF microscopy of glucose-stimulated cells, but then look at the localization of the Rab27 effectors by immunofluorescence staining of fixed cells, where the culture conditions at the time-point of fixation are unknown (e.g. glucose concentration). This makes it difficult to directly compare the results obtained by these two methods. The paper would be much strengthened from live cell imaging of Exophilin-8, melanophilin, granuphilin  and components of the exocyst complex. Such experiments will allow the authors to draw much more accurate conclusions about the identity of different granule subpopulation and how they relate to the different Rab27 effectors. This becomes particularly important for the exocyst components, where the authors data indicate that these would be present on a majority of granules that undergo exocytosis.We have described in Materials and methods that monolayer primary β cells were cultured in RPMI-1640 medium (11.1 mmol/L glucose) before the fixation.ELife, 2017; and melanophilin for Wang et al., Diabetes, 2020). For example, we previously showed by both TIRF and confocal microscopy that granuphilin and melanophilin, which should separately mediate the resident type and the passenger type, respectively, are colocalized on granules . Thus, each effector is not necessarily located on different granule subpopulation. As discussed previously , the fate of each individual granule is not defined by the presence of the particular Rab27 effector per se but appears to be stochastically determined by its interactions with specific proteins and membrane lipids in a local milieu around the granule. As the reviewer noticed, the exocyst component, Sec6, is also distributed to almost all the granules .About the identity of different granule subpopulation related to the different Rab27 effectors, we would first like the reviewer to understand that, although each Rab27 effector regulates a distinct exocytic step, all Rab27 effectors previously examined in pancreatic β cells, including Noc2 that also interacts with Rab2a located in the perinuclear region , are not differentially located on specific granules where they function, but are distributed on almost all insulin granules throughout the cytoplasm via interaction with multiple Rab27a molecules on single granules  and severely inhibits granule exocytosis . Furthermore, we could not perform live cell imaging previously in the case of melanophilin, because mCherry-melanophilin expressed in the presence of endogenous melanophilin was not located on granules, but aberrantly located along the actin microfilaments, and because its expression at the endogenous level in melanophilin-deficient cells display too weak fluorescence to monitor granule exocytosis . Therefore, we always had to express fluorescent Rab27 effectors in the absence of endogenous proteins. This is thought to be particularly critical for the exocyst that generally functions as a heterooctameric protein complex, because exogenous expression of one fluorescent component unbalances the quantities among the components. In support of this view, we found marked effects of SEC10 knockdown on granule exocytosis despite the partial downregulation . Therefore, decent works involving live cell imaging of the exocyst have been performed in knockin cells in both yeast and mammalian cells . Given that our data in Figures 3 and 4 suggest that the holo-exocyst connects only a part of exophilin-8 and melanophilin, we do not want to perform experiments overexpressing one component of the exocyst, which unlikely functions as a good marker of the holo-exocyst and likely yields uncertain and possibly artificial results. We would like to do live cell imaging of the exocyst in primary β cells, after we generate the knockin mouse for one of the components in the future study. We hope that the editors and the reviewers understand that the main purpose of this study is to identify the functional relationship among different Rab27 effectors.We also would like to point out that exophilin-8 and the exocyst are anyway involved in both resident and passenger types of exocytosis, as shown in Figures 2D and 5C, and therefore, their live-cell imagimg would not identify different granule subpopulation mediating the specific type of exocytosis. It should also be noted that TIRF microscopy does not provide direct information in the cell interior. For example, we could not get information where granules mediating the passenger exocytosis, which appear only in one frame prior to fusion, have been originally located before stimulation.However, in the case of granuphilin that molecularly tether granules to the plasma membrane, live cell imaging by TIRF microscopy generates useful information. Given that the evanescent field illuminates fluorescence within 100-200 nm of the coverslip and that the radius of insulin granules is 150-200 nm, granuphilin-positive granules should harbor granuphilin in their hemisphere that is closer to the plasma membrane, and so are likely physically tethered to the plasma membrane via interaction with the closed form of syntaxins. In fact, we previously found that granules visible under TIRF microscopy can be divided into two classes: granuphilin-positive, immobile granules and granuphilin-negative, mobile granules . Therefore, we can discriminate granules physically tethered to the plasma membrane and those just locating nearby. Please also understand that granuphilin-negative granules under TIRF microscopy mean its absence in their hemisphere that is closer to the plasma membrane but may still harbor granuphilin in their opposite hemisphere. To investigate the role of the exocyst in the resident type exocytosis, we performed new experiments in response to the reviewer’s point 5 below.3) The authors show that melanophilin levels are reduced by 50% in exophilin-8 KO β cells, and suggest based on this that Exo8 may be required for stabilization of Melanophilin. This however, complicates the interpretation of the data in this model. How can the authors know that part of the exophilin-8 KO phenotype is not due to the simultaneous reduction in melanophilin expression? Although the authors show that the KO phenotype can be rescued by re-expression of exo-8, they don't show what happens with melanophilin expression under these conditions. It would be essential to show this, and also to test to what extent melanophilin re-expression affect the phenotype of the Exo-8 KO cells.Although melanophilin must be kept at the endogenous level as described above, it is not easy to exactly compensate the decreased amount of endogenous melanophilin in each Exo8KO cell. Therefore, we instead examined the heterozygous MlphKO cells that may express it at a half level of the endogenous melanophilin. Indeed, the level of endogenous melanophilin in the heterozygous cells was 65.4 ± 4.7% of that in WT cells, which was equivalent to the level in Exo8KO cells (59.8 ± 9.0%). The finding that these heterozygous cells did not show reduced insulin secretion  suggests that the decrease in melanophilin expression is unlikely responsible for the reduced insulin secretion in Exo8KO cells.4) In figure 1 – supplement 1 the authors show a strikingly high degree of colocalization between exo-8 and Mlph. However, based on functional data and on the model shown in Figure 7, these two proteins would only be expected to colocalize on a small subset of granules that immediately fuse with the plasma membrane. What is the reason for this discrepancy?As described above in response to the reviewer’s point 2, the localization of each Rab27 effector is not restricted to specific granules. The localization of Rab27 effectors is determined via interaction with multiple Rab27 molecules existing on granules. For example, we previously showed the melanophilin E14A mutant deficient in Rab27 binding are not colocalized with granules at all . Therefore, the two proteins are highly colocalized on each insulin granule, although a subset of these proteins are expected to interact with, as the reviewer points out.5) The authors demonstrate, through biochemical assays, interactions between the exocyst complex and both Exo-8 and Mlph. Based on results from IP-experiments, the authors conclude that Exo-8 interacts with subcomplex I via Sec8 and that Mlph interacts with subcomplex 2 via Exo70. These are interesting results that corroborates earlier studies indicating a link between insulin granule mobilization/secretion and the exocyst complex.However, it is very difficult to envisage how this interaction plays into the regulation of granule mobilization and exocytosis. In particular, it is unclear how the interaction with the exocyst complex affects the localization of the Rab27 effectors and what its role is in Grph-dependent exocytosis. The evidence for the Rab27 effectors associating with the exocyst complex would be strengthened by showing the loss of effectors from Rab27 positive granules following knockdown of Sec8 (for Exo-8) and Exo70 (for Mlph) and showing that the interaction between Exo-8 and Mlph is lost following KD by IP. It would also be relevant to determine to what extent Grph-dependent exocytosis also require the Exocyst complex . To show colocalization with Sec6 as is done now  is not appropriate since it is unclear what granule pool is labelled by this protein and whether its localization is affected by Sec10 KD. In fact, it is unclear to me why the authors chose to reduce the expression of Sec10 and not Sec8/Exo70, which might have allowed the authors to more specifically dissect the Exo-8/Mlph pathways.more specifically dissect the Exo-8/Mlph pathways.In response to the reviewer’s question, we first examined the effects of SEC10 knockdown on the intracellular localization of exophilin-8 and melanophilin, and found that SEC10 knockdown does not affect their colocalization with insulin or Rab27a . It is not surprising, because Rab27 effectors is localized on granules via interaction with Rab27, as described above. Therefore, it is not expected that any effectors will be lost from Rab27 positive granules even if other exocyst component is downregulated.The reason we select SEC10 to downregulate the exocyst is that this component plays a key role to form the holo-exocyst, because previous VIP assays indicate that the two subcomplexes are biochemically linked via the interactions between Sec10 in the subcomplex 2 and Sec8 and/or Sec3 in the subcomplex 1 . If SEC10 knockdown disrupts the interaction between exophilin-8 and melanophilin, the holo-exocyst likely links the two effectors. Consistent with this view, SEC10 knockdown partially disrupted the colocalization with SEC6 of melanophilin, but not that of exophilin-8 .However, according to the reviewer’s suggestion, we also tried to knockdown SEC8 and EXO70. The available siRNAs downregulated SEC8 significantly , but unfortunately not EXO70 see . In factIn response to the reviewer’s question “what the role of the exocyst is in Grph-dependent exocytosis”, we first performed TIRF experiments in GrphKO cells. SEC10 knockdown did not further decrease the number of visible granules in GrphKO cells , as found in GE8DKO cells . However, it markedly decreased the resident type exocytosis , in contrast to the case in GE8DKO cells . To directly assess the influence of exocyst deficiency on granuphilin-mediated, docked granule exocytosis, we then expressed Kuo-Grph in GrphKO cells, as performed in Figure 6D,E. As shown in Figure 7C,D, SEC10 knockdown specifically decreased the fusion probability of granuphilin-negative granules , in contrast that exophilin-8 deficiency markedly decreased that of granuphilin-positive granules . As described in Discussion, these data suggest that the exocyst promote the exocytosis of granuphilin-negative, mobile granules by tethering them to the plasma membrane, as found in other constitutive secretory pathways. In contrast, exophilin-8 appears to promote fusion of granuphilin-mediated docked (tethered) granules by converting the associated, closed form of syntaxins into the open form.Although we could not measure glucose-induced insulin secretion in the above experiments, we hope that the editors and the reviewers understand the vulnerable nature of primary islet cells after isolation. To our knowledge, there is no reports to do such multiple interventions, two times transfections and two kinds of adenovirus infections, for live cell imaging of primary β cells. In general, it is difficult to monitor two kinds of fluorescent proteins in addition to insulin-EGFP to examine their function in granule exocytosis in living β cells, without generating the knockin mouse that expresses multiple fluorescent protein instead of endogenous proteins.6) The components of the exocyst complex can have functions independent of the exocyst. Since the authors only reduce the expression of Sec10, it may be difficult to draw conclusions about the entire complex. More extensive loss of function studies would be required or at a minimum this possibility should be discussed.As described in response to the reviewer’s comment 5), we chose SEC10 knockdown to examine the involvement of the holo-exocyst, and performed additional experiments using SEC8 siRNA . To our knowledge, although the exocyst has been investigated more than quarter century, the literature, including live cell imaging of the exocyst , indicates that it functions as the holo-complex in cells.7) The authors state that exophilin-8 and melanophilin were re-expressed at the endogenous level , which is also what the Western blot shows. However, re-expression of exophilin-8 results in a much larger proportion of cells with peripheral granule accumulation . A likely explanation for this is that not all cells are transduced with the adenovirus. It would therefore be better to not say at the endogenous level but instead just talk about re-expression.It is inevitable that adenovirus infection is somewhat inhomogeneous. We just meant the endogenous level in average.8) Could the authors explain the rational for the choice of glucose concentrations when stimulating the β cells. 2.8 mM is sub-physiological and 25 mM is supra-physiological. More reasonable would have been to increase the glucose concentration from 5 mM to 10 mM.We simply thought that the stronger stimulation makes the phenotypic differences more evident in the mutant cells. The glucose stimulation from 5 or 10 mM may be weak to detect significant differences.9) It is shown that exo-8 is not involved in Grph-mediated exocytosis of docked granules but still somehow involved in exocytosis of docked granules. How can Exo-8 deficiency primarily influence the release of granules that are already docked at the plasma membrane? Based on the proposed mechanism, Exo-8 would be expected to primarily influence exocytosis of granules that are mobilized from the cytosol along actin filaments.No, we did state that exophilin-8 is involved in Grph-mediated exocytosis, as shown in Figure 6. We also proposed that Exo8 promotes the priming reaction to remove a Grph-mediated exocytic clamp by associated proteins such as RIM-BP2, RIM, and Munc13. To support this view, we show that the absence of exophilin-8 inhibits the fusion of granuphilin-positive, docked granules more strongly compared with granuphilin-negative, undocked granules . At present, we have no evidence that Exo8 primarily influences exocytosis of granules that are mobilized from the cytosol along actin filaments, because, under TIRF microscopy, the number of visible granules before stimulation is not changed by exophilin-8 deficiency .10) It would be appropriate to also include Grph KO and WT cells in the experiments presented in Figure 6, so that one can compare the degree of rescue obtained.These experiments are performed to discriminate the effect of exophilin-8 deficiency on granuphilin-positive, docked granules from that on granuphilin-negative, untethered granules. We adjusted the expression level of granuphilin at endogenous level, as shown in Figure 6—figure supplement 1. Furthermore, the actual numbers of fusion events in WT and Exo8KO cells  are equivalent to those found in mimic WT and mimic Exo8KO cells .11) How do the authors propose that actin is involved? Is there an actin-independent (granuphilin) and actin-dependent (melanophilin) form of exocytosis in β cells?We should discriminate the role of actin tracks from that of the actin cortex in vesicle transport. For example, granules may reach the plasma membrane by generating actin tracks by another Rab27 effector, SPIRE, as shown in skin melanocytes . Based on the data in Figure 2, we suggest that melanophilin-mediated passenger type exocytosis is derived from granules captured within the actin cortex by Exophilin-8. Consistently, the increase in the passenger type exocytosis in the absence of granuphilin is markedly reduced to the level found in Exo8KO cells . However, as shown in Figures 2C and 6B, the absence of exophilin-8 does not affect the number of granules visible under TIRF microscopy, which suggests that granules residing close to the membrane are not necessarily derived from granules anchored in the actin cortex.12) Quantify the degree of colocalization between Sec6 and insulin in Figure 3 —figure supplement 1.We have removed this data, because the reviewer mentioned in the comment 5) that Sec6 is not appropriate since it is unclear what granule pool is labelled by this protein and whether its localization is affected by Sec10 KD. We instead examined the effects of SEC10 knockdown on the granule localization of exophilin-8 and melanophilin, and quantified the degree of colocalization .13) Cropped images in figure 1A and 1B are not correctly cropped.Although the images were correctly cropped in the original file, they appear to be improperly cropped during the conversion process to the PDF file. We have corrected them.14) The small decrease in Pearson correlation coefficient reported in Figure 3C should not be artificially inflated by y-axis cropping.We have corrected the y-axis in Figure 4A .15) The authors several times talk about "immediate" binding. Could they please clarify what they mean by this. Is it direct binding?In general, coimmunoprecipitation experiments do not prove direct binding. However, visible IP assays show that only one of the exocyst components can interacts with exophilin-8 or melanophilin, and therefore we used the word “immediate”.16) The authors state that GSIS from the GE8DKO islets exhibit an intermediate level between GrphKO and Exo8KO, but this is not what is shown in Figure 5A .We corrected the sentence as below:GE8DKO cells also exhibited an increase in GSIS compared with Exo8KO cells.17) In Figure 5C, Exo-8 KO is without effect on insulin secretion visualized by granule imaging . This is different from other experiments and complicates interpretations also from the GE8DKO cells. Can the authors please comment on the reason for this.Compared with the experiments of Figure 2D that were performed in mice with the same B6 genetic background, the experiments in Figure 6C  were performed in mice with a mixture of B6 and C3H genetic backgrounds , which may have increased variance of the data and decreased the significance in differences. However, the mean values and differences between WT and Exo8KO cells are similar between these studies.18) How can the authors know that they are recording exocytic events from β cells and not from other islet cell types?Although we and others performing similar TIRF microscopic experiments did not discriminate β cells from other cells beforehand, we previously showed that most (90%) of the monolayer cells from pancreatic islets are β cells. See the examples of WT and melanophilin-deficient cells . Furthermore, we did not include the data of cells that did not respond at all to glucose stimulation, which may be non-β cells or become inert during preparation and/or incubation.Reviewer #2 (Recommendations for the authors):Suggestions for additional experiments or clarifications:– While Exo8 and melanophilin loss-of-function mouse models have been previously characterized , the metabolic phenotype of double knockout ME8DKO mice is not presented here. It would be informative to include some standard in vivo metabolic testing such as IPGTT, body morphometrics, fasting glucose and diabetes incidence on chow or challenge diets, to see if these mice phenocopy Exo8KO and what does the loss of this pathway mean for diabetes propensity.We compared the metabolic phenotypes, such as body weight, blood glucose concentrations during IPGTT and those during ITT including fasting glucose concentrations among WT, MlphKO, Exo8KO, and ME8DKO mice. Each mouse strain displayed correlated insulin secretory capacities in batch , islet perifusion , and TIRF microscopic assays . However, it should be noted that, because these effectors are widely and differentially expressed in other endocrine cells, their deficiency may differently affect secretion of other hormones influencing glucose tolerance, such as incretin and glucagon.– Dynamic insulin secretion as the authors have previously performed  would complement the static secretion data in Figure 1C by showing whether the secretory defect lies in 1st or 2nd phase, and how this might correspond to the release of different insulin pools – resident, passenger, or visitor.We have performed islet perifusion experiments  and compared the time course of insulin secretion with that of granule exocytosis under TIRF microscopy . The passenger type exocytosis is particularly decreased in a later 2nd phase in mutant cells . However, the resident type exocytosis also tended to decrease in a later phase .– The observation that melanophilin-only knockouts (MlphKO) have a milder phenotype than Exo8KO or ME8DKO suggests that there may be effectors other than melanophilin downstream of Exo8. Now that we have double KO data, what are the authors' conclusion regarding role of melanophilin, is it redundant and therefore expendable for Exo8 or Rab27 function? Under what physiologic conditions might this melanophilin-mediated, passenger-type exocytosis pathway be important?As shown in Figures 2A and 2D, MlphKO cells have a milder phenotype compared with Exo8KO or ME8DKO cells, because of the lack of decrease in the resident type exocytosis. However, melanophilin deficiency induces similar decreases in the passenger type exocytosis whether exophilin-8 is present (MlphKO cells) or not (ME8DKO cells), which indicates that melanophilin does not merely play a redundant role, but a unique role in this type of exocytosis. In support of this view, both effectors function through different protein complexes, RIM-BP2-myosin-VIIa and myosin-Va-syntaxin-4, respectively .Reviewer #3 (Recommendations for the authors):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 that1. Mlph works downstream of Myrip/exophilin-8 to mobilise granules for fusion from the actin network to the plasma membrane after stimulation.2. Mlph and exophilin-8 interact via the exocyst3. Down-regulation of exocyst affects exocytosis in cells expressing exophilin-84. Exophilin-8 promotes fusion of granules docked by granuphilin at the membrane5. Exophilin-8 not required for Grph related granule docking at the plasma membraneA 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.In support of 1 they show that:insulin granules are docked at the cortex (a prelude to fusion) in WT and MlphKO, but not Exo8KO and double ME8DKO b-cells .In ME8DKO cells they show that expression of Exo8 but not Mlph restores cortical distribution of granules .Using TIRFM analysis of insulin secretion in pancreatic b-cells that fusion of non-resident granules (passenger and visitor types) is reduced in MlphKO cells compared with controls in response to stimulation .In support of 2 they show that:over-expressed/tagged and endogenous Exo8 and mlph can be co-precipitated in MIN6 cells .exocyst components Sec6 and Sec10 co-IP with Mlph and Exo8 .Depletion of sec10 reduces Mlph association with sec6 positive insulin granules .However, it would be important to show this directly e.g., by showing the results of Mlph/Exo8 IP in exocyst depleted cells to confirm that Mlph/Exo8 interaction is via the exocyst.In support of 3 they show that:using ELISA assays that glucose dependent insulin secretion is reduced in wild-type, but not Exo8KO, cells when Sec10 is KD .In support of 4 they show that:insulin secretion in double granuphilin (Grph)/Exo8 DKO b-cells is reduced compared with single Grph KO cells . Related to this they show that granule number in the TIRF field is affected GrphKO and GE8DKO but not Exo8KO. This seems surprising given that in Figure 1 the authors show that the number of docked granules is increased in Exo8KO cells.In support of 5they use TIRFM to show that granules are docked at the membrane by Exo8KO but not GrphKO or GE8DKO .Overall, their data support the model shown in Figure 6.This is an interesting study but leaves open some important questions regarding insulin secretion and the role of Rab27a and its effectors in this process.The finding that GE8DKO islets release more insulin than wild-type  is striking and suggests that another pathway(s) for insulin secretion exists in these preparations. Can the authors discuss the possible molecular mechanism of this pathway in their manuscript/include this in their model to present a more complete picture of the mechanisms of granule exocytosis?It is possible that other Rab27 effectors compensate for the absence of Granuphilin and Exophilin-8. For example, exophilin-7 is involved in undocked granule exocytosis in the absence of granuphilin, although exophilin-7-knockout β cells display only modestly reduced exocytosis . However, we would like to suggest the possibility that secretory vesicles can fuse efficiently once they are directed to the plasma membrane using machineries such as cytoskeletons and/or the exocyst that are present in almost all cells. For example, in PC12 cells, SNAREs in native plasma membranes are constitutively active even if not engaged in fusion events . Therefore, regulated exocytic pathways are thought to be evolved by acquiring additional gating machinery to prevent spontaneous or inappropriate fusion. We would also like to suggest that granule clustering sites in the actin cortex or along the plasma membrane reflect the presence of such gating steps . We would like to propose that Rab27 effectors play such regulatory roles, rather than essential roles, in exocytosis, as discussed previously . If so, the finding that GE8DKO cells release more insulin than WT cells is not so surprising. I briefly describe our thought in Discussion as below.In summary, our findings are the first to determine the functional hierarchy among different Rab27 effectors expressed in the same cell, although the exact mechanism remains unknown how these different steps are regulated in a coordinated manner.It should be noted, however, that granule exocytosis is not abrogated in β cells lacking any of the single or double Rab27 effectors. This may suggest the existence of other secretory pathways. For example, other Rab27 effectors, such as exophilin-7 , can also promote some types of exocytosis . However, given that granules can arrive close to the plasma membrane and fuse efficiently without prior capture in the actin cortex by exophilin-8 and/or stable docking to the plasma membrane by granuphilin, Rab27 effectors are thought to be evolved to play regulatory roles that prevent spontaneous or inappropriate fusion in regulated secretory pathways, as suggested previously .Related to this while I understand that it may be out of the remit of this project to characterise Grph/Exo8 independent pathways from scratch it would nevertheless be highly relevant to this study on Rab27a effectors for the authors to investigate the possibility that Rab27 and Mlph are involved in this pathway. For example, by generating triple Mlph, Exo8 and Grph8 KO islets or depleting in Mlph in GE8DKO islets and examining the effect of this on exocytosis and how this relates to exocytosis in the absence of Rab27a.If melanophilin has function in the absence of exophilin-8, ME8DKO cells should show additional defects compared with Exo8KO cells, which we could not detect . Furthermore, considering that melanophilin deficiency only affects the passenger type exocytosis , we think it unlikely that additional melanophilin deficiency suppresses the increase in the resident type exocytosis in GrphKO or GE8DKO cells . Furthermore, as described above, constitutive-like exocytic pathways or other effectors such as exophilin-7 may function in the absence of granuphilin and exophilin-8.Regarding the possible different pathways have the authors noticed any differences in the kinetic profile of fusion events as monitored by TIRFM this might provide interesting insight into the relationship and mechanism between the proposed different pathways.Although we have not examined differences in kinetic profiles of fusion events between WT and mutant cells, we previously showed no obvious differences in time courses or amplitudes of fluorescence intense changes during fusion reaction on average, among resident, visitor, and passenger types of exocytosis in WT cells .


Tissue dielectric constant (TDC) measurements are a convenient, reliable, and accurate way to noninvasively access local tissue water content and its change with time or treatment. The method has been widely used for upper extremity lymphedema assessments but less so for lower extremities. For lower extremity assessments, it would be useful to have an estimate of the expected inter-leg TDC differentials of normal non-affected legs to help interpret measured inter-leg differentials when such measurements are done in patients with lower extremity edema or lymphedema. The goals of this study were to determine normal inter-leg TDC differences, quantify TDC temporal changes during 60 minutes of supine lying in a group of 10 middle-aged women (42-57 years), and evaluate the change in TDC values as measured throughout the day from 0800 to 2000 hours in a group of 10 younger women (23-28 years). The overall findings indicate that TDC measurements, as an assessment parameter of lower extremity localized skin-to-fat tissue water, are only minimally dependent on potential confounding factors such as 60-minute supine lying or the time of day at which the measurements are made. If the small sample size of the present study is reflective of the larger population, an inter-leg TDC ratio exceeding a value of 1.2 is suggested as a threshold for judging unilateral lower extremity edema or lymphedema. This possibility needs assessment in women with these conditions. Tissue dielectric constant (TDC) measurements are a convenient, reliable, and accurate way to access local tissue water content and its change since its value strongly depends on skin-to-fat water content -4. The mSubjects2.Two different groups of 10 women participated after having each study explained and signing a Nova Southeastern University Institutional Review Board-approved informed consent (#12180901F). To achieve goal 1, women were recruited from faculty and staff according to the following entry criteria: age 18 or greater; no prior history of lower extremity trauma, surgery, edema, or lymphedema; and willing and able to lie supine for 60 minutes. The group of 10 participants had an age range of 42-57 years and a mean ± standard deviation (SD) of 48.4 ± 5.8 years. To achieve goal 2, 10 women medical students were recruited for participation according to the following entry criteria: age 18 or greater and having no prior history of lower extremity trauma, surgery, edema, or lymphedema. These participants were trained in the TDC measurement procedure by the principal investigator. Their age range was 23-28 years (26.2 ± 1.4 years), and they had a normal body mass index (BMI) of 21.7 ± 2.1 kg/mTDC measurementThe TDC measuring device  determines the dielectric constant (relative permittivity), which is a dimensionless number equal to the ratio of tissue permittivity to vacuum permittivity. Because TDC values mainly depend on tissue water, they provide quantitative indices of skin water content. The measurement is sensitive to both free and bound water . InclusiProcedures for goal 1A site on the medial aspect of both legs located 8 cm proximal to the medial malleolus was marked with a skin marker. These sites were the target TDC measurements. After a supine acclimation interval of 10 minutes, TDC was measured in triplicate on both legs with a measurement probe as illustrated in Figure Procedures for goal 2Self-measurements were done by each participant on a day-night interval of 12 hours at two-hour intervals starting at 0800 and ending at 2000 (seven measurement sets). During this time, the participants did normal activities, mostly studying, with no intervening exercise. Triplicate TDC medial calf measurements were done while seated on the left leg only at a site located 8 cm proximal to the mid-medial malleolus.AnalysesStatistical estimates of the differences in TDC values between paired legs and between the initial and final TDC values after 60 minutes of supine lying  were determined using the nonparametric Wilcoxon test. Investigation of the possible temporal variations in TDC values measured over the 0800 to 2000 time interval  was evaluated via linear regression. In both analyses, a statistical significance was judged based on a p-value < 0.05.TDC bilateral and temporal comparisonsTable TDC variation over timeFigure The focus of the present study was to provide quantitative information on normal lower extremity TDC values including their inter-leg difference and their temporal variation. The main purpose was to provide some initial guidance as to likely variations among healthy women as a reference for the use of TDC measurements in women with unilateral or bilateral edema or lymphedema. A compelling reason for this investigation was that when TDC measurements are used to assess the extent of localized lymphedema and to track and characterize treatment effects, such information would be valuable. The approach adopted was to use two groups of women in which different aspects could be systematically evaluated.In this initial undertaking, the inclusion of only women is consistent with the fact that lower extremity lymphedema is more prevalent in women than in men with its occurrence reported in up to 70% of women who have been treated for gynecological malignancies . In a reAs part of goal 1, inter-leg TDC differences and their change during one hour of supine lying were determined. This information has potential clinical relevance since lower extremity lymphedema treatments and evaluations occur over this time frame. The findings indicate only minor changes in TDC over this interval with no significant change in inter-leg ratios. As part of goal 2, day-to-night TDC variations were assessed via multiple sequential measurements. This information bears on the potential impact of time-of-day influence on TDC values. The results indicate a small decrease of about 5% over the entire 12-hour measurement interval but that the time-of-day variation is small during daylight hours.Although the number of subjects evaluated was not large, the findings do provide insight into the range of expected temporal variations for TDC as measured on the legs of healthy women and normal values of inter-leg TDC ratios. To the extent that the inter-leg TDC ratio herein found reflects that which would be present in a larger non-edematous population, the ratio may provide a useful estimate of the presence of unilateral lymphedema as has been done for upper extremity unilateral lymphedema -14. In tThe principal limitation of this study’s findings is the relatively small number of subjects included and evaluated. A secondary limitation is that because the studied group included only women, the findings for men may differ in any aspect. In future studies, the inclusion of a greater number of subjects of both sexes would seem useful to evaluate the generalizability of the present findings.The findings indicate that TDC values, as an assessment parameter of lower extremity localized skin-to-fat tissue water, minimally depend on potential confounding factors such as the duration of supine lying and the time of day when the measurements are made. Based on a small sample size, an inter-leg TDC ratio exceeding a value of 1.2 is suggested as a potential threshold for judging unilateral lower extremity edema or lymphedema. Verifying this threshold in patients with lower extremity edema or lymphedema is suggested.


However, many techniques have been proposed to get closer to that aim. Here we review the current progress in the field. At first, we describe the most successful technique, the organ culture method, which allows to produce functional haploid cells. However, this method is based on the culturing of intact testis tissue with unknown factors acting inside it. Then we discuss different types of 3D-cultures where specific testicular cell populations may be aggregated and the impact of each cell population may be examined. Unfortunately, germ cell development does not proceed further than the pachytene stage of meiosis there, with rare exceptions. Finally, we describe recent studies that focus on germ cells in a conventional adherent cell culture. Such studies thoroughly examine issues with in vitro meiosis and provide insight into the mechanisms of meiotic initiation.A complete reconstruction of spermatogenesis  The first one expressed GFP in male germ cells from the mid-pachytene stage of meiosis onwards, while the second one started to express GFP during meiotic cell divisions . Testicu complex , confirm complex   and retinol, well-known inductors of spermatogonial differentiation  culture system of testicular somatic cells and germ cells. 3D-culture allows cells to interact with each other and with extracellular matrix in three dimensions and to recapitulate tissue architecture. A 3D-culture of testicular cells with complete spermatogenesis could provide valuable information about critical factors in testicular microenvironment affecting germ cell development and about the role of different populations of somatic cells in spermatogenesis.A further step to more defined conditions for The most successful attempt to initiate spermatogenesis in a 3D culture was made in 2013  by the sIn 2013 , the resAnother 3D culture system was proposed in 2014  with tesSo, the 3D methods described above seem to be far less effective than the organ culture method: most significantly, no functional spermatids were produced when using them. In  some reaW/KITW−V pups at a ratio of 1:1. The two types of cells were co-cultured in a medium supplemented with 10% KSR, RA, BMP-2/4/7, and activin A for 6 days -β; the cells were then cultured for another 4 days. Further analysis of DSBs showed that MEIOB and SPATA22, which cooperatively bind to meiotic DSBs to promote recombination , were prne stage .So, These three studies  have achin vitro spermatogenesis, some key issues remain to be solved. The most successful approach is an organ culture of mouse testis tissue. However, the number of haploid cells in this culture is still low, as compared to in vivo testes (in vitro. Overcoming this problem may increase the efficiency of spermatogenesis in all culture systems.Although many achievements have been made in the field of o testes , and theo testes . Microaro testes . It seemin vitro is unclear, which may be the reason for the low efficiency of in vitro spermatogenesis. This suggestion is also true for other organs, as organoids derived from pluripotent stem cells or fetal progenitors are mainly used to study organogenesis and rarely reach an adult tissue stage, whereas organoids derived from adult cells are considered to be the best for the reproduction of adult tissue functions (in vitro or a technique for efficient and correct meiosis of male germ cells without support of somatic cells.Another issue is connected with the need to use somatic cells. The conception of organoids widely used for other organs and tissues is hardly applicable to the testis. Organoids are 3D-structures that recapitulate basic tissue architectures and functionalities and that develop from stem cells or progenitors . Howeverunctions . The sol


Supportive communities turned out to be an extremely important service for older adults. Policy makers should consider expanding supportive community services for older adults, as it was shown to have a positive correlation with lower healthcare service use, which might be an indicator of better overall health.This study examined social characteristics and their relations to healthcare service demand among older adults during the first COVID-19 lockdown in 2020. The sample was based on a cohort of 103,955 adults over the age of 65. A general index of needs was composed based on healthcare service use data and was predicted in a multi-nominal logistic regression. The frequency of the total needs significantly ( COVID-19 severely impacted older adults, who had to be isolated in order to avoid exposure to the pandemic . During The impact of social factors among older adults as factors influencing the ability to prepare for catastrophic events has been investigated ,12,13. VLoneliness and age are associated with the level of compliance with patient guidelines and the implementation of treatment recommendations . Non-comSocial services and volunteering may play an important role in the mental health of older adults with no social support . The higNatali healthcare solutions [olutions  is a priPrevious studies focused on social characteristics and their relations to healthcare service demand among older adults during the first COVID-19 lockdown. This study used a unique and unexplored dataset to look into this association, employing special social measures in order to draw a clearer picture of healthcare service provision during the time of the pandemic and thereby adding to the existing literature. The purpose of this study was to examine the contribution of different social factors to the healthcare service requirements during the first COVID-19 lockdown. In addition to socio-demographic characteristics, the specific social factors that were examined were living in a community framework, supportive community services, living in nursing homes, and type of housing. We hypothesized a decrease in the total medical needs during the first COVID-19 lockdown compared to the corresponding period in 2019, which would be more pronounced among those with different kinds of social support.This was a longitudinal observational study that followed a cohort of healthcare service provider customers. This study compared two periods: February-April 2019 (P1), before the beginning of the COVID-19 pandemic, and February-April 2020 (P2), during which the first COVID-19 lockdown took place in Israel. In P2, citizens of the State of Israel were subjected to COVID-19 lockdown restrictions, as promulgated by the Ministry of Health in Israel .Natali healthcare solutions has regular subscribers and provides subscribers with medical services according to the type of service to which the subscriber is registered. The healthcare service provider provides a panic (emergency) button service for the call center, doctor home visit services, an ambulance service, and remote telephone consultation. All customers that constituted the population of this study were adults over the age of 65 in Israel. The sample did not undergo a sampling process but was based on all relevant healthcare service provider data. No identification data of the subjects was presented in the research data file. For this study, customers under the age of 65 were removed from the dataset to include only older adults during the two periods under study [er study . A lack Natali healthcare solutions collects data for the purpose of monitoring and supervising healthcare service provider activities. The healthcare service provider data was provided solely for the purpose of the present study, excluding identifying information of the members. Most of the variables relevant to the present study were derived from the healthcare service provider data while other residential variables were based on the CBS. Socio-economic status, level of religiosity, and residential sector were the only relevant sociodemographic characteristics that were provided from the CBS [ the CBS  after maIn situations where there is no risk to human life, the center provides telephone counseling or offers a doctor for a home visit. A supportive community service is also offered in which customers are contacted on a weekly basis for assistance with their needs: questions, consultation, purchase of medicines, purchase of food, and any logistical or medical needs. The aim of the present study was to examine the social characteristics and their impact on the demand for medical services among older adults in Israel during the COVID-19 lockdown in 2020 compared to the corresponding period in 2019. We hypothesized that there would be a decrease in the demand for medical needs during the first COVID-19 lockdown period, which would be more pronounced among those with different kinds of social support.1. Medical calls—The number of times a customer contacted the emergency call center for a medical reason. The value scale was P1 0-114 and P2 0-472. 2. Emergency calls—The number of times a customer contacted the emergency call center. The value scale was P1 0-226 and P2 0-294. 3. Ambulance order—The number of times the customer ordered an ambulance in both periods. These created a scale of values as detailed: P1 0-18 and P2 0-16. 4. Doctor home visit—The number of times a client called a doctor during these periods. The scale of values was P1 0-102 and P2 0-111. These four variables once indexed had an internal consistency of Cronbach alpha α = 0.693 at P1 and α = 0.630 at P2. To construct this index, the four variables were first recoded as dichotomous variables that received values of 0—no service required and 1—required service, and then the four were summed into one complex index for each period on a scale of 0–4. As these four variables had different scales, the action to dichotomize (creating 2 groups of 0—no need or 1—one need or more) had to be implemented in order to unite different scales into one before combining them into one index. The needs for services index constituted the dependent variable for each period separately: pre-COVID-19 (P1) and during the first COVID-19 lockdown (P2). A general index of needs was composed based on four variables: 1. Supportive community services—A service of medical and logistic assistance on a weekly basis. Values are yes—service is supplied or no—not supplied. 2. Living in nursing homes—Whether the service consumer lives in an older adult home (yes/no). 3. Living in community framework—This was taken from the service consumer’s home address and matched with the Israeli CBS [4. Type of housing—(Private residence/building or apartment) If the floor number of the home address was supplied, the type of housing was considered a building. All others were considered private homes. Our social independent variables were tested. aeli CBS  informatAge—This variable was calculated at the starting point of the study (2019) by year of birth. The ages of two participants were missing. In a new age scale, the age group was grouped into three age categories [Marital status—Single/married/divorced/widowed, although 28,742 service users (27.5% of the sample) had a missing marital status. For regression analysis, family status was married and the three values of not married  were combined. Three additional socio-demographic variables were derived from the respondent’s residential address after cross-referencing the information with CBS data [Residential sector—The ethnicity of the community by service user address ; Residential religiosity of the locality by service user address ; and the Socio-economic status of the locality according to CBS coding [Sex—Male/female. tegories ,25: younCBS data : ResidenS coding  in whichData was analyzed using IBM SPSS Statistics 25. First, descriptive statistics for describing the sample by frequency were presented. Then, cross tabulation frequencies between needs in P1 and P2 based on the independent social variables, with a Chi square test for significant differences between groups were implemented. At last, multi-nominal logistic regression results for predicting needs for services in two models: 1. For the comparison of no needs as a reference group for one need and 2. For the comparison of no needs as a reference group for two or more needs. For this purpose, the dependent variable “need for services” was dichotomized by the median. The expected β values shown in the table constitute the RR or relative risk and show the probability of service needs according to the groups of independent variables. The confidence interval (CI) displays the minimum value and the maximum value for the 95% confidence range. Values displayed in bold were significantly higher or lower than 1.Natali healthcare solutions and their needs for medical services during pre-pandemic (P1) and COVID-19 lockdown period (P2) were checked according to social variables. A large older adult sample that contained 103,955 members of p < 0.001), and a decline in needs for services was observed from P1 to P2. From all kinds of housing, living in nursing homes required more services in both study periods  and supportive community services required less services in both study periods . Supportive community services were found to be significantly different between groups of needs in both studied periods. The frequency of needs was lower for customers who used supportive community services in P1 (4.9%) compared to those who did not use them (33.4%) and went down even more in P2 (2.0%) during the COVID-19 lockdown for customers who used supportive community services compared to those who did not (24.2%). For those who live in nursing homes, the frequency of needs was 47.8% in P1 compared to those who do not live in nursing homes (27.4%). Needs for services declined in P2 to 37.4% during the COVID-19 lockdown for customers who live in nursing homes compared to those who do not live in nursing homes (18.9%). In sum, Model 2 shows results in the same direction. The strongest predicting variables were previous needs, supportive community services, and living in a community framework. The probability of having 2 or more needs was 24 times higher if there were needs the year before . The probability of two needs or more was significantly higher  for those who did not have supportive community services. The probability of two needs or more was significantly higher  for those who did not live in a community framework.Natali healthcare solutions. The sample included 63.5% women and the average age of the participants was 80 years old (standard deviation = 7.46). Regarding family status, 61.6% were married and 30.9% were widowers. Most of the sample were living in Jewish (83.9%) and secular (98.4%) communities. According to the Israeli Central Bureau of Statistics [Natali healthcare solutions while around one-quarter of the sample  were living in nursing homes. Regarding the type of housing, 20%  were living in private homes. Epidemics cause social and medical problems and make it imperative to respond quickly during times of emergency . This statistics , particiWe hypothesized a decrease in the total medical needs during the first COVID-19 lockdown compared to the corresponding period in 2019, which would be more pronounced among those with different kinds of social support.Natali healthcare solutions for various social and medical matters was significantly lower compared to the corresponding period in 2019, as was expected. The frequency of the total needs significantly (p < 0.000) declined in P2 (23.8%) compared to P1 (32.8%). In times of crisis, older adults tend to avoid being examined and going to hospitals, preferring to deal with their medical condition on their own [An examination of the findings according to the two study periods showed that during the period of the first COVID-19 lockdown, the frequency of older adult people applying to heir own ,27,28.Natali healthcare solutions is a supportive community, in which service users are contacted on a weekly basis to assist them with questions, consultation, purchase of medicines, purchase of food, and any logistical or medical needs they may have. Our findings show that those who made use of this supportive community service were significantly less likely to demand medical services in both periods. In addition, living in a community framework was significantly associated with less frequent needs. These findings are supported by previous studies [One of the services supplied by  studies ,12 demon studies . Corresp studies ,30,31.The older adult population living in nursing homes represents a high-risk group . This maThe type of housing also has an impact on the consumption of healthcare services. This study found that supportive community services and living in a community framework are protective factors. Regarding differences between types of housing, a few studies found that isolation was particularly difficult in crowded apartments inhabited by low-income individuals, including older adults and those most susceptible to infection. As a result, limited living space contributed to the exacerbation of disease and mental disorders, mainly due to overcrowding ,38,39,40Social variables were found to be significantly associated with lower consumption of healthcare services during the pandemic. Supportive communities turned out to be an extremely important service for older adults, and we recommend that this service is expanded. Policymakers should consider expanding supportive community services for older adults, as it was shown to have a positive correlation with lower healthcare service use, which may be an indicator of better overall health. Further studies should look into the differences between nursing homes and older adult homes that are non-nursing homes, which may be able to explain these findings.This study was based on a very large cohort of older adult people in Israel and designed prospectively to check for risk and protective factors associated with medical needs. Its design and sample allow for stronger confidence in the conclusions of this paper. The limitations of this study prevent us from positing causal relations between the variables as it is observational in nature and cannot prevent the interference of confounder variables. Another limitation may be due to the medical condition of those who live in nursing homes compared to older adult people living in their homes. It is possible that those who live in a nursing home in the first place have worse medical conditions than those living in their homes. A final limitation concerns the uncertainty in the variable of nursing homes since data on differences between nursing homes and older adult homes that are non-nursing homes were not available. This paper presents the need for healthcare services of older adults in Israel during the first COVID-19 lockdown. It reinforces the claim that in times of crisis, older adults tend to avoid receiving healthcare services and prefer to deal with their medical conditions on their own. The main finding is that older adults in supportive community services were significantly less likely to require medical services during the first lockdown. Supportive community services should be expanded to lower the need of older adults for healthcare services, in routine times and in times of crisis.


Annual emissions grow 1% slower in the coming decade than projected in 2015. This slower growth mostly stems from the adoption of new policies and updated expectations on technology uptake and economic growth. However, around one-quarter of these changes are explained by the effects of the COVID-19 pandemic on short-term emissions and economic forecasts. These factors combined result in substantially lower emission projections for India, the European Union plus the UK (EU27 + UK), the Unites States, Russia, Saudi Arabia, and South Africa. We observe a remarkable change in South African projections that changed from a substantial increase to now a decline, driven in part by the planned phase-out of most of its coal-based power. Emissions in India are projected to grow slower than in 2015 and in Indonesia faster, but emissions per capita in both countries remain below 5 tCO2eq in 2030, while those in the EU27 + UK decline faster than expected in 2015 and probably cross the 5 tCO2eq threshold before 2030. Projected emissions per capita in Australia, Canada, Saudi Arabia, and the United States are now lower than projected in 2015 but remain above 15 tCO2eq in 2030. Although emission projections for the G20 improved since 2015, collectively they still slightly increase until 2030 and remain insufficient to meet the Paris Agreement temperature goals. The G20 must urgently and drastically improve adopted policies and actions to limit the end-of-century warming to 1.5 °C.Many years passed since the adoption of the Paris Agreement, which invites countries to determine their own contributions to climate change mitigation efforts. The Agreement does not offer a standard to measure progress but relies on a process of periodic stocktakes to inform ambition-raising cycles. To contribute to this process, we compare 2021 greenhouse gas emission projections up to 2030 against equivalent projections prepared back in 2015. Both sets of projections were prepared using the same bottom-up modelling approach that accounts for adopted policies at the time. We find that 2021 projections for the G20 as a group are almost 15% lower (approximately 6 GtCOThe online version contains supplementary material available at 10.1007/s11027-022-10018-5. More than two decades have passed since major emitting countries had a mandate to limit their greenhouse-gas (GHG) emissions (Kyoto Protocol to the United Nations Framework Convention on Climate Change (UNFCCC), 1997). Since then, global GHG emissions have almost doubled and the effects of climate change intensified IPCC . The lonUnder the Paris Agreement, countries are invited to submit Nationally Determined Contributions (NDCs), which are pledges that reflect countries’ own interpretation of a fair contribution to the challenge of reducing global emissions and keeping end-of-century warming below 1.5 °C UNFCCC . There iSince the Paris Agreement was adopted, several countries updated their NDCs, which vary in content and implied absolute emission levels but collectively result in emissions lower than the original ones  project to which the authors contributed since its inception  using 100-year global warming potentials (GWPs) from the IPCC Fourth Assessment Report (AR4)  IPCC . All CATR4) IPCC .We focused on the progress in reducing emissions in energy, industry, agriculture, and waste sectors since 2015 and excluded land use, land-use change, and forestry (LULULCF) emissions from our analysis. Including LULUCF emissions is important for research that explores whether countries are on track to meet their self-determined targets or to reach net zero emissions  and those developed in the year of the Paris Agreement (2015). We analysed several indicators to analyse this progression. First, we calculated the difference in 2030 emissions to estimate the absolute reduction in projected emissions. This metric clarifies whether the G20 group 2030 emissions are higher or lower and which countries’ estimates explain most of the change. We also analysed full emission trajectories for all countries in terms of absolute and per capita emissions to identify changes in trends and emission-peak years. We calculated the percentage change in emissions in comparison to the year of adoption of the Paris Agreement to improve comparability across countries and progress compared to a base year. Finally, we also calculated the yearly percentage change rates for both sets of projections (2015 and 2021) averaged between 2021 and 2030. This metric focuses on the rate instead the absolute change in the coming decade. We removed both upper and lower fifth percentile of the yearly change rate distribution to avoid including abrupt and significant variations, e.g. those induced by lockdown measures.We analysed key developments that took place in the G20 since 2015, such as the adoption of new policies and the COVID-19 pandemic, and affected emission projections. Diverse initiatives have also led to the availability of more robust and up-to-date historical emission information. Improvements in official inventory reporting, especially by non-Annex-I countries, and the availability of third-party datasets allow for recent trends to be included in our emission estimates  or 15% lower than those projected in 2015  is approximately 200 and 10 MtCO2e in 2010 and 2015, respectively. The COVID-19 pandemic has resulted in substantial changes, which are discussed in more detail below  or at least slowed down (Turkey). Emissions per capita are decreasing but remain above 15 tCO2eq per capita in 2030 in Canada, Australia, the United States, and Saudi Arabia. In the G20, emissions per capita tend to grow at a slower pace than absolute emissions , the distribution of emissions change rates in the 2020s is not significantly different from the change rates in the 2010s Fig . This meHere, we present a non-exhaustive list of factors that can explain changes in emission projections for each G20 country Table .Table 1N2eq (range: 1.3–1.6 GtCO2eq) lower in 2030 due to COVID-19. This corresponds to a 3.6% (range: 3.1–4.2%) reduction compared to emission projections excluding the effect of COVID-19. Our results are within that range of other studies, which find that COVID-19 policy responses and economic slowdown could reduce annual global emissions by 1–5 GtCO2eq, or 1.5–8.5%, in 2030 . In 2021, data was on average 2 years old . In most G20 countries, pre-2020 emissions are substantially different between the two sets of projections.Post-2020 emission projections are influenced by the adoption of additional policies, especially those that reduce energy-related emissions. Since 2015, energy-related emissions have been periodically analysed by international organisations, such as the International Energy Agency (IEA). The IEA often revises their estimates to account for lower energy demand and higher rate of uptake of renewables  publish official projections that consistently update the list of policies included as well as their effect on future emissions. New or additional scenarios became available for all countries analysed since 2015. This results from more transparent and frequent communication of climate change mitigation progress by non-Annex I countries that now submit Biennial Update Reports (BURs) to the UNFCCC. In ten G20 countries, most recent projections include additional sources.The periodic evaluation of progress is a fundamental element of the Paris Agreement ambition-raising mechanism. The official Global Stocktake focuses on global emission projections and collective progress UNFCCC , but ultThe G20 covers a large share of global emissions, and their emission projections bear a strong influence on global progress. We find that emissions of the G20 as a group are projected to increase up to 0.6% per year between 2021 and 2030. An increase in the emissions of group covering such as high share of global emissions is certainly misaligned with the goals of the Paris Agreement IPCC . Global 2 emissions published by the IEA in their World Energy Outlook reports are also reviewed and often included in the CAT analysis. They show emissions lower than projected in 2015, except for Russia and China. Our 2021 estimates for Russia are lower than 2015 due to a revision in historical methane emissions, which are outside IEA’s scope. The IEA show an overall increase in energy-related CO2 emissions for approximately 10% for the same period analysed in this study. Our results for China indicate emissions have not changed substantially, but the IEA indicates an increase of approximately 5% in energy-related CO2 emissions. The IEA scenario is used as part of the upper range of our analysis but is adjusted downwards to better reflect key national energy policies, such as the target to meet 20% non-fossil energy share in 2025 . For all other countries analysed, IEA emission projections are also expected to be lower than projected in 2015.Since the CAT analysis builds on or reviews many studies, the comparison to other literature is not trivial. Other country-specific analyses used as input to the CAT analysis show progress in the same direction and of similar magnitude to ours. Latest official projections for Australia and Canada show that emissions in both countries are approximately 40% and 30% lower compared to the 2015 projections, respectively  drop in estimated emissions between 2019 and 2020. Pandemic-induced policy responses and economic slowdown also reduce projected emissions in almost all countries and explains approximately one-quarter of the reduction in 2030 emissions observed in the G20. In most recent projections, Australia, Canada, the EU27 + UK, Japan, South Korea, South Africa, and United States are expected to reduce their emissions below 2015 levels by 2030. Even though revised representation of historical effects and the pandemic influence our estimates, the adoption of additional policies explains lower emissions. Renewable uptake has been faster than projected before, efficiency improvements result in lower energy demand, and coal phase-out policies result in substantial shifts in emission projections of some countries. These factors combined result in changes in most countries’ 2021 projections when compared to those developed in 2015:South Africa presents the most significant change in emission projections in the coming decade. The country has improved from substantial annual increase on its emissions to an annual decrease between 2021 and 2030.The EU27 + UK and Japan were already expected to decrease their emissions in the coming decade but are expected to decrease it faster in 2021 projections. South Korea is likely to slightly decrease its emissions in the coming decade.The United States, Canada, and Australia were expected to increase their emissions and now show a slightly declining trend. This is a positive development, but their emissions per capita remain among the highest in the G20.China’s 2030 emissions remain at similar levels than projected in 2015. The faster historical emission growth is offset by slower emission growth in the coming decade. Emissions are now expected to peak before 2030.Argentina, Brazil, Mexico, Russia, and Saudi Arabia now show lower projected emissions in 2030 than estimated in 2015 but are expected to still increase their emissions. The implementation of Saudi Arabia’s renewable targets could result in plateauing emissions before 2030.2eq) in 2030.Turkey slowed their substantial expected emission growth, but emissions increase up to 2030. Emissions in India and Indonesia also increase but remain at low per capita levels Below is the link to the electronic supplementary material.