Impaired insulin release is a hallmark of type 2 diabetes and is closely related to chronically e... more Impaired insulin release is a hallmark of type 2 diabetes and is closely related to chronically elevated glucose concentrations, known as “glucotoxicity.” However, the molecular mechanisms by which glucotoxicity impairs insulin secretion remain poorly understood. In addition to known kiss-and-run and kiss-and-stay fusion events in INS-1 cells, ultrafast Hessian structured illumination microscopy (Hessian SIM) enables full fusion to be categorized according to the newly identified structures, such as ring fusion (those with enlarged pores) or dot fusion (those without apparent pores). In addition, we identified four fusion intermediates during insulin exocytosis: initial pore opening, vesicle collapse, enlarged pore formation, and final pore dilation. Long-term incubation in supraphysiological doses of glucose reduced exocytosis in general and increased the occurrence of kiss-and-run events at the expense of reduced full fusion. In addition, hyperglycemia delayed pore opening, vesicl...
SummaryThe medial entorhinal cortex (MEC) creates a map of local space, based on the firing patte... more SummaryThe medial entorhinal cortex (MEC) creates a map of local space, based on the firing patterns of grid, head direction (HD), border, and object-vector (OV) cells. How these cell types are organized anatomically is debated. In-depth analysis of this question requires collection of precise anatomical and activity data across large populations of neurons during unrestrained behavior, which neither electrophysiological nor previous imaging methods fully afford. Here we examined the topographic arrangement of spatially modulated neurons in MEC and adjacent parasubiculum using miniaturized, portable two-photon microscopes, which allow mice to roam freely in open fields. Grid cells exhibited low levels of co-occurrence with OV cells and clustered anatomically, while border, HD and OV cells tended to intermingle. These data suggest that grid-cell networks might be largely distinct from those of border, HD and OV cells and that grid cells exhibit strong coupling among themselves but we...
In the conserved autophagy pathway, autophagosomes (APs) engulf cellular components and deliver t... more In the conserved autophagy pathway, autophagosomes (APs) engulf cellular components and deliver them to the lysosome for degradation. Before fusing with the lysosome, APs have to close via an unknown mechanism. We have previously shown that the endocytic Rab5-GTPase regulates AP closure. Therefore, we asked whether ESCRT, which catalyzes scission of vesicles into late endosomes, mediates the topologically similar process of AP sealing. Here, we show that depletion of representative subunits from all ESCRT complexes causes late autophagy defects and accumulation of APs. Focusing on two subunits, we show that Snf7 and the Vps4 ATPase localize to APs and their depletion results in accumulation of open APs. Moreover, Snf7 and Vps4 proteins complement their corresponding mutant defects in vivo and in vitro. Finally, a Rab5-controlled Atg17–Snf7 interaction is important for Snf7 localization to APs. Thus, we unravel a mechanism in which a Rab5-dependent Atg17–Snf7 interaction leads to rec...
Despite the well-known role of the GLP-1 receptor (GLP-1R) in potentiating glucose-stimulated ins... more Despite the well-known role of the GLP-1 receptor (GLP-1R) in potentiating glucose-stimulated insulin secretion (GSIS) and as a therapeutic drug target in diabetes treatment, how GLP-1R is activated under physiological and pathological conditions remains elusive. Against preconceptions in the field, we prove that glucagon released from pancreatic α-cells serves as the principal ligand for GLP-1R on β-cells and is essential for GSIS in normal mice. Activation of the cognate glucagon receptor (GCGR) by glucagon amplifies GSIS evoked by intermediate concentrations of glucose only, as high glucose bypasses GCGR in increasing cytosolic cAMP from the same downstream pool. As the endogenous dual-receptor agonist, glucagon switches from GLP-1R to GCGR in β-cells of mice fed a high-fat diet, which significantly enhances GSIS and reduces body weight. Therefore, developing dual-receptor agonists that target balanced activation of GCGR and GLP-1R in vivo may better preserve glycemic control than the activation of GLP-1R alone.
Journal of visualized experiments : JoVE, Jul 23, 2017
Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. How... more Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy may incur acute whole blood reactions and result in poor islet engraftment. Here, we report a robust protocol for the transplantation of islets at the extrahepatic transplantation site-the epididymal fat pad (EFP)-in a diabetic mouse model. A protocol to isolate and purify islets at high yields from C57BL/6J mice is described, as well as a transplantation method performed by seeding islets onto a decellularized scaffold (DCS) and implanting them at the EFP site in syngeneic C57BL/6J mice rendered diabetic by streptozotocin. The DCS graft containing 500 islets reversed the hyperglycemic condition within 10 days, while the free islets without DCS required at least 30 days. The normoglycemia was maintained for up to 3 months until the graft was explanted. In conclusion, DCS enhanced the engraftment of islets into the extrahepatic site ...
Rationale: In cardiac dyads, junctional Ca 2+ directly controls the gating of the ryanodine recep... more Rationale: In cardiac dyads, junctional Ca 2+ directly controls the gating of the ryanodine receptors (RyRs), and is itself dominated by RyR-mediated Ca 2+ release from the sarcoplasmic reticulum. Existing probes do not report such local Ca 2+ signals because of probe diffusion, so a junction-targeted Ca 2+ sensor should reveal new information on cardiac excitation–contraction coupling and its modification in disease states. Objective: To investigate Ca 2+ signaling in the nanoscopic space of cardiac dyads by targeting a new sensitive Ca 2+ biosensor (GCaMP6f) to the junctional space. Methods and Results: By fusing GCaMP6f to the N terminus of triadin 1 or junctin, GCaMP6f-triadin 1/junctin was targeted to dyadic junctions, where it colocalized with t-tubules and RyRs after adenovirus-mediated gene transfer. This membrane protein-tagged biosensor displayed ≈4× faster kinetics than native GCaMP6f. Confocal imaging revealed junctional Ca 2+ transients (Ca 2+ nanosparks) that were ≈50×...
Sexually reproducing organisms acquire genetic diversity through meiotic recombination during mei... more Sexually reproducing organisms acquire genetic diversity through meiotic recombination during meiosis I, which is initiated via programmed DNA double-strand breaks (DSBs) induced by Spo11-containing machinery in each meiotic cell. The combination of programmed DSB sites in each meiotic cell must be diverse, which requires a certain degree of randomness in the distribution of DSBs. The formation of programmed DSBs requires a preestablished loop-axis structure of chromatin. Here, we demonstrate that the axial element protein Mer2 undergoes liquid-liquid phase separation in vitro and in vivo through its intrinsically disordered C-terminal domain. A DNA binding motif within its central domain is responsible for bringing DNA into Mer2 liquid droplets and Mer2-DNA complex could assemble into filamentous structures extending from the droplets. These results suggest that phase separation of Mer2 drives the formation of a droplet-loop structure of meiotic chromatin to facilitate and to diver...
Structured illumination microscopy (SIM) is widely applied due to its high temporal and spatial r... more Structured illumination microscopy (SIM) is widely applied due to its high temporal and spatial resolution imaging ability. sCMOS cameras are often used in SIM due to their superior sensitivity, resolution, field of view, and frame rates. However, the unique single-pixel-dependent readout noise of sCMOS cameras may lead to SIM reconstruction artefacts and affect the accuracy of subsequent statistical analysis. We first established a nonuniform sCMOS noise model to address this issue, which incorporates the single-pixel-dependent offset, gain, and variance based on the SIM imaging process. The simulation indicates that the sCMOS pixel-dependent readout noise causes artefacts in the reconstructed SIM superresolution (SR) image. Thus, we propose a novel sCMOS noise-corrected SIM reconstruction algorithm derived from the imaging model, which can effectively suppress the sCMOS noise-related reconstruction artefacts and improve the signal-to-noise ratio (SNR).
Despite its wide application in live-cell super-resolution (SR) imaging, structured illumination ... more Despite its wide application in live-cell super-resolution (SR) imaging, structured illumination microscopy (SIM) suffers from aberrations caused by various sources. Although artifacts generated from inaccurate reconstruction parameter estimation and noise amplification can be minimized, aberrations due to the scattering of excitation light on samples have rarely been investigated. In this paper, by simulating multiple subcellular structure with the distinct refractive index (RI) from water, we study how different thicknesses of this subcellular structure scatter incident light on its optical path of SIM excitation. Because aberrant interference light aggravates with the increase in sample thickness, the reconstruction of the 2D-SIM SR image degraded with the change of focus along the axial axis. Therefore, this work may guide the future development of algorithms to suppress SIM artifacts caused by scattering in thick samples.
Incretin-potentiated glucose-stimulated insulin secretion (GSIS) is critical to maintaining eugly... more Incretin-potentiated glucose-stimulated insulin secretion (GSIS) is critical to maintaining euglycemia, of which GLP-1 receptor (GLP-1R) on β-cells plays an indispensable role. Recently, α-cell-derived glucagon but not intestine-derived GLP-1 has been proposed as the critical hormone that potentiates GSIS via GLP-1R. However, the function of glucagon receptors (GCGR) on β-cells remains elusive. Here, using GCGR or GLP-1R antagonists, in combination with glucagon, to treat single β-cells, α-β cell clusters and isolated islets, we found that glucagon potentiates insulin secretion via β-cell GCGR at physiological but not high concentrations of glucose. Furthermore, we transfected primary mouse β-cells with RAB-ICUE (a genetically encoded cAMP fluorescence indicator) to monitor cAMP level after glucose stimulation and GCGR activation. Using specific inhibitors of different adenylyl cyclase (AC) family members, we revealed that high glucose concentration or GCGR activation independently ...
Cyclooctatetraene-conjugated cyanine dyes represent an effective strategy to improve biocompatibi... more Cyclooctatetraene-conjugated cyanine dyes represent an effective strategy to improve biocompatibility under light in live-cell fluorescence imaging and analysis of mitochondria.
Single-molecule localization microscopy (SMLM) has the highest spatial resolution among the exist... more Single-molecule localization microscopy (SMLM) has the highest spatial resolution among the existing super-resolution (SR) imaging techniques, but its temporal resolution needs further improvement. An sCMOS camera can effectively increase the imaging rate due to its large field of view and fast imaging speed. Using an sCMOS camera for SMLM imaging can significantly improve the imaging time resolution, but the unique single pixel-dependent readout noise of sCMOS cameras severely limits their application in SMLM imaging. This paper develops a Hessian-based SMLM (Hessian-SMLM) method that can correct the variance, gain and offset of a single pixel of a camera and effectively eliminate the pixel-dependent readout noise of sCMOS cameras, especially when the signal-to-noise ratio is low. Using Hessian SMLM to image mEos3.2-labeled actin was able to significantly reduce the artifacts due to camera noise.
Impaired insulin release is a hallmark of type 2 diabetes and is closely related to chronically e... more Impaired insulin release is a hallmark of type 2 diabetes and is closely related to chronically elevated glucose concentrations, known as “glucotoxicity.” However, the molecular mechanisms by which glucotoxicity impairs insulin secretion remain poorly understood. In addition to known kiss-and-run and kiss-and-stay fusion events in INS-1 cells, ultrafast Hessian structured illumination microscopy (Hessian SIM) enables full fusion to be categorized according to the newly identified structures, such as ring fusion (those with enlarged pores) or dot fusion (those without apparent pores). In addition, we identified four fusion intermediates during insulin exocytosis: initial pore opening, vesicle collapse, enlarged pore formation, and final pore dilation. Long-term incubation in supraphysiological doses of glucose reduced exocytosis in general and increased the occurrence of kiss-and-run events at the expense of reduced full fusion. In addition, hyperglycemia delayed pore opening, vesicl...
SummaryThe medial entorhinal cortex (MEC) creates a map of local space, based on the firing patte... more SummaryThe medial entorhinal cortex (MEC) creates a map of local space, based on the firing patterns of grid, head direction (HD), border, and object-vector (OV) cells. How these cell types are organized anatomically is debated. In-depth analysis of this question requires collection of precise anatomical and activity data across large populations of neurons during unrestrained behavior, which neither electrophysiological nor previous imaging methods fully afford. Here we examined the topographic arrangement of spatially modulated neurons in MEC and adjacent parasubiculum using miniaturized, portable two-photon microscopes, which allow mice to roam freely in open fields. Grid cells exhibited low levels of co-occurrence with OV cells and clustered anatomically, while border, HD and OV cells tended to intermingle. These data suggest that grid-cell networks might be largely distinct from those of border, HD and OV cells and that grid cells exhibit strong coupling among themselves but we...
In the conserved autophagy pathway, autophagosomes (APs) engulf cellular components and deliver t... more In the conserved autophagy pathway, autophagosomes (APs) engulf cellular components and deliver them to the lysosome for degradation. Before fusing with the lysosome, APs have to close via an unknown mechanism. We have previously shown that the endocytic Rab5-GTPase regulates AP closure. Therefore, we asked whether ESCRT, which catalyzes scission of vesicles into late endosomes, mediates the topologically similar process of AP sealing. Here, we show that depletion of representative subunits from all ESCRT complexes causes late autophagy defects and accumulation of APs. Focusing on two subunits, we show that Snf7 and the Vps4 ATPase localize to APs and their depletion results in accumulation of open APs. Moreover, Snf7 and Vps4 proteins complement their corresponding mutant defects in vivo and in vitro. Finally, a Rab5-controlled Atg17–Snf7 interaction is important for Snf7 localization to APs. Thus, we unravel a mechanism in which a Rab5-dependent Atg17–Snf7 interaction leads to rec...
Despite the well-known role of the GLP-1 receptor (GLP-1R) in potentiating glucose-stimulated ins... more Despite the well-known role of the GLP-1 receptor (GLP-1R) in potentiating glucose-stimulated insulin secretion (GSIS) and as a therapeutic drug target in diabetes treatment, how GLP-1R is activated under physiological and pathological conditions remains elusive. Against preconceptions in the field, we prove that glucagon released from pancreatic α-cells serves as the principal ligand for GLP-1R on β-cells and is essential for GSIS in normal mice. Activation of the cognate glucagon receptor (GCGR) by glucagon amplifies GSIS evoked by intermediate concentrations of glucose only, as high glucose bypasses GCGR in increasing cytosolic cAMP from the same downstream pool. As the endogenous dual-receptor agonist, glucagon switches from GLP-1R to GCGR in β-cells of mice fed a high-fat diet, which significantly enhances GSIS and reduces body weight. Therefore, developing dual-receptor agonists that target balanced activation of GCGR and GLP-1R in vivo may better preserve glycemic control than the activation of GLP-1R alone.
Journal of visualized experiments : JoVE, Jul 23, 2017
Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. How... more Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy may incur acute whole blood reactions and result in poor islet engraftment. Here, we report a robust protocol for the transplantation of islets at the extrahepatic transplantation site-the epididymal fat pad (EFP)-in a diabetic mouse model. A protocol to isolate and purify islets at high yields from C57BL/6J mice is described, as well as a transplantation method performed by seeding islets onto a decellularized scaffold (DCS) and implanting them at the EFP site in syngeneic C57BL/6J mice rendered diabetic by streptozotocin. The DCS graft containing 500 islets reversed the hyperglycemic condition within 10 days, while the free islets without DCS required at least 30 days. The normoglycemia was maintained for up to 3 months until the graft was explanted. In conclusion, DCS enhanced the engraftment of islets into the extrahepatic site ...
Rationale: In cardiac dyads, junctional Ca 2+ directly controls the gating of the ryanodine recep... more Rationale: In cardiac dyads, junctional Ca 2+ directly controls the gating of the ryanodine receptors (RyRs), and is itself dominated by RyR-mediated Ca 2+ release from the sarcoplasmic reticulum. Existing probes do not report such local Ca 2+ signals because of probe diffusion, so a junction-targeted Ca 2+ sensor should reveal new information on cardiac excitation–contraction coupling and its modification in disease states. Objective: To investigate Ca 2+ signaling in the nanoscopic space of cardiac dyads by targeting a new sensitive Ca 2+ biosensor (GCaMP6f) to the junctional space. Methods and Results: By fusing GCaMP6f to the N terminus of triadin 1 or junctin, GCaMP6f-triadin 1/junctin was targeted to dyadic junctions, where it colocalized with t-tubules and RyRs after adenovirus-mediated gene transfer. This membrane protein-tagged biosensor displayed ≈4× faster kinetics than native GCaMP6f. Confocal imaging revealed junctional Ca 2+ transients (Ca 2+ nanosparks) that were ≈50×...
Sexually reproducing organisms acquire genetic diversity through meiotic recombination during mei... more Sexually reproducing organisms acquire genetic diversity through meiotic recombination during meiosis I, which is initiated via programmed DNA double-strand breaks (DSBs) induced by Spo11-containing machinery in each meiotic cell. The combination of programmed DSB sites in each meiotic cell must be diverse, which requires a certain degree of randomness in the distribution of DSBs. The formation of programmed DSBs requires a preestablished loop-axis structure of chromatin. Here, we demonstrate that the axial element protein Mer2 undergoes liquid-liquid phase separation in vitro and in vivo through its intrinsically disordered C-terminal domain. A DNA binding motif within its central domain is responsible for bringing DNA into Mer2 liquid droplets and Mer2-DNA complex could assemble into filamentous structures extending from the droplets. These results suggest that phase separation of Mer2 drives the formation of a droplet-loop structure of meiotic chromatin to facilitate and to diver...
Structured illumination microscopy (SIM) is widely applied due to its high temporal and spatial r... more Structured illumination microscopy (SIM) is widely applied due to its high temporal and spatial resolution imaging ability. sCMOS cameras are often used in SIM due to their superior sensitivity, resolution, field of view, and frame rates. However, the unique single-pixel-dependent readout noise of sCMOS cameras may lead to SIM reconstruction artefacts and affect the accuracy of subsequent statistical analysis. We first established a nonuniform sCMOS noise model to address this issue, which incorporates the single-pixel-dependent offset, gain, and variance based on the SIM imaging process. The simulation indicates that the sCMOS pixel-dependent readout noise causes artefacts in the reconstructed SIM superresolution (SR) image. Thus, we propose a novel sCMOS noise-corrected SIM reconstruction algorithm derived from the imaging model, which can effectively suppress the sCMOS noise-related reconstruction artefacts and improve the signal-to-noise ratio (SNR).
Despite its wide application in live-cell super-resolution (SR) imaging, structured illumination ... more Despite its wide application in live-cell super-resolution (SR) imaging, structured illumination microscopy (SIM) suffers from aberrations caused by various sources. Although artifacts generated from inaccurate reconstruction parameter estimation and noise amplification can be minimized, aberrations due to the scattering of excitation light on samples have rarely been investigated. In this paper, by simulating multiple subcellular structure with the distinct refractive index (RI) from water, we study how different thicknesses of this subcellular structure scatter incident light on its optical path of SIM excitation. Because aberrant interference light aggravates with the increase in sample thickness, the reconstruction of the 2D-SIM SR image degraded with the change of focus along the axial axis. Therefore, this work may guide the future development of algorithms to suppress SIM artifacts caused by scattering in thick samples.
Incretin-potentiated glucose-stimulated insulin secretion (GSIS) is critical to maintaining eugly... more Incretin-potentiated glucose-stimulated insulin secretion (GSIS) is critical to maintaining euglycemia, of which GLP-1 receptor (GLP-1R) on β-cells plays an indispensable role. Recently, α-cell-derived glucagon but not intestine-derived GLP-1 has been proposed as the critical hormone that potentiates GSIS via GLP-1R. However, the function of glucagon receptors (GCGR) on β-cells remains elusive. Here, using GCGR or GLP-1R antagonists, in combination with glucagon, to treat single β-cells, α-β cell clusters and isolated islets, we found that glucagon potentiates insulin secretion via β-cell GCGR at physiological but not high concentrations of glucose. Furthermore, we transfected primary mouse β-cells with RAB-ICUE (a genetically encoded cAMP fluorescence indicator) to monitor cAMP level after glucose stimulation and GCGR activation. Using specific inhibitors of different adenylyl cyclase (AC) family members, we revealed that high glucose concentration or GCGR activation independently ...
Cyclooctatetraene-conjugated cyanine dyes represent an effective strategy to improve biocompatibi... more Cyclooctatetraene-conjugated cyanine dyes represent an effective strategy to improve biocompatibility under light in live-cell fluorescence imaging and analysis of mitochondria.
Single-molecule localization microscopy (SMLM) has the highest spatial resolution among the exist... more Single-molecule localization microscopy (SMLM) has the highest spatial resolution among the existing super-resolution (SR) imaging techniques, but its temporal resolution needs further improvement. An sCMOS camera can effectively increase the imaging rate due to its large field of view and fast imaging speed. Using an sCMOS camera for SMLM imaging can significantly improve the imaging time resolution, but the unique single pixel-dependent readout noise of sCMOS cameras severely limits their application in SMLM imaging. This paper develops a Hessian-based SMLM (Hessian-SMLM) method that can correct the variance, gain and offset of a single pixel of a camera and effectively eliminate the pixel-dependent readout noise of sCMOS cameras, especially when the signal-to-noise ratio is low. Using Hessian SMLM to image mEos3.2-labeled actin was able to significantly reduce the artifacts due to camera noise.
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