Analysis of synaptic-like microvesicle exocytosis of B-cells using a live imaging technique

Abstract

Pancreatic β-cells play central roles in blood glucose homeostasis. Beside insulin, these cells release neurotransmitters and other signaling molecules stored in synaptic-like microvesicles (SLMVs). We monitored SLMV exocytosis by transfecting a synaptophysin-pHluorin construct and by visualizing the cells by Total Internal Reflection Fluorescence (TIRF) microscopy. SLMV fusion was elicited by 20 mM glucose and by depolarizing K(+) concentrations with kinetics comparable to insulin secretion. SLMV exocytosis was prevented by Tetanus and Botulinum-C neurotoxins indicating that the fusion machinery of these organelles includes VAMP-2/-3 and Syntaxin-1, respectively. Sequential visualization of SLMVs by TIRF and epifluorescence microscopy showed that after fusion the vesicle components are rapidly internalized and the organelles re-acidified. Analysis of single fusion episodes revealed the existence of two categories of events. While under basal conditions transient fusion events prevailed, long-lasting episodes were more frequent upon secretagogue exposure. Our observations unveiled similarities between the mechanism of exocytosis of insulin granules and SLMVs. Thus, diabetic conditions characterized by defective insulin secretion are most probably associated also with inappropriate release of molecules stored in SLMVs. The assessment of the contribution of SLMV exocytosis to the manifestation of the disease will be facilitated by the use of the imaging approach described in this study.

Figure 1. Two types of secretory vesicles in the pancreatic β-cell line MIN6B1.

A. Confocal images showing double labeling of endogenous synaptophysin (marker of SLMVs) and insulin (marker of LDCVs) in the pancreatic β-cell line MIN6B1. Lower panels present details of the same images at higher magnification. Note that in the merge images there is no colocalization between the two signals. B. TIRF images of a 40 nm red fluorescent bead, a representative SLMV expressing synaptophysin-mCherry, a 400 nm red fluorescent bead and a representative LDCV. C. Average radial sweeps of 20 TIRF images. Red lines are the means of the measured values. The 40-nm beads are significantly smaller than LDCVs but display apparent sizes similar to SLMVs. In contrast, LDCVs exhibit apparent sizes similar to 400-nm beads. Error bars indicate SD. Bar, 0.5 µm. Intracellular distribution of synaptophysin-pHluorin. D. MIN6 cells were transfected with synaptophysin-pHluorin and immunolabeling performed with antibodies against GFP, endogenous insulin or synaptophysin. Images were acquired with confocal microscope and quantitative analysis carried out using the co-localization module in the Imaris 7.6 software (Bit plane). On the top: the left panel shows the localization of synaptophysin-pHluorin revealed using the GFP antibody (green). The middle panel shows immunolabeling against endogenous insulin (red) and the right panel the merged images. The panels on the bottom present higher magnifications of the top pictures. The analysis of the images shows 8.8±6.8% (n = 5 cells) co-localization between the green and the red signals. E. same experiment as D. but with immunolabeling against synaptophysin (red). The analysis of the images indicates 96.9±2.4% (n = 5 cells) co-localization of the two signals. Bars: 12 µm.

Figure 2. Stimulation of exocytosis and endocytosis of SLMVs in MIN6B1 cells with different secretagogues: whole-cell studies.

A. Time-course of cocktail-induced (glucose 20 mM; KCl 30 mM; forskolin 10 µM, IBMX 100 µM) pHluorin fluorescence intensity changes analyzed by TIRF illumination. The curve represents the whole-cell pHluorin fluorescence signal, expressed as ΔF/F₀ obtained averaging results from 13 cells. B. Same as A. but induced by KCl 30 mM. The curve is obtained by averaging results from 9 cells. Data points are collected every 200 ms and represent mean values ± SD.

Figure 3. Effect of Somatostatin and Epinephrine on exocytosis of SLMVs.

A. Time-course of cocktail-induced (glucose 20 mM; KCl 30 mM; forskolin 10 µM, IBMX 100 µM) pHluorin fluorescence intensity in the absence or in the presence of somatostatin (500 nM) obtained with TIRF illumination. The curve represents the whole-cell pHluorin fluorescence signal, expressed as ΔF/F₀ obtained averaging results from 5 and 11 cells, respectively. B. same experiment as A. but in the absence or in the presence of epinephrine (1 µM). The curve represents the whole-cell pHluorin fluorescence signal, expressed as ΔF/F₀ obtained averaging results from 4 and 7 cells, respectively. C–D. Histograms represent the amplitudes (ΔF/F₀) of the cocktail-induced pHluorin fluorescence intensity change in the absence or in the presence of somatostatin or epinephrine.

Figure 4. Inhibition of SLMV exocytosis by clostridial neurotoxins.

A. Time-course of cocktail-induced (glucose 20 mM; KCl 30 mM; forskolin 10 µM, IBMX 100 µM) pHluorin fluorescence intensity obtained with TIRF illumination in cell transfected with a control plasmid or with tetanus neurotoxin-tomato (TeTX). The curve represents the whole-cell pHluorin fluorescence signal, expressed as ΔF/F₀ obtained averaging results from 3 and 9 cells, respectively. B. The same as A. but in cells transfected with a control plasmid or with GFP-botulinum neurotoxin C (BoNT/C). Since the GFP-tagged BoNT/C light chain is uniformly distributed in the cytoplasm, the fluorescence signal originating from this construct in the TIRFM evanescent field was negligible. The curve represents the whole-cell pHluorin fluorescence signal, expressed as ΔF/F₀ obtained averaging results from 5 and 6 cells, respectively. C–D. Histograms represent the amplitudes ΔF/F₀) of the cocktail-induced pHluorin fluorescence intensity change in cells transfected with control plasmids or with TeTX or BoNT/C.

Figure 5. Characteristics of stimulation-evoked exo-endocytosis and reacidification of SLMVs in MIN6B1 cells: whole-cell studies.

A. Time-course of cocktail-induced (glucose 20 mM; KCl 30 mM; forskolin 10 µM, IBMX 100 µM) pHluorin fluorescence intensity obtained with TIRF and EPI illumination. The curve represents the whole-cell pHluorin fluorescence signal, expressed as ΔF/F₀ obtained averaging results from 5 cells. B. Same as A. but in the presence of BafA1 (2.5 µM; n = 4 cells). The rate of spontaneous alkalinization (TIRF 0.08±0.006 ΔF/F₀; EPI 0.096±0.01 ΔF/F₀) was calculated in the pre-stimulus period and subtracted from the curves. Not that the curve under EPI represents a cumulative curve of exocytosis. C. Cumulative curve of estimated endocytosis (movement out of the TIRF field) obtained by subtracting the normalized curves of the pHluorin fluorescence signal measured under TIRF and under EPI both in the presence of BafA1 (curves in B). Curves in B were normalized to the maximum fluorescence obtained under EPI and TIRF, respectively in the presence of BafA1 for each cell. D. Cumulative curve of estimated reacidification obtained by subtracting the normalized curves of the pHluorin fluorescence signal obtained under EPI in the absence of BafA1 (curve in A) from the curve in the presence of the drug (curve in B). Curves in A and B were normalized to the maximum fluorescence obtained under EPI in the presence of BafA1 for each cell.

Figure 6. Stimulation triggers SLMV exocytosis in MIN6B1 cells: single vesicle studies.

A-B Temporal distribution of the fusion events on a 6000 pixel area (95 µm²)/300 ms before and after the bath application of the cocktail (glucose 20 mM; KCl 30 mM; forskolin 10 µM, IBMX 100 µM) or of KCl (30 mM). Note the significant increase in the frequency of pHluorin-mediated fusion events after bath application of both cocktail and KCl (n = 4 cells, from 0 to 75 sec, p<0.05 multiple comparisons, Dunnett test).

Figure 7. SLMV exocytosis triggered by 20 mM glucose: single vesicle studies.

Temporal distribution of the fusion events before and after the bath application of glucose (20 mM). Note the significant increase in the frequency of pHluorin-mediated fusion events after bath application of glucose (n = 5 cells, from 600 sec to 1800 sec, p<0.05 multiple comparisons, Dunnett test).

Figure 8. Two modes of exo-endocytosis: single vesicle studies.

A. Serial TIRF images illustrate typical type 1 and type 2 fusion events of SLMVs expressing syn-pHluorin. Time 0 represents the fusion event. Each frame corresponds to 150 milliseconds. The syn-pHluorin labelled vesicle start to fuse with the plasma membrane at 0. B. Histograms representing the ratio between type 1 and type 2 fusion events under basal conditions or in the presence of different stimuli (cocktail, KCl and glucose) (n = 5 cells, t-test, p<0.05).