Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets

Summary

We describe a protocol for visualization of insulin exocytosis in intact islets using pHluorin, a pH-sensitive green fluorescent protein. Isolated islets are infected with adenovirus encoding pHluorin coupled to the vesicle cargo neuropeptide Y. This allows for the detection of insulin granule fusion events by confocal microscopy.

Abstract

Insulin secretion plays a central role in glucose homeostasis under normal physiological conditions as well as in disease. Current approaches to study insulin granule exocytosis either use electrophysiology or microscopy coupled to the expression of fluorescent reporters. However most of these techniques have been optimized for clonal cell lines or require dissociating pancreatic islets. In contrast, the method presented here allows for real time visualization of insulin granule exocytosis in intact pancreatic islets. In this protocol, we first describe the viral infection of isolated pancreatic islets with adenovirus that encodes a pH-sensitive green fluorescent protein (GFP), pHluorin, coupled to neuropeptide Y (NPY). Second, we describe the confocal imaging of islets five days after viral infection and how to monitor the insulin granule secretion. Briefly, the infected islets are placed on a coverslip on an imaging chamber and imaged under an upright laser-scanning confocal microscope while being continuously perfused with extracellular solution containing various stimuli. Confocal images spanning 50 µm of the islet are acquired as time-lapse recordings using a fast-resonant scanner. The fusion of insulin granules with the plasma membrane can be followed over time. This procedure also allows for testing a battery of stimuli in a single experiment, is compatible with both mouse and human islets, and can be combined with various dyes for functional imaging (e.g., membrane potential or cytosolic calcium dyes).

Introduction

Insulin is produced by the beta cells of the pancreatic islet and it is a key regulator of glucose metabolism¹. Death or dysfunction of beta cells disturbs glucose homeostasis and leads to diabetes². Insulin is packed in dense-core granules that are released in a Ca²⁺-dependent manner³. Elucidating how insulin granule exocytosis is regulated is essential to fully understand what determines insulin secretion and opens new avenues for the identification of novel therapeutic targets for the treatment of diabetes.

Insulin exocytosis has been extensively studied using electrophysiological approaches, such as membrane capacitance measurements, and microscopic approaches in combination with fluorescent molecules. Membrane capacitance measurements have good temporal resolution and allow single cell recordings. However, changes in the capacitance reflect the net surface change of the cell and do not capture individual fusion events or distinguish insulin granule fusion from other non-insulin secretory vesicles³. Microscopic approaches, such as two-photon or total internal reflection fluorescence (TIRF) microscopy in combination with fluorescent probes and vesicle cargo proteins, provide additional detail. These techniques capture single exocytotic events and also the pre- and post-exocytotic stages and can be used for studying exocytotic patterns in populations of cells³.

Fluorescent reporters can be of three types: 1) extracellular, 2) cytoplasmic, or 3) vesicular. 1) Extracellular reporters are polar tracers (e.g., dextrans, sulforhodamine B (SRB), lucifer yellow, pyranine) that can be introduced through the extracellular milieu⁴. The use of polar tracers allows for the investigation of the fusion pore in a population of cells and captures various intercellular structures such as blood vessels. However, they do not report on vesicle cargo behavior. 2) Cytoplasmic reporters are fluorescent probes coupled to membrane-associated proteins that face the cytoplasm and are involved in docking and exocytosis. Examples include members of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family that have been successfully used in neuroscience for studying neurotransmitter release⁵. Such proteins have multiple binding partners and are not insulin-granule specific. 3) Vesicular reporters are fluorescent probes fused to vesicular cargo proteins that allow for the investigation of cargo-specific vesicle behavior. Insulin-granule specific cargo proteins include insulin, c-peptide, islet amyloid polypeptide, and NPY among others^6,7. NPY is only present in insulin containing granules, and is co-released with insulin, making it an excellent partner for a fluorescent reporter⁸.

The fusion of different fluorescent proteins to NPY has been previously employed to study various aspects of exocytosis in neuroendocrine cells, such as the requirement of specific synaptotagmin isoforms^9,10 and how the time-course of release depends on the actin cytoskeleton and on myosin II^11,12. In this study, we chose pHluorin as the fluorescent reporter, which is a modified GFP that is non-fluorescent at the acidic pH inside dense core granules but becomes brightly fluorescent upon exposure to the neutral extracellular pH¹³. Mature insulin granules have an acidic pH below 5.5. Once the granule fuses with the plasma membrane and opens, its cargo is exposed to the neutral extracellular pH of 7.4, allowing the use of the pH-sensitive proteins pHluorin as a reporter^7,14.

In view of the pH sensitive nature of pHluorin and the selective expression of NPY in insulin granules, the NPY-pHluorin fusion construct can be used to study various properties of insulin granule exocytosis. The viral delivery of the fusion construct ensures high transfection efficiency and works on primary beta cells or cell lines as well as on isolated islets. This method can also be used as a guideline for studying exocytosis in any other cell type with NPY-containing vesicles. It can also be combined with any transgenic mouse model to study effects of certain conditions (knockdowns, overexpression, etc.) on exocytosis. This technique has been previously used to characterize spatial and temporal patterns of insulin granule secretion in beta cell populations in human islets¹⁵.

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Protocol

The animal ethics committee of the University of Miami has approved all the experiments.

1. Viral Infection of Intact Isolated Human or Mouse Pancreatic Islets

1. Islet culture: prepare the islet culture media: Connaught Medical Research Laboratories (CMRL) 1066, 10% (v/v) FBS and 2 mM L-Glutamine.
   1. Human pancreatic islets are obtained from the Integrated Islet Distribution Program (NIDDK, NIH). Upon arrival, transfer the islets (~ 500 islet equivalents) to 35-mm non-tissue culture treated Petri dishes with 2 mL CMRL culture media at 37 °C, 5%/95% CO₂/O₂ for 24 h before viral infection.
   2. Mouse pancreatic islets can be isolated following previously established protocols¹⁶. Following isolation, culture ~ 200 islet equivalents in 35-mm non-tissue culture treated Petri dishes with 2 mL CMRL culture media at 37 °C, 5%/95% CO₂/O₂ for 24 h before viral infection.
      NOTE: Avoid using transgenic mice with GFP or YFP reporters expressed on islets to avoid fluorescence overlap with NPY-pHluorin.
2. Virus preparation
   NOTE: The NPY-pHluorin fusion was cloned into the pcDNA3 vector¹⁰ and subcloned into an adenoviral vector for adenoviral production [adenovirus serotype 5 (DE1/E3)] by a recombinant adenovirus manufacturing company. The virus was aliquoted and stored at -80 °C. The viral stock is provided by the company at titers of 10¹² to 10¹³ viral particles (~ 3 x 10¹⁰ - 3 x 10¹¹ PFU).
   1. For in vitro islet infection, use 10⁶ PFU/mL, resulting in an approximate multiplicity of infection (MOI) of around 2 (see Discussion for details)
3. Viral infection of pancreatic islets
   Caution: Working with adenoviruses requires Biosafety Level 2 (BSL2) procedures and certification. Check with the Institutional Biosafety Officer for guidance and training on BSL2 procedures.
   1. Prepare human/mouse islets as described above.
   2. Add 5 - 10 µL of stock virus to each 35-mm Petri dish containing human/mouse islets in 2 mL of CMRL culture media (with 10% FBS and 2 mM L-Glutamine).
      NOTE: Adjust the volume of the virus used according to the viral titer as provided by the company datasheet.
   3. Culture the islets in virus-containing media at 37 °C/5% CO₂ for 24 h.
   4. After 24 h, aspirate the virus-containing media and replace with 2 mL CMRL culture media (with 10% FBS and 2 mM L-Glutamine).
   5. Culture the islets for 4 - 6 days at 37 °C/5% CO₂, replacing the media every 3 days.
   6. After 4 - 6 days of culturing, expect around 30% of islet cells to be infected. Islets can then be used for live imaging experiments.

2. Confocal Imaging of Infected Islets

NOTE: Refer to the Table of Materials for the materials and equipment required for confocal imaging.

1. Reagent preparation and experimental setup
   1. Prepare extracellular solution: add 125 mM NaCl, 5.9 mM KCl, 2.56 mM CaCl₂, 1 mM MgCl₂, 25 mM HEPES, 0.1% BSA, pH 7.4, sterile filtered.
      NOTE: This buffer is normally prepared without glucose and can be stored at 4 °C for up to 1 month. Glucose is added on the day of the experiment to reach the desired final concentration.
      1. Prepare basal glucose (3 mM) medium: add 75 µL of 2 M glucose stock to 50 mL of extracellular solution.
      2. Hyperglycemic (16 mM) medium: add 400 µL of 2 M glucose stock to 50 mL of extracellular solution
   2. Dilute any additional stimulus (e.g., KCl or adenosine triphosphate (ATP)) in extracellular solution containing 3 mM glucose.
   3. Before starting an experiment, pretreat the coverslips with poly-D-lysine by adding 30 µL of poly-D-Lysine solution (1 mg/mL) to the coverslip for 1 h and thoroughly rinsing it with H₂O.
      NOTE: The poly-lysine coated coverslips can be stored at room temperature for up to 6 months.
   4. At least 1 h before the experiment, using a 1 mL pipette, transfer the islets to a 35-mm Petri dish containing extracellular solution with 3 mM glucose. Keep the islets at 37 °C and 5% CO₂.
      NOTE: If needed, the plasma membrane can be labeled in this step. To label the plasma membrane, add 2 µM di-8-ANEPP dye to the extracellular solution with 3 mM glucose. Incubate the islets in dye solution for 1 h at 37 °C/5% CO₂. The plasma membrane dye can be excited at 488 nm and detected at 620 nm.
   5. min before starting an experiment, attach the coverslip to the imaging chamber by sealing it with vacuum silicone grease. Fix the imaging chamber to the imaging platform. Using a pipette, place 20 - 30 islets on the poly-D-Lysine-treated area of the coverslip and let the islets adhere to the surface for 20 min.
      NOTE: It is important not to let the coverslip dry completely to avoid islet damage.
   6. While the islets are adhering to the coverslip, prepare the perfusion system by thoroughly rinsing it with water. Add each solution to a different channel: 3 mM glucose (channel 1), 16 mM glucose (channel 2), 16 mM glucose with 100 µM 3-isobutyl-1-methylxanthine (IBMX) and 10 µM forskolin (channel 3), 25 mM KCl in 3 mM glucose (channel 4), 10 µM ATP in 3 mM glucose (channel 5). Remove all the bubbles from the system by opening each channel separately and letting the solution flow for a few minutes, and make sure that the flow is consistent (0.5 mL/min) and the tubing is not leaking.
   7. Connect the single inline solution heater to the perfusion outlet tube and adjust the temperature of the outflowing buffer to 37 °C.
   8. Prepare the suction pump. Remove all the bubbles from the system, and make sure that the flow is consistent and the tubing is not leaking.
   9. Once the islets adhere to the coverslip surface, gently fill up the imaging chamber with the extracellular solution containing 3 mM glucose. Avoid washing the islets away from the surface of the coverslip.
   10. Place the imaging platform with the islets onto the microscope stage and connect it to the perfusion system and suction pump.
   11. Turn on the flow and constantly perfuse the islets with the 3G extracellular solution. The system is now ready for confocal imaging.
2. Confocal imaging
   1. Locate the islets in the microscope field with lower magnification. Once focused on the islets, switch to higher magnification objectives (e.g., 63X water immersion objective (63X/0.9 NA)).
   2. Open the acquisition using software (Table of Materials) and activate the resonant scanner mode.
   3. Select the XYZT imaging mode and configure the acquisition settings as follows:
      1. Turn on the Argon laser and the 488 nm laser line and adjust the laser power to 50% for pHluorin excitation.
      2. Collect emission at 505 - 555 nm.
      3. Choose a resolution of 512 x 512 pixels. Press the "Live" button to start imaging and adjust the gain levels (typical gain is around 600 V).
      4. Set the begin and end of the z-stack: focus on the top of the islet and choose "begin" and move to the last plane that can be focused and choose "end". Use a z-step size of 5 µm. The software will automatically calculate the number of confocal planes.
      5. Set the time interval for acquisition of each z-stack close to 1.5 - 2 s and choose the option "Acquire until stopped" for continuous imaging.
      6. Press the "start" button to initialize.
   4. Use various stimulation protocols to induce granule exocytosis by perfusing the islets with the desired stimuli. Stimulation protocols can be customized to fit the desired scientific purpose (see below).
3. Stimulation protocols
   NOTE: In every stimulation protocol, start by recording at least 2 min of islet background activity during constant perfusion with extracellular solution containing 3 mM glucose. Perfuse with a stimulant for the desired period of time. The order of stimulants, duration of stimulation as well as duration of recordings can be customized to fit the desired scientific purposes. Make sure to wash islets thoroughly with extracellular solution containing 3 mM glucose before starting a new stimulation. Below find the sample stimulation protocols that have been used to demonstrate the method capabilities.
   1. Stimulate with ammonium chloride (NH₄Cl) as a positive control for pHluorin pH sensitivity and viral infection efficiency (Figure 3): 3 mM glucose (2 min) → 50 mM NH₄Cl (2 min) in 3 mM glucose → 3 mM glucose (2 min)
      NOTE: In the NH₄Cl solution, replace NaCl on an equimolar basis.
   2. Stimulate insulin granule exocytosis by increasing the glucose concentration (Figure 5 and Figure 6): 3 mM glucose (2 min) → 16 mM glucose (15-30 min) → 3 mM glucose (2 min
      NOTE: In order to see several bursts of activity, perfuse the islets continuously with the 16G solution for at least 15 min.
      NOTE: In order to increase the consistency of secretory responses¹⁷, users may add cAMP-raising agents (100 µM IBMX and 10 µM forskolin) to both the 3G and 16G solutions. This does not change the temporal patterns of granule secretion. For details see¹⁵ and Discussion.

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Results

The entire workflow of the technique is shown in Figure 1. Briefly, mouse or human islets can be infected with adenovirus encoding NPY-pHluorin and imaged, after few days in culture, under a confocal microscope. As granules fuse with plasma membrane and open, an increase in fluorescence is observed and can be quantified (Figure 1). To determine if NPY-pHluorin is indeed a suitable tool to monitor insulin granule dynamics, infecte...

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Discussion

This manuscript describes a technique that can be used to visualize exocytosis of insulin granules in beta cells within intact pancreatic islets by confocal microscopy. It uses NPY-pHluorin as the fluorescent reporter cloned into adenovirus to ensure a high transfection efficiency.

Although the method was highly efficient in our hands, it might require some modifications that primarily depend on two parameters: 1) the quality of islet preparation, and 2) the titer of viral stock with optimizat...

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Materials

Name	Company	Catalog Number	Comments
Upright laser-scanning confocal microscope	Leica Microsystems, Wetzlar, Germany	TCS-SP5	includes LAS AF, the image acquisition software
Imaging chamber	Warner instruments	RC-26
Imaging chamber platform	Warner instruments	PH-1
22 x 40 glass coverslips	Daiggerbrand	G15972H
Vacuum silicone grease	Sigma	Z273554-1EA
Multichannel perfusion system	Warner instruments	VC-8
Single inline solution heater	Warner instruments	SH-27B
Temperature controller	Warner instruments	TC-324C
Peristaltic Suction pump	Pharmacia	P-1
35 mm Petri dish, non-tissue culture treated	VWR	10861-586
CMRL Medium, no glutamine	ThermoFisher	11530037
FBS, heat inactivated	ThermoFisher	16140071
L-Glutamine 200 mM	ThermoFisher	25030081
5 M NaCl solution	Sigma	S5150
3 M KCl solution	Sigma	60135
1 M CaCl2 solution	Sigma	21115
1 M MgCl2 solution	Sigma	M1028
Bovine Serum Albumin	Sigma	A2153
1 M HEPES solution	Sigma	H0887
Vacuum filter	VWR	431098
D-Glucose	Sigma	G8270
Poly-D-lysine hydrobromide	Sigma-aldrich	P6407
Di-8-ANNEP	ThermoFisher	D3167
3-isobutyl-1-methylxanthine (IBMX)	Sigma	I5879
Forskolin	Sigma	F3917