Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Summary

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

Abstract

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human islet samples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

Introduction

Pancreatic beta cells produce the insulin required to maintain normal glucose homeostasis, and their failure results in the development of all forms of diabetes. Beta cells retain a robust mitochondrial capacity to generate the energy required to couple glucose metabolism with insulin release. Recently, it has become apparent that the maintenance of functional mitochondrial mass is of pivotal importance for optimal beta cell function^1,2,3. In order to sustain functional mitochondrial mass, beta cells rely on quality control mechanisms to remove dysfunctional, damaged, or aging mitochondria⁴. We and others have previously demonstrated that beta cells rely on a specialized form of mitochondrial turnover, called mitochondrial autophagy (or mitophagy), to maintain mitochondrial quality control in both rodent and human islets^1,2,5. Unfortunately, however, there was no simple method to detect mitophagy, or endogenously expressed mitophagy components, in human pancreatic beta cells.

We have recently shown that upstream regulation of mitophagy in beta cells relies on formation of a protein complex comprising the E3 ligases CLEC16A and NRDP1 and the deubiquitinase USP8¹. NRDP1 and USP8 have been shown independently to affect mitophagy through action on the key mitophagy initiator PARKIN^6,7. NRDP1 targets PARKIN for ubiquitination and degradation to switch off mitophagy⁶, and USP8 specifically deubiquitinates K6-linked PARKIN to promote its translocation to mitochondria⁷. Proximity ligation assay (PLA) technology has been a recent advance in the field of protein interaction biology⁸, allowing visualization of endogenous protein interactions in situ in single cells, and is not limited by scarce sample material. This methodology is particularly enticing for human islet/beta cell biology, due to the sparsity of sample availability, coupled to the need for understanding physiologically relevant protein complexes within heterogeneous cell types.

Utilizing the PLA approach, we are able to observe key endogenous mitophagy complexes in primary human pancreatic beta cells and neuronal cell lines, and demonstrate the effects of a diabetogenic environment on the mitophagy pathway¹. In summary, the overarching goal of this protocol is to analyze specific mitophagy protein complexes in tissues lacking abundant material, or where conventional protein-interaction studies are not possible.

Protocol

Use of de-identified donor human pancreatic islets is via an Institutional Review Board (IRB) exemption and in compliance with University of Michigan IRB policy. Human pancreatic islets were provided by the NIH/NIDDK-sponsored Integrated Islet Distribution Program (IIDP).

1. Human islet sample preparation

1. Single cell dissociation
   1. Culture human islet samples (4000–6000 islet equivalents/10 mL media) for at least 1 day at 37 °C in pancreatic islet media (PIM(S)) media supplemented with 1 mM glutamine (PIM(G)), 100 units/mL antimycotic-antibiotic, 1 mM sodium pyruvate and 10 % fetal bovine serum (FBS) or human AB serum.
   2. Use a dissection light microscope at 3x magnification to count individual human islets from culture. Pick 40 islets per treatment/condition of interest (such as glucolipotoxicity) into 1.5 mL tubes in islet media.
   3. Centrifuge islets at 400 x g for 1 min at 10 °C to sediment.
   4. Wash samples, by brief inversion of tubes at room temperature, twice with 1 mL phosphate buffered saline (PBS) containing 50 μM PR619 (a deubiquitinase inhibitor; to preserve ubiquitin dependent protein interactions), with centrifugation between each wash at 400 x g for 1 min at 10 °C.
   5. Dissociate islets into single cells with 125 μL of 0.25% trypsin containing 50 μM PR619 by incubating prewarmed trypsin (37 °C) for 3 min with islet pellets. Gently disperse islets during this time with periodic gentle pipetting using a 200 μL pipette.
   6. Quench the trypsin with 1 mL warmed (37 °C) PIM(S) media containing 50 μM PR619, then sediment cells by centrifuging at 400 x g for 1 min.
   7. Wash cells by centrifugation at 400 x g for 1 min, twice, with PBS + 50 μM PR619.
   8. Finally, resuspend cells in 150 μL PBS containing 50 μM PR619.
2. Single cell adherence and fixation
   1. After resuspension, spin the cell solution onto frosted, charged, microscope slides using a cytocentrifuge at 28 x g for 10 min.
   2. Following cytocentrifugation, outline the cellular area with a hydrophobic pen (see the Table of Materials) to minimize antibody/PLA solution volumes needed. Fix cells with 4% paraformaldehyde in PBS for 15 min at room temperature.
      CAUTION: PFA is hazardous and must be handled with care.
   3. For best results, carry out staining/PLA immediately, or within 24 h. If necessary, store samples in PBS at 4 °C until staining for no longer than 48 h.

2. Immunohistochemistry

1. Blocking
   1. Wash cells twice with 1x PBS for 5 min at room temperature.
   2. Block cell solution, to eliminate background staining, with 10% donkey serum in PBS containing 0.3% detergent (see the Table of Materials) for 1 h at room temperature.
2. Staining
   1. Incubate cells with primary mouse or rabbit antibodies against USP8 (1:250) and NRDP1 (1:250) respectively (see the Table of Materials) diluted in phosphate buffered saline with detergent (PBT, see Table of Materials), at 4 °C overnight, to detect mitophagy complex signaling via PLA.
   2. Co-incubate cells with a marker specific for beta cell identification that was not raised in mouse or rabbit so as to not interfere with PLA signal. In this case incubate cells with anti-goat PDX1 (1:500) in PBT. Incubate all primary antibodies overnight at 4 °C, using plastic film on top of the solution to prevent evaporation.

3. Proximity ligation assay

1. PLA probe and counterstain
   1. Prepare the PLA probe solution according to manufacturer’s instructions, i.e., make 20 μL of probe solution per sample by preparing a final concentration of 1:5 solution of both anti-mouse and anti-rabbit probe solution in PBT, and incubating for 20 min at room temperature.
   2. After overnight incubation, wash cells twice with 1x PBS for 5 min each, on a rocker.
   3. Just before incubation with probe solution, add anti-goat Cy5 secondary at a final concentration of 1:600 to the probe solution (for detection of endogenous PDX1). Add 20 μL probe solution to each cell condition, cover gently with plastic film and incubate at 37 °C for 1 h.
2. Ligation
   1. Wash cells twice, at room temperature, with Buffer A (see the Table of Materials for the recipe) for 5 min each, on a rocker.
   2. Prepare ligation solution (part of the detection reagents, see Table of Materials) according to manufacturer’s instructions. For this, dilute ligation stock (5x) 1:5 in diethyl pyrocarbonate (DEPC)-treated water. Immediately before incubation add 0.025 U/mL ligase. Add 20 μL ligation solution to cells. Cover with plastic film and incubate at 37 °C for 30 min.
3. Amplification
   1. Wash cells twice at room temperature with Buffer A for 2 min each.
   2. Make amplification solution (part of the detection reagents, see Table of Materials) according to manufacturer’s instructions. Dilute amplification buffer (5x) 1:5 in DEPC-treated water and keep in the dark until use. Add a 1:80 dilution of polymerase (part of the detection reagents, see Table of Materials) to the solution immediately prior to adding the solution to the cells.
   3. Add 20 μL of amplification solution to the cells. Cover with plastic film and place in the dark at 37 °C for between 1 h 40 min to 2 h for maximum signal.
4. Preparation for imaging
   1. Wash cells twice with Buffer B (see the Table of Materials for recipe) for 10 min at room temperature, on a rocker.
   2. Finally, wash cells once in 0.01x Buffer B, for 2 min at room temperature on a rocker.
   3. Mount samples by adding a drop of mounting media containing 4′,6-diamidino-2-phenylindole (DAPI) and carefully placing a coverslip over the samples using a scalpel blade to press out any bubbles formed. Seal coverslips using clear nail polish around the edges.
   4. Image samples on a microscope capable of capturing multiple focal planes, such as a laser scanning confocal microscope, or an inverted fluorescence microscope with deconvolution capabilities, taking at least 9 different focal plane images.
      1. Capture images at 100x magnification, with approximately 0.45 μm z-stack height. Capture PLA at 550 nm excitation, 570 nm emission; counter-stain at 650 nm excitation, 670 nm emission; and DAPI at 405 nm excitation, 450 nm emission.
   5. To ensure fluorescence image background signals and stray light are minimized for downstream analysis of PLA events (especially on widefield microscopes), process images utilizing a two-dimensional deconvolution (nearest neighbor) algorithm through a standard image processing software of choice.
      NOTE: For Olympus use CellSens, Nikon use NIS-Elements, Leica use LAS X, Zeiss – ZEN, Metamorph, MATLAB, Huygens Software, as well as several Plugins available through Image-J. For analysis, the total number of interactions over all the z-stacks should be analyzed using Image-J software, ensuring only Pdx-1 positive cells are analyzed (section 3.5).
5. Quantification of PLA interactions
   1. Open the free Image-J software application, and open the PLA image. Start from the first sharply in-focus PLA image.
   2. Click the image tab, and select adjust, then threshold (Figure 1A). Adjust the threshold to remove all non-specific PLA signal, make a note of the threshold adjustment and try to keep it consistent throughout analysis.
   3. Click the process tab, and select binary, then make binary (Figure 1B). Use the binary image to measure particles, by clicking on the “analyze” tab and pressing analyze particles (Figure 1C).
   4. For analysis make sure the settings are as follows: Size = 0–Infinity, Circularity = 0–1.0, Show = Outlines, check boxes for Display results, and Clear results (Figure 1D). Click OK. Take note of the number of particles analyzed in a spreadsheet denoting sample, and z-stack position.
   5. Repeat steps 3.5.2–3.5.4 until all in-focus z-stacks for the sample have been analyzed. Sum the total number of particles for the sample in the spreadsheet to quantify total number of interactions.

Results

We conducted initial experiments in the MIN6 pancreatic beta cell line and the SH-SY5Y neuroblastoma cell line SH-SY5Y, to optimize and confirm both the specificity of the antibodies and the visualized protein interactions. MIN6 cells were plated onto coverslips at 30,000 cells/mL and left to adhere for 48 h, SH-SY5Y cells were plated onto coverslips at 15,000 cells/mL and left to adhere for 24 h. The PLA protocol was then followed as above, beginning at Step 1.2.3. To ensure the specific...

Discussion

Here we describe a simple and efficient approach to use NRDP1:USP8 PLA in tissues/cells of interest to quantify formation of upstream mitophagy complexes. We previously confirmed the formation of the CLEC16A-NRDP1-USP8 mitophagy complex in pancreatic beta cells by several methodologies, including co-immunoprecipitation experiments, cell-free interaction studies, and in vitro as well as cell-based ubiquitination assays, and demonstrated how this complex drives regulated mitophagic flux^1,...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25% trypsin-EDTA 1X	Life Technologies	25200-056
Antibiotic-Antimycotic	Life Technologies	15240-062
Block solution	Homemade		Use 1X PBS, add 10 % donkey serum, and 0.3% Triton X-100 detergent.
Buffer A	Homemade		To make 1L: Mix 8.8g NaCl, 1.2g Tris base, 500ul Tween-20, with 750mL ddH20. pH to 7.5 with HCl, and fill to 1L. Filter solution and store at 4C. Bring to RT before experimental use
Buffer B	Homemade		To make 1L: Mix 5.84g NaCl, 4.24g Tris base, 26g Tris-HCl with 500mL ddH20. pH to 7.5, and fill to 1L. Filter solution and store a 4C. Bring to RT before experimental use
Cy5-conjugated AffiniPure donkey anti-goat	Jackson Labs	705-175-147
Detection Reagents Red	Sigma- Aldrich	DU092008-100RXN	Kit containing: ligation solution stock (5X), ligase, amplification solution stock (5X) and polymerase.
DuoLink PLA probe anti-mouse MINUS	Sigma- Aldrich	DU092004-100RXN
DuoLink PLA probe anti-rabbit PLUS	Sigma- Aldrich	DU092002-100RXN
Fetal bovine serum
Goat polyclonal anti-PDX1 (clone A17)	Santa Cruz	SC-14664	RRID: AB_2162373
HEPES (1M)	Life Technologies	15630-080
MIN6 pancreatic cell line	Gift from D. Stoffers		Mouse insulinoma cell line, utilized for cell-based assays.
Mouse monoclonal anti-USP8 antibody (clone US872)	Sigma- Aldrich	SAB200527
Pap-pen	Research Products International	195505
Parafilm			Use to seal antibody and probe solutions on your cells to prevent evaporation when using small solution volumes.
PBT (phosphate buffered saline with triton)	Homemade		To make 50mL: 43.5mLddH2O, 5mL 10X PBS, 0.5mL 10X BSA(100mg/mL solution), 1mL 10% triton X-100 solution in ddH20)
Penicillin-Streptomycin (100X)	Life Technologies	15140-122
Phosphate buffered saline, 10X	Fisher Scientific	BP399-20
PIM(ABS) Human AB serum	Prodo Labs	PIM-ABS001GMP
PIM(G) (glutamine)	Prodo Labs	PIM-G001GMP
PIM(S) media	Prodo Labs	PIM-S001GMP
PR619	Apex Bio	A812
Prolong Gold antifade reagent with DAPI	Life Technologies (Molecular Probes)	P36935
Rabbit polyclonal anti-FLRF/RNF41 (Nrdp1)	Bethyl Laboratories	A300-049A	RRID: AB_2181251
SH-SY5Y cells	Gift from L. Satin		Human neuroblastoma cell line, utilized for cell-based assays.
Sodium Pyruvate (100X)	Life Technologies	11360-070
Triton X-100	Fisher Scientific	BP151-100
Tween-20	Fisher Scientific	BP337-100
Water for RNA work (DEPC water)	Fisher Scientific	BP361-1L