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 agin....

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 se....

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