The authors acknowledge funding support from JDRF (CDA-2016-189 and SRA-2018-539), the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (R01-DK-108921), the Brehm family, and the Anthony family. The JDRF Career Development Award to S.A.S. is partly supported by the Danish Diabetes Academy, which is supported by the Novo Nordisk Foundation.

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
<ol>
	<li>Single cell dissociation
	<ol>
		<li>Culture human islet samples (4000&#8211;6000 islet equivalents/10 mL media) for at least 1 day at 37 &#176;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.</li>
		<li>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.</li>
		<li>Centrifuge islets at 400 x g for 1 min at 10 &#176;C to sediment.</li>
		<li>Wash samples, by brief inversion of tubes at room temperature, twice with 1 mL phosphate buffered saline (PBS) containing 50 &#956;M PR619 (a deubiquitinase inhibitor; to preserve ubiquitin dependent protein interactions), with centrifugation between each wash at 400 x g for 1 min at 10 &#176;C.</li>
		<li>Dissociate islets into single cells with 125 &#956;L of 0.25% trypsin containing 50 &#956;M PR619 by incubating prewarmed trypsin (37 &#176;C) for 3 min with islet pellets. Gently disperse islets during this time with periodic gentle pipetting using a 200 &#956;L pipette.</li>
		<li>Quench the trypsin with 1 mL warmed (37 &#176;C) PIM(S) media containing 50 &#956;M PR619, then sediment cells by centrifuging at 400 x g for 1 min.</li>
		<li>Wash cells by centrifugation at 400 x g for 1 min, twice, with PBS + 50 &#956;M PR619.</li>
		<li>Finally, resuspend cells in 150 &#956;L PBS containing 50 &#956;M PR619.</li>
	</ol>
	</li>
	<li>Single cell adherence and fixation
	<ol>
		<li>After resuspension, spin the cell solution onto frosted, charged, microscope slides using a cytocentrifuge at 28 x g for 10 min.</li>
		<li>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.</li>
		<li>For best results, carry out staining/PLA immediately, or within 24 h. If necessary, store samples in PBS at 4 &#176;C until staining for no longer than 48 h.</li>
	</ol>
	</li>
</ol>
2. Immunohistochemistry
<ol>
	<li>Blocking
	<ol>
		<li>Wash cells twice with 1x PBS for 5 min at room temperature.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Staining
	<ol>
		<li>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 &#176;C overnight, to detect mitophagy complex signaling via PLA.</li>
		<li>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 &#176;C, using plastic film on top of the solution to prevent evaporation.</li>
	</ol>
	</li>
</ol>
3. Proximity ligation assay
<ol>
	<li>PLA probe and counterstain
	<ol>
		<li>Prepare the PLA probe solution according to manufacturer&#8217;s instructions, i.e., make 20 &#956;L 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.</li>
		<li>After overnight incubation, wash cells twice with 1x PBS for 5 min each, on a rocker.</li>
		<li>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 &#956;L probe solution to each cell condition, cover gently with plastic film and incubate at 37 &#176;C for 1 h.</li>
	</ol>
	</li>
	<li>Ligation
	<ol>
		<li>Wash cells twice, at room temperature, with Buffer A (see the Table of Materials for the recipe) for 5 min each, on a rocker.</li>
		<li>Prepare ligation solution (part of the detection reagents, see Table of Materials) according to manufacturer&#8217;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 &#956;L ligation solution to cells. Cover with plastic film and incubate at 37 &#176;C for 30 min.</li>
	</ol>
	</li>
	<li>Amplification
	<ol>
		<li>Wash cells twice at room temperature with Buffer A for 2 min each.</li>
		<li>Make amplification solution (part of the detection reagents, see Table of Materials) according to manufacturer&#8217;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.</li>
		<li>Add 20 &#956;L of amplification solution to the cells. Cover with plastic film and place in the dark at 37 &#176;C for between 1 h 40 min to 2 h for maximum signal.</li>
	</ol>
	</li>
	<li>Preparation for imaging
	<ol>
		<li>Wash cells twice with Buffer B (see the Table of Materials for recipe) for 10 min at room temperature, on a rocker.</li>
		<li>Finally, wash cells once in 0.01x Buffer B, for 2 min at room temperature on a rocker.</li>
		<li>Mount samples by adding a drop of mounting media containing 4&#8242;,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.</li>
		<li>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.
		<ol>
			<li>Capture images at 100x magnification, with approximately 0.45 &#956;m 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.</li>
		</ol>
		</li>
		<li>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 &#8211; 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).</li>
	</ol>
	</li>
	<li>Quantification of PLA interactions
	<ol>
		<li>Open the free Image-J software application, and open the PLA image. Start from the first sharply in-focus PLA image.</li>
		<li>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.</li>
		<li>Click the process tab, and select binary, then make binary (Figure 1B). Use the binary image to measure particles, by clicking on the &#8220;analyze&#8221; tab and pressing analyze particles (Figure 1C).</li>
		<li>For analysis make sure the settings are as follows: Size = 0&#8211;Infinity, Circularity = 0&#8211;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.</li>
		<li>Repeat steps 3.5.2&#8211;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.</li>
	</ol>
	</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+regulation+of+&#946;-cell+function:+Maintaining+the+momentum+for+insulin+release.">Mitochondrial regulation of &#946;-cell function: Maintaining the momentum for insulin release.</a> Molecular Aspects of Medicine. , (2015).</li><li>Hattori, N., Saiki, S., Imai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+by+mitophagy.">Regulation by mitophagy.</a> The International Journal of Biochemistry &amp; Cell Biology. 53, 147-150 (2014).</li><li>Soleimanpour, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetes+Susceptibility+Genes+Pdx1+and+Clec16a+Function+in+a+Pathway+Regulating+Mitophagy+in+beta-Cells.">Diabetes Susceptibility Genes Pdx1 and Clec16a Function in a Pathway Regulating Mitophagy in beta-Cells.</a> Diabetes. 64 (10), 3475-3484 (2015).</li><li>Zhong, L., Tan, Y., Zhou, A., Yu, Q., Zhou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RING+Finger+Ubiquitin-Protein+Isopeptide+Ligase+Nrdp1/FLRF+Regulates+Parkin+Stability+and+Activity.">RING Finger Ubiquitin-Protein Isopeptide Ligase Nrdp1/FLRF Regulates Parkin Stability and Activity.</a> Journal of Biological Chemistry. 280 (10), 9425-9430 (2005).</li><li>Durcan, T. 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A., Deering, T. G., Mirmira, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+feat+of+metabolic+proportions:+Pdx1+orchestrates+islet+development+and+function+in+the+maintenance+of+glucose+homeostasis.">A feat of metabolic proportions: Pdx1 orchestrates islet development and function in the maintenance of glucose homeostasis.</a> Molecular Genetics and Metabolism. 92 (1), 43-55 (2007).</li><li>Poitout, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucolipotoxicity+of+the+pancreatic+beta+cell.">Glucolipotoxicity of the pancreatic beta cell.</a> Biochimica Biophysica Acta. 1801 (3), 289-298 (2010).</li><li>Pearson, G., Soleimanpour, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+ubiquitin-dependent+mitophagy+complex+maintains+mitochondrial+function+and+insulin+secretion+in+beta+cells.">A ubiquitin-dependent mitophagy complex maintains mitochondrial function and insulin secretion in beta cells.</a> Autophagy. 14 (7), 1160-1161 (2018).</li><li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+transcriptomes+reveal+characteristic+features+of+human+pancreatic+islet+cell+types.">Single-cell transcriptomes reveal characteristic features of human pancreatic islet cell types.</a> EMBO Reports. 17 (2), 178-187 (2016).</li><li>DiGruccio, M. 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The authors have nothing to disclose.

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 flux1,...

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 function1,2,3. 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 islets1,2,5. 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 PARKIN6,7. 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&#160;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.

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

visualization of endogenous mitophagy complexes in situ in human pancreatic beta cells utilizing proximity ligation assay

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&#160;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.

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...

Watch this Scientific Journal Video about Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay at JoVE.com

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

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.

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...

visualization-endogenous-mitophagy-complexes-situ-human-pancreatic

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

5.8K Views.  University of Michigan, Ann Arbor. 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.

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

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

In situ Quantification of Pancreatic Beta-cell Mass in Mice

Tracing changes of specific cell populations in health and disease is an important goal of biomedical research. The process of monitoring pancreatic beta-cell proliferation and islet growth is particularly challenging. We have developed a method to capture the distribution of beta-cells in the intact pancreas of transgenic mice with fluorescence-tagged beta-cells with a macro written for ImageJ (rsb.info.nih.gov/ij/). Following pancreatic dissection and tissue clearing, the entire pancreas is captured as a virtual slice, after which the GFP-tagged beta-cells are examined. The analysis includes the quantification of total beta-cell area, islet number and size distribution with reference to specific parameters and locations for each islet and for small clusters of beta-cells. The entire distribution of islets can be plotted in three dimensions, and the information from the distribution on the size and shape of each islet allows a quantitative and qualitative comparison of changes in overall beta-cell area at a glance.

In situ Quantification of Pancreatic Beta-cell Mass in Mice

The following protocol outlines the process of pancreatic dissection for virtual slice imaging, and the subsequent quantification of all GFP-tagged beta-cells in the entire pancreas.

Tracing changes of specific cell populations in health and disease is an important goal of biomedical research. The process of monitoring pancreatic ...

Biology

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

Neuroscience

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Characterization of Neuronal Lysosome Interactome with Proximity Labeling Proteomics

Lysosomes frequently communicate with a variety of biomolecules to achieve the degradation and other diverse cellular functions. Lysosomes are critical to human brain function, as neurons are postmitotic and rely heavily on the autophagy-lysosome pathway to maintain cellular homeostasis. Despite advancements in the understanding of various lysosomal functions, capturing the highly dynamic communications between lysosomes and other cellular components is technically challenging, particularly in a high-throughput fashion. Here, a detailed protocol is provided for the recently published endogenous (knock-in) lysosome proximity labeling proteomic method in human induced pluripotent stem cell (hiPSC)-derived neurons. 
Both lysosomal membrane proteins and proteins surrounding lysosomes within a 10-20 nm radius can be confidently identified and accurately quantified in live human neurons. Each step of the protocol is described in detail, i.e., hiPSC-neuron culture, proximity labeling, neuron harvest, fluorescence microscopy, biotinylated protein enrichment, protein digestion, LC-MS analysis, and data analysis. In summary, this unique endogenous lysosomal proximity labeling proteomics method provides a high-throughput and robust analytical tool to study the highly dynamic lysosomal activities in live human neurons.

A neuronal lysosome proximity labeling proteomics protocol is described here to characterize the dynamic lysosomal microenvironment in human induced pluripotent stem cell-derived neurons. Lysosomal membrane proteins and proteins that interact with lysosomes (stably or transiently) can be accurately quantified in this method with excellent intracellular spatial resolution in live human neurons.

Lysosomes frequently communicate with a variety of biomolecules to achieve the degradation and other diverse cellular functions. Lysosomes are critical to ...

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), are crucial hubs for cellular signaling and cell fate. Particularly, these inter-organelle contact sites allow the transfer of calcium from the ER to mitochondria through the voltage-dependent anion channel (VDAC)/glucose-regulated protein 75 (GRP75)/inositol 1,4,5-triphosphate receptor (IP3R) calcium channeling complex. While this subcellular compartment is under intense investigation in both physiological and pathological conditions, no simple and sensitive method exists to quantify the endogenous amount of ER-mitochondria contact in cells. Similarly, MAMs are highly dynamic structures, and there is no suitable approach to follow modifications of ER-mitochondria interactions without protein overexpression. Here, we report an optimized protocol based on the use of an in situ proximity ligation assay to visualize and quantify endogenous ER-mitochondria interactions in fixed cells by using the close proximity between proteins of the outer mitochondrial membrane (VDAC1) and of the ER membrane (IP3R1) at the MAM interface. Similar in situ proximity ligation experiments can also be performed with the GRP75/IP3R1 and cyclophilin D/IP3R1 pairs of antibodies. This assay provides several advantages over other imaging procedures, as it is highly specific, sensitive, and suitable to multiple-condition testing. Therefore, the use of this in situ proximity ligation assay should be helpful to better understand the physiological regulations of ER-mitochondria interactions, as well as their role in pathological contexts.

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Here, we describe a procedure to visualize and quantify with high sensitivity the endogenous interactions between the endoplasmic reticulum and mitochondria in fixed cells. The protocol features an optimized in situ proximity ligation assay targeting the inositol 1,4,5-triphosphate receptor/glucose-regulated protein 75/voltage-dependent anion channel/cyclophilin D complex at the mitochondria-associated membrane interface.

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), ...

Visualizing Mitophagy with Fluorescent Dyes for Mitochondria and Lysosome

Mitochondria, being the powerhouses of the cell, play important roles in bioenergetics, free radical generation, calcium homeostasis, and apoptosis. Mitophagy is the primary mechanism of mitochondrial quality control and is generally studied using microscopic observation, however in vivo mitophagy assays are difficult to perform. Evaluating mitophagy by imaging live organelles is an alternative and necessary method for mitochondrial research. This protocol describes the procedures for using the cell-permeant green-fluorescent mitochondria dye MitoTracker Green and the red-fluorescent lysosome dye LysoTracker Red in live cells, including the loading of the dyes, visualization of the mitochondria and the lysosome, and expected outcomes. Detailed steps for the evaluation of mitophagy in live cells, as well as technical notes about microscope software settings, are also provided. This method can help researchers observe mitophagy using live-cell fluorescent microscopy. In addition, it can be used to quantify mitochondria and lysosomes and assess mitochondrial morphology.

Mitophagy is the primary mechanism of mitochondrial quality control. However, the evaluation of mitophagy in vivo is hindered by the lack of reliable quantitative assays. Presented here is a protocol for the observation of mitophagy in living cells using a cell-permeant green-fluorescent mitochondria dye and a red-fluorescent lysosome dye.

Mitochondria, being the powerhouses of the cell, play important roles in bioenergetics, free radical generation, calcium homeostasis, and apoptosis. ...

Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence

Autophagy is a specialized catabolic process that selectively degrades cytoplasmic components, including proteins and damaged organelles. Autophagy allows cells to physiologically respond to stress stimuli and, thus, maintain cellular homeostasis. Cancer cells might modulate their autophagy levels to adapt to adverse conditions such as hypoxia, nutrient deficiency, or damage caused by chemotherapy. Ductal pancreatic adenocarcinoma is one of the deadliest types of cancer. Pancreatic cancer cells have high autophagy activity due to the transcriptional upregulation and post-translational activation of autophagy proteins.
Here, the PANC-1 cell line was used as a model of pancreatic human cancer cells, and the AR42J pancreatic acinar cell line was used as a physiological model of highly differentiated mammalian cells. This study used the immunofluorescence of microtubule-associated protein light chain 3 (LC3) as an indicator of the status of autophagy activation. LC3 is an autophagy protein that, in basal conditions, shows a diffuse pattern of distribution in the cytoplasm (known as LC3-I in this condition). Autophagy induction triggers the conjugation of LC3 to phosphatidylethanolamine on the surface of newly formed autophagosomes to form LC3-II, a membrane-bound protein that aids in the formation and expansion of autophagosomes. To quantify the number of labeled autophagic structures, the open-source software FIJI was utilized with the aid of the &#34;3D Objects Counter&#34; tool.
The measure of the autophagic levels both in physiological conditions and in cancer cells allows us to study the modulation of autophagy under diverse conditions such as hypoxia, chemotherapy treatment, or the knockdown of certain proteins.

The goal of this protocol is to determine autophagic levels in pancreatic cancer and pancreatic acinar cells through LC3 immunofluorescence and LC3 dot quantification.

Autophagy is a specialized catabolic process that selectively degrades cytoplasmic components, including proteins and damaged organelles. Autophagy allows ...

Quantitative Analysis of Mitophagy in Murine Pancreatic &#946;-Cells

Complementary Approaches to Interrogate Mitophagy Flux in Pancreatic &#946;-Cells

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient insulin release. Advanced quantitative assessments of mitophagy often require the use of genetic reporters. The mt-Keima mouse model, which expresses a mitochondria-targeted pH-sensitive dual-excitation ratiometric probe for quantifying mitophagy via flow cytometry, has been optimized in &#946;-cells. The ratio of acidic-to-neutral mt-Keima wavelength emissions can be used to robustly quantify mitophagy. However, using genetic mitophagy reporters can be challenging when working with complex genetic mouse models or difficult-to-transfect cells, such as primary human islets. This protocol describes a novel complementary dye-based method to quantify &#946;-cell mitophagy in primary islets using MtPhagy. MtPhagy is a pH-sensitive, cell-permeable dye that accumulates in the mitochondria and increases its fluorescence intensity when mitochondria are in low pH environments, such as lysosomes during mitophagy. By combining the MtPhagy dye with Fluozin-3-AM, a Zn2+ indicator that selects for &#946;-cells, and Tetramethylrhodamine, ethyl ester (TMRE) to assess mitochondrial membrane potential, mitophagy flux can be quantified specifically in &#946;-cells via flow cytometry. These two approaches are highly complementary, allowing for flexibility and precision in assessing mitochondrial quality control in numerous &#946;-cell models.

Author Spotlight: Assessment of Mitophagy Flux in Pancreatic &#946;-Cells Using Effective and Robust Complementary Approaches 

This protocol outlines two methods for the quantitative analysis of mitophagy in pancreatic &#946;-cells: first, a combination of cell-permeable mitochondria-specific dyes, and second, a genetically encoded mitophagy reporter. These two techniques are complementary and can be deployed based on specific needs, allowing for flexibility and precision in quantitatively addressing mitochondrial quality control.

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient ...

Visualization and Quantification of Endogenous Intra-Organelle Protein Interactions at ER-Mitochondria Contact Sites by Proximity Ligation Assays

Membrane contact sites (MCSs) are areas of close membrane proximity that allow and regulate the dynamic exchange of diverse biomolecules (i.e., calcium and lipids) between the juxtaposed organelles without involving membrane fusion. MCSs are essential for cellular homeostasis, and their functions are ensured by the resident components, which often exist as multimeric protein complexes. MCSs often involve the endoplasmic reticulum (ER), a major site of lipid synthesis and cellular calcium storage, and are particularly important for organelles, such as the mitochondria, which are excluded from the classical vesicular transport pathways. In the last years, MCSs between the ER and mitochondria have been extensively studied, as their functions strongly impact cellular metabolism/bioenergetics. Several proteins have started to be identified at these contact sites, including membrane tethers, calcium channels, and lipid transfer proteins, thus raising the need for new methodologies and technical approaches to study these MCS components. Here, we describe a protocol consisting of combined technical approaches, that include proximity ligation assay (PLA), mitochondria staining, and 3D imaging segmentation, that allows the detection of proteins that are physically close (&gt;40 nm) to each other and that reside on the same membrane at ER-mitochondria MCSs. For instance, we used two ER-anchored lipid transfer proteins, ORP5 and ORP8, which have previously been shown to interact and localize at ER-mitochondria and ER-plasma membrane MCSs. By associating the ORP5-ORP8 PLA with cell imaging software analysis, it was possible to estimate the distance of the ORP5-ORP8 complex from the mitochondrial surface and determine that about 50% of ORP5-ORP8 PLA interaction occurs at ER subdomains in close proximity to mitochondria.

The need for new approaches to study membrane contact sites (MCSs) has grown due to increasing interest in studying these cellular structures and their components. Here, we present a protocol that integrates previously available microscopy technologies to identify and quantify intra-organelle and inter-organelle protein complexes that reside at MCSs.

Membrane contact sites (MCSs) are areas of close membrane proximity that allow and regulate the dynamic exchange of diverse biomolecules (i.e., calcium ...

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

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Staining Protocols for Human Pancreatic Islets

In situ Quantification of Pancreatic Beta-cell Mass in Mice

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Characterization of Neuronal Lysosome Interactome with Proximity Labeling Proteomics

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Visualizing Mitophagy with Fluorescent Dyes for Mitochondria and Lysosome

Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence

Complementary Approaches to Interrogate Mitophagy Flux in Pancreatic β-Cells

Visualization and Quantification of Endogenous Intra-Organelle Protein Interactions at ER-Mitochondria Contact Sites by Proximity Ligation Assays