Name: visualization of endogenous mitophagy complexes in situ in human pancreatic beta cells utilizing proximity ligation assay
Uploaded: 2019-05-02T14:00:00
Description: 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

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 is...

<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. M., 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=USP8+regulates+mitophagy+by+removing+K6-linked+ubiquitin+conjugates+from+parkin.">USP8 regulates mitophagy by removing K6-linked ubiquitin conjugates from parkin.</a> The EMBO Journal. 33 (21), 2473-2491 (2014).</li><li>Gauthier, T., Claude-Taupin, A., Delage-Mourroux, R., Boyer-Guittaut, M., Hervouet, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proximity+Ligation+In+situ+Assay+is+a+Powerful+Tool+to+Monitor+Specific+ATG+Protein+Interactions+following+Autophagy+Induction.">Proximity Ligation In situ Assay is a Powerful Tool to Monitor Specific ATG Protein Interactions following Autophagy Induction.</a> PLoS ONE. 10 (6), e0128701 (2015).</li><li>Silva Xavier, D. a., G, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cells+of+the+Islets+of+Langerhans.">The Cells of the Islets of Langerhans.</a> Journal of Clinical Medicine. 7 (3), 54 (2018).</li><li>Steiner, D. J., Kim, A., Miller, K., Hara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+islet+plasticity:+Interspecies+comparison+of+islet+architecture+and+composition.">Pancreatic islet plasticity: Interspecies comparison of islet architecture and composition.</a> Islets. 2 (3), 135-145 (2010).</li><li>Cabrera, O., 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=The+unique+cytoarchitecture+of+human+pancreatic+islets+has+implications+for+islet+cell+function.">The unique cytoarchitecture of human pancreatic islets has implications for islet cell function.</a> Proceedings of the National Academy of Sciences. 103 (7), 2334 (2006).</li><li>Brissova, M., 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=Assessment+of+Human+Pancreatic+Islet+Architecture+and+Composition+by+Laser+Scanning+Confocal+Microscopy.">Assessment of Human Pancreatic Islet Architecture and Composition by Laser Scanning Confocal Microscopy.</a> Journal of Histochemistry and Cytochemistry. 53 (9), 1087-1097 (2005).</li><li>Babu, D. 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. R., 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=Comprehensive+alpha,+beta+and+delta+cell+transcriptomes+reveal+that+ghrelin+selectively+activates+delta+cells+and+promotes+somatostatin+release+from+pancreatic+islets.">Comprehensive alpha, beta and delta cell transcriptomes reveal that ghrelin selectively activates delta cells and promotes somatostatin release from pancreatic islets.</a> Molecular Metabolism. 5 (7), 449-458 (2016).</li><li>Lawlor, N., 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+identify+human+islet+cell+signatures+and+reveal+cell-type&#8211;specific+expression+changes+in+type+2+diabetes.">Single-cell transcriptomes identify human islet cell signatures and reveal cell-type&#8211;specific expression changes in type 2 diabetes.</a> Genome Research. 27 (2), 208-222 (2017).</li><li>Dorrell, C., 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=Human+islets+contain+four+distinct+subtypes+of+&#946;+cells.">Human islets contain four distinct subtypes of &#946; cells.</a> Nature Communications. 7, 11756 (2016).</li><li>Dorrell, C., 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=Isolation+of+major+pancreatic+cell+types+and+long-term+culture-initiating+cells+using+novel+human+surface+markers.">Isolation of major pancreatic cell types and long-term culture-initiating cells using novel human surface markers.</a> Stem Cell Research. 1 (3), 183-194 (2008).</li><li>Jayaraman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+the+detection+of+viable+human+pancreatic+beta+cells+by+flow+cytometry+using+fluorophores+that+selectively+detect+labile+zinc,+mitochondrial+membrane+potential+and+protein+thiols.">A novel method for the detection of viable human pancreatic beta cells by flow cytometry using fluorophores that selectively detect labile zinc, mitochondrial membrane potential and protein thiols.</a> Cytometry Part A. 73 (7), 615-625 (2008).</li><li>Arda, H. E., 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=Age-Dependent+Pancreatic+Gene+Regulation+Reveals+Mechanisms+Governing+Human+&#223;-Cell+Function.">Age-Dependent Pancreatic Gene Regulation Reveals Mechanisms Governing Human &#223;-Cell Function.</a> Cell Metabolism. 23 (5), 909-920 (2016).</li><li>Allen, G. F. G., Toth, R., James, J., Ganley, I. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+iron+triggers+PINK1/Parkin-independent+mitophagy.">Loss of iron triggers PINK1/Parkin-independent mitophagy.</a> EMBO Reports. 14 (12), 1127-1135 (2013).</li><li>Villa, E., Marchetti, S., Ricci, J. -. 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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 ...

This protocol allows us to monitor endogenous mitophagy complexes in human tissues such as beta cells, facilitating mitophagy research in pivotal translationally-relevant tissues. One advantage of this technique is its ability to visualize important mitophagy complexes in vivo in tissues with a rare availability or in small sample numbers. After isolation according to standard protocols, culture four to six times 10 to the third human islet equivalents in 10 milliliters of complete pancreatic islet medium for at least one day at 37 degrees Celsius.The next day, use a dissection light microscope at a three times magnification to count individual human islets within the culture. Pick 40 islets per treatment or condition of interest into 1.5 milliliter tubes of islet medium. Sediment the islets by centrifugation and resuspend the pellet in one milliliter of PBS supplemented with 50 micromolar PR619.Invert the tube and collect the islets by centrifugation for a second wash in fresh PBS plus deubiquitinase inhibitor. After the second centrifugation, dissociate the islets into single cells with 125 microliters of 0.25%trypsin plus inhibitor for three minutes at 37 degrees Celsius with periodic gentle pipetting. At the end of the incubation, arrest the reaction with one milliliter of 37 degree Celsius warmed pancreatic islet medium supplemented with PR619.Collect the islets by centrifugation for two washes in fresh PBS plus inhibitor per wash. After the second wash, resuspend the dissociated islets in 150 microliters of fresh PBS plus inhibitor. For adherence and fixation of the individual islets, cytospin the cell solution onto frosted charged microscope slides and outline the cellular area with a hydrophobic pen.Then fix the encircled cells with 4%paraformaldehyde and PBS for 15 minutes at room temperature. For immunohistochemical analysis of the islet samples, wash the cells with two five-minute washes in PBS at room temperature, followed by non-specific binding blocking with 10%donkey serum and PBS plus 0.3%detergent for one hour at room temperature. At the end of the blocking incubation, coincubate the cells with primary mouse antibody against ubiquitin-specific peptidase 8, primary rabbit antibody against neuregulin receptor degradation protein-1, and a marker specific for beta cell identification that was not raised in mouse or rabbit so as not to interfere with the proximity ligation assay signal overnight at four degrees Celsius.The next morning, wash the samples with two five-minute washes in PBS on a rocker. Add anti-goat Cy5 secondary antibody to a freshly prepared proximity ligation assay solution. After the second wash, label each islet sample with 20 microliters of probe solution and recover the slides with plastic film for a one-hour incubation at 37 degrees Celsius.At the end of the incubation, wash the cells with two five-minute washes in buffer A on a rocker followed by incubation with 20 microliters of freshly prepared ligation solution supplemented with 0.25 units per microliter of ligase per slide for a 30-minute incubation at 37 degrees Celsius. At the end of the ligation, wash the cells two times with buffer A for two minutes per wash. Cover each sample with 20 microliters of amplification solutions supplemented with polymerase for a 100-to 120-minute incubation at 37 degrees Celsius.To prepare the cells for imaging, wash the samples two times in fresh buffer A for 10 minutes per wash on a rocker followed by one wash in 0.1x buffer B for two minutes on the rocker. After the last wash, mount the slides with a drop of mounting medium containing DAPI and carefully place a coverslip over each sample using a scalpel to press out any bubbles. Then seal the coverslips with clear nail polish around the edges.To image the samples, place a slide onto the stage of a microscope capable of capturing multiple focal planes and select the 100 times magnification. Set the Z-stack height to approximately 0.45 micrometers and the appropriate stain, counterstain, and live cell signal excitation and emission parameters. Obtain at least nine different focal plane images.To ensure that the fluorescence image background signals and stray light are minimized, process the images utilizing a two-dimensional deconvolution nearest neighbor algorithm using a standard image processing software of choice. To quantify the proximity ligation assay interactions, open ImageJ and open the proximity ligation assay image. Starting from the first sharply in-focus image, click Image and select Adjust and Threshold.Adjust the threshold to remove all of the non-specific proximity ligation assay signal and make a note of the threshold adjustment to try to keep the signal consistent throughout analysis. Click Process and select Binary and Make Binary. Confirm that Size is set to zero to infinity, Circularity is set to zero to one, Show is set to Outlines and check the Display results and Clear results boxes.Note the number of particles analyzed in a spreadsheet denoting the sample and Z-stack position. Then click Analyze and Analyze Particles to measure the particles. When all of the in-focus Z-stacks for the sample have been analyzed, send the total number of particles for the sample in the spreadsheet to quantify the total number of interactions.The antibodies used in the proximity assay are specific for the ligands and do not demonstrate any overlap. As observed, the dispersion protocol is highly efficient at obtaining single cells in the microscope field of view for downstream analysis and the beta cell staining is retained following proximity ligation assay staining in primary human islets. Using the demonstrated techniques, it could be determined that the interaction of neuregulin receptor degradation protein-1 and ubiquitin-specific peptidase 8 is decreased following a 48-hour exposure to palmitate, highlighting the feasibility of this assay for assessing key endogenous mitophagy factors following diabetogenic stimuli.The efficient and gentle dispersion of human islets is essential for ensuring quantification of the correct cell populations downstream. PLA allows for the assessment of important mitophagy complexes in human islet samples allowing the analysis of mitophagy and biologically-important human patient samples moving the field of diabetes research forward.

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

Research

JoVE Journal

Medicine

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 ...

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of micrometastases by radiologic imaging and the success in their therapeutic eradication remain limited.
While animal models have proven to be invaluable tools for cancer research1, the monitoring/visualization of micrometastases remains a challenge and inaccurate evaluation of metastatic spread in preclinical studies potentially leads to disappointing results in clinical trials2. Consequently, there is great interest in refining the methods to finally allow reproducible and reliable detection of metastases down to the single cell level in normal tissue. The main focus therefore is on techniques, which allow the detection of tumor cells in vivo, like micro-computer tomography (micro-CT), positron emission tomography (PET), bioluminescence or fluorescence imaging3,4. We are currently optimizing these techniques for in vivo monitoring of primary tumor growth and metastasis in different osteosarcoma models. Some of these techniques can also be used for ex vivo analysis of metastasis beside classical methods like qPCR5, FACS6 or different types of histological staining. As a benchmark, we have established in the present study the stable transfection or transduction of tumor cells with the lacZ gene encoding the bacterial enzyme &beta;-galactosidase that metabolizes the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to an insoluble indigo blue dye7 and allows highly sensitive and selective histochemical blue staining of tumor cells in mouse tissue ex vivo down to the single cell level as shown here. This is a low-cost and not equipment-intensive tool, which allows precise validation of metastasis8 in studies assessing new anticancer therapies9-11. A limiting factor of X-gal staining is the low contrast to e.g. blood-related red staining of well vascularized tissues. In lung tissue this problem can be solved by in-situ lung perfusion, a technique that was recently established by Borsig et al.12 who perfused the lungs of mice under anesthesia to clear them from blood and to fix and embed them in-situ under inflation through the trachea. This method prevents also the collapse of the lung and thereby maintains the morphology of functional lung alveoli, which improves the quality of the tissue for histological analysis. In the present study, we describe a new protocol, which takes advantage of a combination of X-gal staining of lacZ-expressing tumor cells and in-situ perfusion and fixation of lung tissue. This refined protocol allows high-sensitivity detection of single metastatic cells in the lung and enabled us in a recent study to detect "dormant" lung micrometastases in a mouse model13, which was originally described to be non-metastatic14.

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

The novel protocol reported in the present study allows selective detection of lung metastases at single cell resolution in mice by combined in-situ lung perfusion and fixation and X-Gal staining of lacZ-tagged tumor cells.

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of ...

Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells

Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within pancreatic islets and act in a coordinated fashion to maintain glucose homeostasis. It is becoming increasingly clear that interactions between transmembrane proteins on the surfaces of adjacent beta cells are important determinants of beta-cell function.
Elucidation of the roles of particular transcellular interactions by knockdown, knockout or overexpression studies in cultured beta cells or in vivo necessitates direct perturbation of mRNA and protein expression, potentially affecting beta-cell health and/or function in ways that could confound analyses of the effects of specific interactions. These approaches also alter levels of the intracellular domains of the targeted proteins and may prevent effects due to interactions between proteins within the same cell membrane to be distinguished from the effects of transcellular interactions.
Here a method for determining the effect of specific transcellular interactions on the insulin secreting capacity and responsiveness of beta cells is presented. This method is applicable to beta-cell lines, such as INS-1 cells, and to dissociated primary beta cells. It is based on coculture models developed by neurobiologists, who found that exposure of cultured neurons to specific neuronal proteins expressed on HEK293 (or COS) cell layers identified proteins important for driving synapse formation. Given the parallels between the secretory machinery of neuronal synapses and of beta cells, we reasoned that beta-cell functional maturation might be driven by similar transcellular interactions. We developed a system where beta cells are cultured on a layer of HEK293 cells expressing a protein of interest. In this model, the beta-cell cytoplasm is untouched while extracellular protein-protein interactions are manipulated. Although we focus here primarily on studies of glucose-stimulated insulin secretion, other processes can be analyzed; for example, changes in gene expression as determined by immunoblotting or qPCR.

Transcellular protein interactions are important determinants of pancreatic beta-cell function. Detailed here is a method&#x2014;adapted from a coculture model of synaptogenesis&#x2014;for investigating how specific transmembrane proteins influence insulin secretion. Transfected HEK293 cells express proteins of interest; beta cells do not need to be transfected or otherwise directly perturbed.

Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within ...

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

Drosophila has illuminated our understanding of the genetic basis of normal development and disease for the past several decades and today it continues to contribute immensely to our understanding of complex diseases 1-7. Progression of tumors from a benign to a metastatic state is a complex event 8 and has been modeled in Drosophila to help us better understand the genetic basis of this disease 9. Here I present a simple protocol to genetically induce, observe and then analyze the progression of tumors in Drosophila larvae. The tumor induction technique is based on the MARCM system 10 and exploits the cooperation between an activated oncogene, RasV12 and loss of cell polarity genes (scribbled, discs large and lethal giant larvae) to generate invasive tumors 9. I demonstrate how these tumors can be visualized in the intact larvae and then how these can be dissected out for further analysis. The simplified protocol presented here should make it possible for this technique to be utilized by investigators interested in understanding the role of a gene in tumor invasion.

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

Cooperation between an activated oncogene like RASV12 and mutations in cell polarity genes like scribbled, result in tumor growth in Drosophila where tumor cells also display invasive behaviors. Here a simple protocol for the induction and observation of the benign and invasive tumors is presented.

Drosophila has illuminated our  understanding of the genetic basis of normal development and disease for the  past several decades and today it continues ...

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the pathophysiological role of neuroplasticity in GI disorders remains unknown. Novel experimental models which allow simulation and modulation of GI neuroplasticity may enable enhanced appreciation of the contribution of neuroplasticity in particular GI diseases such as pancreatic cancer (PCa) and chronic pancreatitis (CP). Here, we present a protocol for simulation of pancreatic neuroplasticity under in vitro conditions using newborn rat dorsal root ganglia (DRG) and myenteric plexus (MP) neurons. This dual-neuron approach not only permits monitoring of both organ-intrinsic and -extrinsic neuroplasticity, but also represents a valuable tool to assess neuronal and glial morphology and electrophysiology. Moreover, it allows functional modulation of supplied microenvironmental contents for studying their impact on neuroplasticity. Once established, the present neuroplasticity assay bears the potential of being applicable to the study of neuroplasticity in any GI organ.

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuronal plasticity is an increasingly recognized, but insufficiently understood feature of the gastrointestinal (GI) tract. Here, in the example of human pancreatic disorders, we present an in vitro neuroplasticity assay for the study of neuronal plasticity in the GI tract at both morphological and functional level.

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

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 ...

High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in atherosclerosis development. However, an important factor in early stages of atheroma plaque formation is cholesterol efflux capacity to HDL (the ability of HDL particles to accept cholesterol from macrophages) in order to avoid foam cell formation. This is a key step in avoiding the accumulation of cholesterol in the endothelium and a part of reverse cholesterol transport (RCT) to eliminate cholesterol through the liver. Cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool that can be used as biomarker for atherosclerosis. Traditionally, [3H]-cholesterol has been used in cholesterol efflux assays. In this study, we aim to develop a safer and faster strategy using fluorescent labelled-cholesterol (NBD-cholesterol) in a cellular assay to trace the cholesterol uptake and efflux process in THP-1-derived macrophages. Finally, we optimize and standardize the NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Measurement of in vitro cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool as a biomarker for atherosclerosis. In the present study, we optimize and standardize a fluorescent NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.