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.

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.