In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

This method captures transient protein interactions in organisms like bacteria and yeast and in different cell types. Here we monitor transiently formed specific chaperone complexes that are involved in cellular proteostasis. The main advantage of this technique is the ability to generate information on dynamics and sub-cellular localization of protein assemblies in prokaryotic and eukaryotic cells.

Adaptations of this technique in unicellular organisms like bacteria and yeast, allows researchers to use it as a powerful tool to investigate diseases related to microbial infections. This technique could potentially be employed to analyze protein interactions in almost all organisms by simply changing the conditions at the sample preparation phase of the protocol. It is important to select good quality antibodies that are specific and tested in either immunohistochemistry, immunofluorescence, ELISA, or immunoprecipitations.

Begin by pre-treating 10-well diagnostic slides with poly-L-Lysine. Add 100 microliters of sterile and filtered 0.0001%poly-L-Lysine to each well, and incubate the slides for 30 minutes in a sterile laminar flow hood. Then, wash each well with 50 microliters of sterile water to remove excess poly-L-Lysine.

Add approximately 15, 000 cells per well depending on the cell type, and grow the cells in a sterile humid incubator at 37 degrees Celsius with 5%carbon dioxide to a 60 to 80%confluency. After 24 hours, remove the growth medium by placing a tissue paper at the edge of each well, and wash the cells three times with 50 microliters of PBS. Then fix the cells by adding 50 microliters of freshly prepared 4%paraformaldehyde in PBS to each well and incubating the slide at room temperature for 10 minutes.

After fixation, wash the slide three times by submerging the slide in a slide-staining jar with PBS. To permeabilize the membranes of the attached cells, submerge the slide in 100 milliliters of 0.5%Triton-X100 in PBS, and incubate the cells for 10 minutes at room temperature. Then wash the slides three times in TBS-T.

After the last wash, remove excess buffer with a tissue paper. Grow and dilute S.cerevisiae cells according to manuscript directions, then transfer them to a 50-milliliter centrifuge tube and pellet by centrifugation at 665 times g for three minutes. Remove the supernatant and resuspend the cells in five milliliters of fresh medium.

Then add 550 microliters of 37%formaldehyde for a final concentration of 4%and incubate the culture at room temperature for 15 minutes to fix the cells. After the incubation, pellet the cells, remove the supernatant, and resuspend the pellet in one milliliter of freshly prepared 4%paraformaldehyde in wash buffer. Incubate the cells at room temperature for 45 minutes.

Meanwhile, prepare the diagnostic slides by adding 100 microliters of 0.0001%poly-L-Lysine solution to each well and incubating the slides for 30 minutes at room temperature. Then wash away the excess poly-L-Lysine with ultrapure water and let the slides air-dry. After the incubation step, wash the cells twice with one milliliter of wash buffer by centrifugation.

Remove the supernatant and resuspend the cells in wash buffer. Pellet the cells after the last wash, and remove the supernatant and resuspend the cells in one milliliter of wash buffer containing 1.2 molar sorbitol. To digest the cell wall, pellet the cells again and resuspend the cells in 250 microliters of freshly prepared Lyticase solution.

Incubate the cells at 30 degrees Celsius for 15 minutes with gentle shaking. After the digestion step, wash the cells three times in wash buffer and resuspend the cells in 250 microliters of wash buffer containing 1.2 molar sorbitol. Add 20 microliters of re-suspended cells to each poly-L-Lysine-coated well, and allow the fixed and permeabilized cells to attach for 30 minutes.

Wash the wells three times with 50 microliters of wash buffer to remove non-adherent cells, and proceed with the proximity ligation assay. Initiate the proximity ligation assay, or PLA protocol, by adding a drop of the blocking buffer to each well and incubate the slide in a humid chamber for 30 minutes at 37 degrees Celsius. Remove the blocking buffer by placing a tissue paper at the edge of each well, and wash the cells twice in 100 milliliters of wash buffer A.Next, add 40 microliters of the primary antibody solution to each well, and incubate the slide for 60 minutes at 37 degrees Celsius in a humid chamber.

Remove the primary antibody solution and wash the slides twice in wash buffer A.After the wash, add 40 microliters of the secondary antibody solution to each well and incubate the slide for 60 minutes at 37 degrees Celsius in a humid chamber. Then remove the solution and wash the slides twice in wash buffer A.Then add 40 microliters of the DNA ligation mixture which contains the connector oligonucleotides and DNA ligase, to each well, and incubate the slide in the humid chamber at 37 degrees Celsius for 30 minutes. After incubation, remove the ligation mixture and wash the slides twice with wash buffer A.Add 40 microliters of DNA amplification mixture which contains the DNA polymerase and the fluorescently labeled oligonucleotides to each well, and incubate for 100 minutes in the humid chamber at 37 degrees Celsius.

Remove the amplification mixture and wash the slides in 100 milliliters of wash buffer B for 10 minutes per wash. Then wash the slides in 100 milliliters of wash buffer B diluted one to 100 in ultrapure water. Add 20 microliters of DAPI-containing mounting medium per well.

Cover the wells of the slide with a cover slip and seal with nail polish. This protocol was successfully used to monitor transient chaperone assemblies formed between distinct J-domain proteins and Hsp70 chaperone and J-domain proteins in HeLa, S.cerevisiae, and E.coli cells. Human class A and class B J-domain proteins were targeted with highly specific primary antibodies.

Each of the red fluorescent puncti represents a protein complex formed between two distinct J-domain proteins in HeLa cells. Similar mixed-class J-domain protein complexes are also observed in the unicellular eukaryote S.cerevisiae, or baker's yeast. Some of the fluorescent puncti generated from PLA were more difficult to resolve as individual foci, due to the small cell size.

Complex formation between class A and B J-domain proteins were not observed in E.coli cells, which is consistent with biochemical studies performed with purified proteins. But other chaperone assemblies involving J-domain proteins and the Hsp70 chaperone were captured using the PLA protocol. Technical controls lacking a primary antibody against one of the interacting J-domain proteins or chaperones in the otherwise complete PLA reactions showed little or no fluorescence puncti formation in the cells, indicating a lack of false positive signal amplification.

To obtain reliable results using the proximity ligation assay, verify beforehand that the antibodies used are of sufficient quality. Furthermore, take precaution in avoiding over-digestion of cells containing a cell wall.