SUMO is an essential and highly conserved, small ubiquitin-like modifier protein. In this protocol we are describing the use of a stress-tolerant recombinant SUMO-trapping protein (kmUTAG) to visualize native, untagged SUMO conjugates and their localization in a variety of cell types.
The significance of this protocol is the use of a novel fluorescent SUMO-trapping UTAG protein for the detection of the protein modifier SUMO in various eukaryotic cells. The main advantage of this technique is a straightforward protocol for the antibody-free detection of SUMO in a variety of cells, including mammalian tissue culture and nematode gonads. SUMO plays important roles in cancer and neurodegenerative disorders, and this underscores the need for robust and reliable tools for the detection and functional analysis of SUMO-modified proteins.
Because SUMO is highly conserved, the fluorescent SUMO-trapping UTAG protein can be used to label conjugated SUMO in many different organisms. This video demonstrates its use in mammalian cells. In addition, the full text describes its use in nematode oocytes.
The visual demonstration of this technique provides critical hints for the treatment of cells before staining and SUMO detection. To begin, grow tissue culture cells of choice on 22-millimeter round cover slips in six-well TC plates until 70 to 80%confluent. Wash the cells briefly with one milliliter of DPBS.
To fix the cells, add two milliliters of fresh 4%PFA and DPBS to each well. Incubate the cells for 20 minutes at room temperature. Wash the fixed cells three times for five minutes in one milliliter of DPBS while nutating the plate.
To permeabilize the cells, incubate for 15 minutes with 1%Triton X-100 in DPBS. Wash the cells again, as before. Incubate the cells with 500 microliters of 1 molar glycine HCL pH 2 for 10 seconds.
Then neutralize the pH immediately with 500 microliters of 10X SUMO Protease buffer, or SPB. After washing the cells as before, remove the cover slips from the well and place them in a humidity chamber. For UTAG-FL staining only, mix one microgram of UTAG-FL with 100 microliters of 1X SPB containing five millimolar TCEP in a tube.
Then, pipette the mix onto the cells on the cover slip and incubate at room temperature for one hour in the humidity chamber. To wash the cover slips, pipette 200 microliters of DPBS on each cover slip and leave in place for 10 minutes. Repeat the wash two more times.
Remove the cover slips from the last wash and invert onto a pre-cleaned microscopy slide with a drop of mounting medium. Store the samples overnight in a 20 degree Celsius freezer before viewing under the microscope. Visualize the cells using the appropriate filter sets for DAPI and Texas Red.
Please see the full text for a protocol on how to label SUMO and fixed nematode gonads. While labeling mammalian cells is pretty straightforward, labeling nematode oocytes requires prior training in gonad dissections. KmUTAG-FL incubated with fixed PMT2 cells showed a distinct nuclear staining.
Both diffuse nuclear staining and distinct nuclear foci were visible. Nuclear localization was confirmed using co-staining with DAPI. Consistent with the SUMO trapping activity of KmUTAG-FL, the nuclear localization pattern was reminiscent of SUMO23 staining.
Co-staining with anti-SUMO23 8A2 antibody confirmed the co-localization of KmUTAG-FL with the SUMO23 signal. This validates the efficacy of KmUTAG-FL to detect SUMO23 in mammalian cells. Isolated gonads from oocyte-producing adult hermaphrodite c.
elegans nematodes were labeled with anti-SUMO antibody. Consistent with previous reports, anti-SUMO antibody initially localized to the nuclear plasm of late myotic prophase oocytes. It then redistributed to the central ring complex of the paired homologs as the nuclear envelope broke down and the chromosomes congressed towards the metaphase plate.
In parallel preparations, gonads labeled with KmUTAG-FL revealed similar patterns. KmUTAG-FL shifted from labeling the nucleoplasm to concentrating on the ring complex as oocytes began and completed the process of nuclear envelope breakdown. These results validate KmUTAG-FL as a useful tool for the analysis of myosis and SUMO-related processes in c.
elegans and possibly other nematodes. While performing fixation, it is critical to use fresh paraformaldehyde and a short fixation time to keep SUMO natively folded so that it can be recognized by the KmUTAG. Since SUMO's tertiary structure is highly conserved, it is very likely that SUMO variants from additional model and non-model systems can be analyzed with the KmUTAG-FL reagent.
Currently, we are using this novel fluorescent SUMO-trapping UTAG protein to study the function of SUMO during cellular stress. Finally, please note that all steps using the fixative paraformaldehyde should be performed in a laboratory safety hood and this reagent must be disposed of properly.
