Blog Post by Dr. Tamara Maiuri
“What did you discover that you didn’t set out to discover?”Richard Feynman
Sometimes you find the coolest things when you’re not looking for them. In this case, I wanted to test the effect of PARP or PARG inhibition on the recruitment of huntingtin to sites of micro-irradiation.
Rationale side note:
-PARP produces poly ADP ribose (PAR) in response to DNA damage; PARG breaks it down
-We know that huntingtin binds PARylated proteins in cells, and we’ve identified a PAR-binding motif within the huntingtin sequence that binds PAR in vitro, but we still don’t know why huntingtin binds PAR.
It’s been awhile since I’ve done a micro-irradiation “stripe” experiment (what with the pandemic and all), so I must not have adjusted the settings properly on the microscope: I was surprised to find no DNA damage stripes after fixing the cells, staining for huntingtin, and returning to the same cells on the scope.
Where are my stripes?
I decided to look for them the old-fashioned way: with my eyes. That is, through the eyepiece instead of using the automated microscope stage and computer screen.
In the cells treated with PARG inhibitor, something caught my eye–not stripes, but unusual staining in mitotic cells. We know that huntingtin localizes to centrosomes and mitotic spindle poles, and mitotic cells are very obvious when looking at huntingtin (phospho-N17)-stained cells. But this was something I’d never seen before: huntingtin was “coating” the mitotic chromosomes. Very cool.
I looked for mitotic cells in the PARP inhibitor-treated sample and found that huntingtin staining was excluded from the condensed chromosomes (nothing crazy here–this is what mitotic cells normally look like in the absence of inhibitors). In this case, huntingtin is in red and PARP1 is in green:
What does this mean?
This is strong evidence that huntingtin binds PAR in cells: when the break-down of PAR is inhibited by PARG inhibitor, PAR accumulates on mitotic chromosomes. A quick search on BenchSci returned these images of anti-PAR immunofluorescence (one from 1981!):
Cool to note that the PAR in those old images also lives at nuclear puncta in non-mitotic cells. We’ve seen this too, and identified the puncta as SC35-positive “nuclear speckles”, where huntingtin also lives.
I’ve since repeated the experiment to confirm the effect of PARG inhibition on huntingtin localization to mitotic chromosomes and deposited the results to Zenodo.
While this data still doesn’t tell us why huntingtin binds PAR, it’s another (rather pretty) piece of the puzzle.