ASC Speck formation

One of the possible projects is understanding how protein sequence variants impact inflammasome formation. There are multiple possible assays for this, but a classic one is observing the formation of ASC specks, where a nucleation of activated sensor (say, MEFV / Pyrin or NLRP3) nucleates the oligomerization of all of the ASC in the cell into a single gigantic filament.

To try to assess this, we have a construct where ASC is directly fused to mCherry. There is clear speck formation by microscopy, as you can see here.

Clearly there is a bunch of really bright puncta when MEFV and ASC-mCherry is present (right), very few such puncta when no MEFV is there (middle), and no puncta at all when there is no mCherry-tagged ASC (left).

That said, any high-throughput compatible experiment can’t simply rely on microscopy (without a bunch of extra infrastructure), and it’s easier to make it a FACS compatible assay. Thus, the big question was whether we could see a difference in flow. Here are the results:

As you can tell, the diffuse cells (left) have a streak of points below the slope = 1 diagonal,  the ASC only cells (diffuse with a handful of puncta) have a similar streak of points but with a small shadow of points along the slope = 1 diagonal (probably the completely spontaneous ASC speck formation cells; middle), and the MEFV and ASC-mCherry overexpressing cells are almost exclusively puncta forming and also almost exclusively have points running down that slope = 1 diagonal. Thus, it does seem we can distinguish ASC speck formation using flow cytometry.

To make things even easier, one can turn it into a ratiometic density plot, where I’ve divided the red fluorescence height by the red fluorescence area. The differences are relatively subtle, so you have to make sure your axes are zoomed into the right region, but once you do, you can definitely see that there is a different distribution for the ASC speck cells. Cool!