Localization of protein under dietary restriction.

Model Organism: Worms Scientific Discussion
1

We took some confocal images of a mCherry tagged line under well fed and dietary restriction (DR). To my surprise, the protein (which I don’t wish to reveal) goes from being diffuse under well-fed conditions to being to being punctuate and fibrous under DR. The attached images are of the head region under the two conditions. Visible light images are provided for reference. The fibrils also appear elsewhere in the body, they are not limited to the head.

Does anyone have a suggestion as to where this protein is localizing to and/or what structures these ‘fibrils’ represent? Oh - and have a happy new years!

DR images

https://rollinslab.mdibl.org/wp-content/uploads/2019/12/mCH-DR-fluor.jpg

https://rollinslab.mdibl.org/wp-content/uploads/2019/12/mCH-DR-vis.jpg

AL images

https://rollinslab.mdibl.org/wp-content/uploads/2019/12/mCH-fluor.jpg

https://rollinslab.mdibl.org/wp-content/uploads/2019/12/mCh-AL-vis.jpg

2

Does the transcriptional fusion have the same puncta? If not, it could be either autophagosome ( which can be induced by DR) or granule complex (which can be induced by stress condition,and is your puncta reversible?)

3

Thanks for the insight! We don’t have a transcriptional reporter. The images above are from our gene of interest being endogenously tagged using CRISPR-cas9. The puncta don’t co-localize with lgg-1::GFP, a common autophagosome marker. The puncta do appear to be reversible upon refeeding. I will look into stress granule markers, that is a plausible explanation!

4

Lysosomes can be tubular like this, especially in the hypodermis.

5

In general, RFP or its modified tag like mcherry can easily form such puncta structure, comparing with GFP.

6

It looks like there is a mix of real signal plus extracellular punctae that indeed look like granules.

I would focus on those few that look like the outline of a cell-- a few look like in-focus cell structures with signal around the membrane.

To minimize the granule formation look at the DR sooner after starting to starve them before the worms get old or die. You can tell the worms were not ideal specimens by much fluid is inside the body near the pharynx in all specimens for some reason.

And then get better DIC images, if there is a cell body there you may find some evidence that it’s at the membrane. The cells that you can see in your DIC clearly don’t look like they match up with the red membrane pattern… try some different DIC focal planes as well. Even though it should be par focal DIC/fluorescence, you may need to refocus to see a cell bod on DIC and membrane stain.