Woops! .png with no background ruined EVERYTHING. Here is a readable version:

Special mention to Xiaolei Su, that led us to this adventure in immune cell biology.
Thanks to @hfspo.bsky.social for funding this new exciting @franbottanelli.bsky.social lab adventure! :)

So yes, protrusions do it better! We hope this work sparks new ideas on how membrane architecture shapes signalling.
Thanks for reading, and feel free to reach out with questions or thoughts!

13. Even when we used a non-signalling CAR mutant, CD45 was still excluded at protrusions.
So the enhanced CD45 exclusion is not signalling-dependent.

12. We observed that exclusion at protrusion contacts happened earlier and more strongly when compared to CD45 exclusion at flat membranes

11. The answer is the increased of CD45 exclusion at protrusion mediated contacts

10. Interestingly, Lck doesn’t rearrange at the macroscale. It stays uniformly distributed even during activation, without any specific enrichment in protrusions at any point
So what drives the enhanced protrusion CAR activation?

9. So… why? We go on to observe the positive and negative regulators of the process: the kinase Lck and the phosphatase CD45.

8. Then comes LAT, clustering downstream of ZAP-70.
LAT clusters appear faster and more intensely in protrusions compared to main body membrane regions

7. The CAR clusters are activated immediately and the increased CAR enrichment in protrusions is accompanied with an increased ZAP-70 recruitment to these structures

6. The result: CARs enrich faster and stronger in protrusion contacts than flat membranes

5. In collaboration with Amin from the @ewerslab.bsky.social, we developed an image analysis pipeline to distinguish between protrusions and main body protein rearrangements

4. The logic works both ways: protrusive structures from the breast cancer cell are also able to push into the T cell and create a contact that accumulates CARs!

3. But the game changes when T cells meet their targets.
First contacts happen via protrusions.
Within seconds, CARs cluster specifically at these sites. In here, you can see a Jurkat T cell expressing a HER2-CAR (yellow) and a membrane marker (cyan) contacting a breast cancer cell (magenta).

2. In resting T cells, none of these signalling proteins were enriched in protrusions...

We CRISPR-edited Jurkat T cells to tag key signalling proteins Lck, ZAP-70, LAT, and CD45 at their endogenous loci.
This means no overexpression or immunolabelling artefacts!
Then we used live-cell STED and confocal imaging to track them in 4D

1. T cells are covered with actin-rich protrusions that play a key role in antigen scanning and discrimination. While growing evidence highlights their importance in immune signalling, it is unclear how they influence the reorganisation of signalling proteins when T cells engage with a target.