Carmen?

preLighters go live! This Thursday - preLights – How do membrane actin protrusions initiate to T-cells signalling? – What does RhoA have to do with nuclear envelope tensions in stem cells? – Does advanced microscopy help study mechano-signalling –...

preLighters go live!

A light and lively face-to-face conversation between the preLighters @fadelvalle.bsky.social & Vibha Singh and the researchers probablycarmen.bsky.social & Eva Mejia-Ramirez will be hosted at the @focalplane.bsky.social webinar.

4 Sept, 14.00-15.00 BST

Tune in!

One week to go!

Register now for our #FocalPlaneFeatures webinar with @prelights.bsky.social.

us02web.zoom.us/webinar/regi...

FocalPlane features... meets preLights Thursday 4 September 2025, 14:00 BST (UTC+1) FocalPlane logo Picture of Carmen Rodilla-Ramírez T cell protrusions enable fast, localised initiation of CAR signalling Carmen Rodilla-Ramírez (Freie Universität Berlin) Picture of Eva Mejia-Ramirez Targeting RhoA activity rejuvenates aged hematopoietic stem cells Eva Mejia-Ramirez (The Bellvitge Institute for Biomedical Research) preLights logo Hosted by preLighters Felipe Del Valle Batalla and Vibha Singh @focalplane.bsky.social @prelights.bsky.social #FocalPlaneFeatures #preprints

#FocalPlaneFeatures meets @prelights.bsky.social

@fadelvalle.bsky.social & Vibha Singh have picked 2 fantastic #cellbio preprints & will be presenting their preLights alongside research talks from @probablycarmen.bsky.social & Eva Mejia-Ramirez.

Register: focalplane.biologists.com/2025/08/20/f...

Great to see our preprint from @probablycarmen.bsky.social and co highlighted in the @focalplane.bsky.social list of studies using #microscopy to answer bio questions questions! www.biorxiv.org/content/10.1...

Microscopy preprints - applications in biology - FocalPlane Microscopy preprints - applications in biology - News

📢Our preprint list is up on FocalPlane 🔬

This week we featured preprints that use #microscopy to answer questions in #biology. Check them out and let us know about any recent preprints that we should add to the list!
focalplane.biologists.com/2025/08/12/m...

Protocol for fast antibiotic resistance-based gene editing of mammalian cells with CRISPR-Cas9 Protein tagging with CRISPR-Cas9 enables the investigation of protein function in its native environment but is limited by low homology-directed repair (HDR) efficiency. Here, we present a protocol for fast antibiotic resistance-based gene editing with CRISPR-Cas9 (FAB-CRISPR), which streamlines N/C-terminal tagging using an antibiotic resistance cassette for rapid selection and enrichment of gene-edited cells. We describe in detail guide RNA and HDR donor plasmid cloning, transfection of editin...

Protocol for fast antibiotic resistance-based gene editing of mammalian cells with CRISPR-Cas9 #protocol #starprotocols #cellpress

Protocol for fast antibiotic resistance-based gene editing of mammalian cells with CRISPR-Cas9 Protein tagging with CRISPR-Cas9 enables the investigation of protein function in its native environment but is limited by low homology-directed repai…

Thrilled to share our step-by-step protocol for endogenous protein tagging with CRISPR/Cas9, featuring an antibiotic resistance cassette to boost knock-in efficiencies 🧫🧬
Now out in STAR protocols: www.sciencedirect.com/science/arti...

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.

Check out our new preprint on the role of T cell actin-rich protrusion in CAR signalling initiation.
Spoiler alert: Protrusions do it better
www.biorxiv.org/content/10.1...

Fast Antibiotic resistance-Based gene editing of mammalian cells with CRISPR-Cas9 (FAB-CRISPR) Protein tagging with CRISPR-Cas9 enables the investigation of protein function in its native environment but is limited by low homology-directed repair (HDR) efficiency causing low knock-in rates. We ...

Long in the making but our protocol for gene editing is online! FAB CRISPR is super easy and allows the generation of N and C terminal fusions with TurboID, SNAP, Halo and mStayGold. A single cloninf step to generate HDR templates! arxiv.org/abs/2502.12675 1/n

T cells Thursday? Let's start it! We are a group of "accidental immunologists" diving into the mechanisms of membrane organization of signaling molecules in immune cells. Jurkat T cell expressing endogenously tagged ZAP70 (cyan) and a HER2 CAR (yellow), interacting with a SK-BR-2 breast cancer cell