New JCS snapshot - Cytotoxic T lymphocytes adapt to the loss of Cdc42
@adamrochussen.bsky.social presents the key findings from their recent JCS paper with Claire Ma and Gillian Griffiths @griffithslab.bsky.social.
journals.biologists.com/jcs/article/...

You can find a great summary of the work by @jcellsci.bsky.social here: journals.biologists.com/jcs/article/...

And a profile of first author @adamrochussen.bsky.social here: doi.org/10.1242/jcs....

Huge thanks to @thecimr.bsky.social for supporting this work!

If you find a difference in phenotype between knockout and inhibitor/knockdown, transcriptional adaptation may be the culprit. This remarkable phenomenon demonstrates the robustness & complexity of cells, but can hamper CRISPR-based investigations if you aren't aware of it!

doi.org/10.1242/jcs....

This phenomenon is called "transcriptional adaptation", as coined by @stainierlab.bsky.social

Consistent with their NMD-mediated mechanism, we found targeting the promoter of Cdc42 avoids TA in CTLs. It also seems to be specific for CDC42 among Rho GTPases in CTLs.

Multi-omics revealed that the knockout cells were compensating for the loss of CDC42 by transcriptionally upregulating genes that could enhance T cell function, including by upregulating the activity of related Rho GTPases!

CRISPR/Cas9-mediated gene deletion normally requires several days of recovery before assaying cellular function.

When we performed killing assays daily after delivery of RNPs, we found that the knockout cells initially pheno-copied inhibited cells, but then regained functionality over time

Inhibiting CDC42 with a chemical inhibitor (CASIN) gave the opposite result: worsened CTL function.

Meanwhile, the knockout cells showed *enhanced* function regardless of inhibitor treatment ...

Confusingly, we found the knockout cells performed *better* than control cells across killing, degranulation, migration, and signalling.

CDC42 is a Rho GTPase known to control cell polarity and cytoskeletal dynamics in many cell types.

It is necessary for embryonic viability in mice, and pathogenic variants cause immunodeficiency diseases in humans.

To determine its role in CTLs, we targeted the Cdc42 gene via CRISPR/Cas9

Sometimes, knocking out a gene isn't a good way to figure out the function of that gene ...

In our new @jcellsci.bsky.social paper, we report potent transcriptional adaptation by cytotoxic T lymphocytes (CTLs) in response to CRISPR/Cas9-mediated deletion of the important polarity protein CDC42 🧵⬇️

As promised at my seminar at NCBS, here is the video showing what happens to the centrosome in a “confused T cell” that is contacting two targets with equal signals.

You're seeing the actin cytoskeleton of cytotoxic T lymphocytes as they crawl across glass. These guys move at around 0.15 μm/s. Movie taken by PhD student @adamrochussen.bsky.social on our spinning disk confocal microscope.

Welcome to the dynamic world of T cells!