T-cell protrusions enable fast, localised initiation of chimeric antigen receptor signalling.

Actin-rich protrusions densely cover the surface of T cells and are well characterised for their role in migration. Recent studies have uncovered their contribution to antigen surveillance and immune signalling. To further explore how protrusions initiate signalling pathways mediating T-cell activation, we performed live-cell imaging of endogenously tagged proteins in HER2-specific chimeric antigen receptor (CAR) T cells targeting HER2⁺ breast-cancer cells. Quantitative STED microscopy allowed us to monitor protein rearrangement and to correlate it with membrane topology over time. Before activation, key signalling proteins (including Lck, CD45, LAT, and the CAR) were not enriched in protrusions. Upon contact with target cells, rapid protein reorganisation occurred preferentially within protrusions, initiating signalling. HER2-CAR clustering, accompanied by ZAP-70 and LAT recruitment, was enhanced in protrusions. While Lck distribution remained unchanged, exclusion of the phosphatase CD45 was enhanced at protrusion-cell contacts, independently of the CAR signalling domain. Overall, signalling machinery rearranged faster and more effectively at protrusive contacts than at main plasma membrane regions. Together, our data re-frame protrusions as sites of enhanced receptor activation by exclusion and clustering dynamics rather than by pre-enrichment of the signalling machinery.