Data from Shalek lab (Rubin et al., bioRxiv 2024)

Abstract: The disordered adapter protein linker for activation of T cells (LAT) propagates signals downstream of the T cell receptor. To interrogate how LAT initiates multiple downstream pathways, we developed a single-cell screening approach which identified widespread functional segments including protein interaction motifs and blocks of negative charge. Regardless of their position in LAT, individual segments generally confer defects across all downstream signaling pathways. To understand the underlying mechanism, we used molecular biology, computational modeling, and imaging to demonstrate that disruption of LAT interaction with a single partner protein indirectly disrupts other partner interactions, likely through the dual roles of these proteins as effectors of downstream pathways and bridging factors between LAT molecules. Overall, we describe an approach for interrogating sequence-function relationships for proteins with complex activities.

In this experiment, LAT knockout Jurkat T cells were transduced with a library of open reading frames (ORFs) and stimulated to trigger T cell activation for 30 minutes. We then harvested cells and performed 10x single-cell ATAC-seq with Spear-ATAC for ORF barcode detection.

Metadata annotation “orf_call” indicates ORF genotype delivered to cells.

Metadata annotation “dataset”indicates one of two technical replicate 10x channels.