Extended Data Fig. 9: Moving mEos3.2 probe to the extracellular terminus slows diffusion (D) and increases the population of confined diffusers (α) for both trLAT and trCD4.

a, Diffusion coefficients (D) for the mobile component and the fraction of molecules in the confined state (α) are plotted versus Lo enrichment in GPMVs. Points for D and α are averages and s.e.m. over values extracted from single cells, and Lo enrichment is mean and s.e.m. replotted from Fig. 2b. Most points are replotted from Fig. 4a. Constructs with extracellular mEos3.2 are shown as open symbols and arrows highlight the change for trLAT and trCD4 when the probe is moved from the cytoplasmic side of the transmembrane anchor (trLAT and trCD4) to the extracellular space (trLATo and trCD4o). Cell numbers for each condition are shown in Supplementary Fig. 3 other than trLATo (n = 5) and trCD4o (n = 3). b, Probe enrichment at BCR clusters for trLAT and trCD4 anchors with intracellular or extracellular probes. Large points indicate mean and s.e.m. over values in individual cells, with cell numbers indicated. Repositioning the probe does not significantly impact probe enrichment at BCR clusters as evaluated using a two-tailed t-test. For these anchors, positioning mEos3.2 on the extracellular face dramatically impacts anchor surface expression, leading to large statistical errors.