Fig. 7: Nanoscopic segregation of CD4 WT and CD45 on the surface of resting Jurkat cells.

a Representative two-dimensional images of Jurkat cell surface sequentially analyzed for CD45 (magenta; left panel) and CD4 WT (green; middle panel) by SMLM. The right panel represents an overlaid image. CD4 was visualized as mEos2 fusion protein (PALM) after transient transfection of cells, and the surface CD45 was labeled using Alexa Fluor 647-conjugated MEM-28 antibody (dSTORM). ROIs 1-2 were zoomed to indicate details of proteins’ distribution (right side). b Intensity line-profiles were measured along the transparent gray regions indicated in ROIs 1–2 (as in a). Green line represents CD4 WT and magenta CD45 signals. c Schematic illustration of microvillus with indicated structural segments: tip, shaft and the basis. d Two-dimensional SMLM images of a selected Jurkat cell captured during its association with the optical surface which was sequentially analyzed for CD45 and CD4 WT (as in a). The accumulation of CD4 WT on the tips of large membrane protrusions was observed in 10 out of 32 imaged Jurkat cells. ROIs 3–4 were zoomed to show the details of membrane protrusions with accumulated CD4 WT on their tips (right side). e Intensity line-profiles were measured along the transparent gray regions indicated in ROIs 3–4. The arrows indicate the onset of line-profiles. Scale bars, 5 µm. Images from three independent experiments are shown (n = 32). f Schematic illustration of the organization of CD4 WT, CD4 CS1 and CD45 on the protrusions as indicated by the nanoscopy.