Actin rings are close to the plasma membrane. (a) Top: Confocal image of GPI-GFP; middle: live-STED image of actin rings in progenitor-derived neuronal cells; bottom: merge of actin and GPI-GFP. Right images are zoom-ins of box on the left. Scale bars on the left are 1 µm. Scale bars on the right are 500 nm. (b) Line profile of box of confocal image of GPI-GFP shown in (a) on the right. (c) Line profile of box of live-STED image of actin rings shown in (a) on the right. (d) Schematic representation of the calculation of the distance between GPI-GFP and actin rings: The distance of the peaks shown in (b) and (c) is measured. The difference between the distances is used to calculate the distance between actin rings and GPI-GFP. (e) Violin plot of the measured distances between GPI-GFP and the PM stained via Cholesterol-Star Red (ΔGPI/Chol, n = 47 regions from eight images), GPI-GFP and actin rings stained via SIR-actin (ΔGPI/actin, n = 31 regions from 12 images), and a bead control (beads, n = 46 regions from seven images). Lines indicate mean. Normality was tested using Shapiro–Wilk Test and rejected (ΔGPI/Chol P = 0.00054***, ΔGPI/actin P = 0.076 ns, Δbeads P = 3.6E-5****). Significance was tested using Mann–Whitney Test (ΔGPI/Chol versus ΔGPI/actin P = 0.0098**, ΔGPI/actin versus beads P = 8.7E-8****, ΔGPI/Chol versus beads P = 2.2E-5****).