Fig. 5: A5 small aggregates and 2D-lattice can mediate the order of underlying membrane.
From: Annexin-V stabilizes membrane defects by inducing lipid phase transition
a, b CLSM fluorescence images of GUVs (DOPC/DOPS 50:50, focusing at the equatorial plane) staining with environment-sensitive dye, Laurdan, recorded in the conditions of a A5-free or b full A5 2D-lattice coverage, respectively. Three spectral channels were used to monitor the fluorescence of Laurdan (Ch1: 412–463 nm; Ch2: 472–535 nm) or A5-Alexa 647, respectively. Based on the Equation 1, the generalized polarization (GP) image of GUVs can be determined using the averaged fluorescence images of Ch1 and Ch2 (n = 20). c GP Histograms of two A5-free GUVs (blue) and a GUV fully covered by A5 (orange) as shown in a, b, respectively. The presence of A5 2D-lattice shifts the most probable peak in GP histogram toward to a higher value (black arrow), which specifies the membrane becomes more ordered. d The mean GP value of the A5-free GUVs (blue, 0.01 ± 0.04) or A5-covered GUVs (orange, 0.17 ± 0.03), respectively. Each data represent for mean ± s.d. among different GUVs with n ≥ 10. e, f Representative CLSM fluorescence image frames (e) and time-dependent fluorescence intensity traces (f) showing the impacts of A5 2D-lattice self-assembly on a GUV, following the subsequent additions of A5 at 114.4 s and 468.0 s, respectively (indicated by red triangles in f). g Time-lapse analysis of GP value (blue curve), background corrected, normalized A5 intensity (red curve), and diameter of the GUV (green curve). This GP value is calculated using the sum of fluorescence intensity in two spectral channels of Laurdan. Although the first A5 addition makes GUV partially covered by A5 with ~10% coverage, a small increasement in GP value can still be observed at ~395 s (blue triangle, GP = 0.07, ~280 s after A5 addition). The subsequent second A5 addition increases the GP value up to 0.169 at 592.8 s, which is highly compatible with the mean GP value observed for A5-covered GUVs in d. Upon two A5 additions, the diameter of the GUV decreases especially during the second A5 addition, along with simultaneous GP value increases. Statistical analysis on the shrinkage of GUVs diameter is provided in Supplementary Fig. 3.
