Poly-L-lysine-induced morphology changes in mixed anionic/zwitterionic and neat zwitterionic-supported phospholipid bilayers

Abstract

Poly-L-lysine-induced morphological changes in liquid phase supported bilayers consisting of mixed anionic/zwitterionic and neat zwitterionic headgroup phospholipids were studied with atomic force microscopy and epifluorescence microscopy. Results obtained from these studies indicate that poly-L-lysine can induce domains, defects, and aggregate structures on both mixed bilayers and strictly zwitterionic bilayers. The structures formed on liquid phase supported bilayers were observed to be immobile from a timescale of 50 ms to several minutes. We propose that poly-L-lysine of sufficient length interacts with the mica substrate and phospholipids to create the stationary structures noted.

FIGURE 1

(A) Pure 10 mol % DOPS/90 mol % DOPC. (B–C) After addition of 125 μM PLL. (D–E) After addition of 600 μM. F 600 μM PLL added to mica alone. Arrows point to defects revealing the mica substrate. Stars highlight spherical aggregates, and a line is used to denote PLL-induced domains.

FIGURE 2

(A) DMPC gel phase. An arrow points to a defect extending to the mica substrate. (B) DMPC liquid phase. (C) Liquid phase DMPC after addition of 50 μM PLL. A star encircles two spherical aggregates. (D) Liquid phase DPPC after addition of 50 μM PLL. A line is drawn over a defect, and a star is used to denote a spherical aggregate. (E) DMPC cooled from liquid phase to gel phase after 50 μM PLL addition. A line is drawn over a defect. (F) DPPC cooled from liquid phase to gel phase after 50 μM PLL addition. A line is drawn over a defect. (A, E) 15°C. (C) 45°C. (D) 56°C. (F) 24°C.

FIGURE 3

(A) Control study PBS buffer over quartz. (B) Image of 0.1 mol % NBD-DOPS/9.9 mol % DOPS/90 mol % DOPC. (C) 100 μM PLL solution over DOPS/DOPC bilayer. Arrows point to bright fluorescent spots, indicating NBD-DOPS segregation. (D) Control study 100 μM PLL in PBS over quartz. All Images are 50 × 50 μm.

FIGURE 4

(A) T = 0 after 100 μM PLL addition. (B) T = 2.5 s after 100 μM PLL addition. (C) 5 s after 100 μM PLL addition. (D–E) AFM images taken 10 min apart. (F) Cross section analysis of domain movement in AFM images. Arrows in panels A–C illustrate that the bright fluorescent spots representing segregated NBD-DOPS are immobile. Lines in panels D–E represent cross section comparisons which are shown in panel F. Panels A–C are select fluorescent images from a movie of 100 frames with a capture time of 50 ms. Fluorescent image size is 100 × 100 μm. AFM image size is 2 × 2 μm.

FIGURE 5

(A) Five minutes after PLL addition. An arrow points to a PLL-induced domain. (B) Forty minutes after PLL addition. (C) Magnified image after PLL incubation. The line represents a cross section over a PLL domain. Images A–C of 10 mol % DOPS/90 mol % DOPC bilayer. (D) Five minutes after PLL addition. Arrows point to domains formed. (E) Twenty minutes after PLL addition. (F) Forty minutes after PLL addition. A line spans a defect formed in the bilayers, and an arrow points to a spherical aggregate seen.