Domain Growth, Shapes, and Topology in Cationic Lipid Bilayers on Mica by Fluorescence and Atomic Force Microscopy

Abstract

Domain formation in mica-supported cationic bilayers of dipalmitoyltrimethylammoniumpropane (DPTAP) and dimyristoyltrimethylammoniumpropane (DMTAP), fluorescently doped with an NBD (((7-nitro-2-1, 3-benzoxadiazol-4-yl)amino)caproyl) phospholipid, was investigated with fluorescence microscopy and atomic force microscopy. Heating above the acyl chain melting temperature and cooling to room temperature resulted in nucleation and growth of domains with distinguishable patterns. Fractal patterns were found for DPTAP, whereas DMTAP domains were elongated and triangular with feathery edges. Reducing the cooling rate or probe concentration for DPTAP bilayers resulted in larger, filled-in domains with more rounded edges. However, for DMTAP, cooling rates mainly affected size and only slightly modified domain morphology. In a saline environment, the domains were dark, and the surrounding continuous region was bright and thus contained the fluorescent probe. However, as the salt concentration was decreased, the dark regions percolated (connected), resulting in bright domains. Atomic force microscopy scans along domain edges revealed that the dark regions in fluorescence images were approximately 1.4 nm thicker than the light regions. Additionally, the dark regions were of bilayer thickness, approximately 4 nm. Comparison of these results in bilayers to well-documented behavior in Langmuir monolayers has revealed many similarities (and some differences) and is therefore useful for understanding our observations and identifying possible growth mechanisms that may occur in domain formation in cell membranes or supported membrane systems.

Combinatorial microscopy for the study of protein–membrane interactions in supported lipid bilayers: Order parameter measurements by combined polarized TIRFM/AFM

“…2D-F) revealed that this probe's association was strongest with the phase that exhibited an average height $1.3 ± 0.3 nm greater than the other phase. This height difference is usually attributed to the difference in lipid acyl chain splay, tilt and/or packing between the neighboring membrane phases (Connell and Smith, 2006;Dufrene and Lee, 2000;Dunn, 1997, 1998;McKiernan et al, 2000;Yang et al, 1994). The taller of these two regions was therefore identified as the more ordered cholesterol-rich L o phase, and confirmed the previously observed phase partitioning behavior of DiI-C 20 .…”

“…SLBs composed of either lipid mixture 1 or 2 were created by the method of vesicle fusion (Brian and McConnell, 1984;McKiernan et al, 2000;Mou et al, 1994). Briefly, 100 lL of the SUV suspension and 2.9 ml of pH 7.4 aqueous buffer were sequentially deposited onto the freshly cleaved mica previously affixed to the glass of the Willco dish.…”

Mechanisms of antimicrobial peptide action: Studies of indolicidin assembly at model membrane interfaces by in situ atomic force microscopy

“…The Xuid cell was then heated to 70°C for three minutes, and then allowed to cool to room temperature. This heating-cooling step facilitated the nucleation and growth of micron-size gel-phase domains, as has been reported by others (McKiernan et al, 2000). The Xuid cell was then washed sequentially with lipid-and protein-free 10 mM Hepes buVer, pH 7.4, containing 4 mM CaCl 2 , 150 mM NaCl, followed by 10 mM Hepes buVer, pH 7.4, containing 4 mM EDTA, 150 mM NaCl, and 10 mM Hepes buVer, pH 7.4, containing 150 mM NaCl to remove excess solution liposomes prior to confocal-AFM imaging.…”

Cationic peptide-induced remodelling of model membranes: Direct visualization by in situ atomic force microscopy

“…Occasionally, the AFM was placed in a 65°C oven for $15 min and then allowed to cool down to room temperature before imaging. This heating and cooling step was necessary for the formation of supported bilayers from the 50 mol% DSPC/50 mol% DOPC lipid mixtures, facilitating the nucleation and growth of micron-size gel-phase domains, as has been reported by others (McKiernan et al, 2000).…”