Atomic Force Microscopy Characterization of Palmitoylceramide
and Cholesterol Effects on Phospholipid Bilayers: A Topographic and
Nanomechanical Study
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Abstract
Supported
planar bilayers (SPBs) on mica substrates have been studied
at 23 °C under atomic force microscopy (AFM)-based surface topography
and force spectroscopy with two main objectives: (i) to characterize
palmitoylceramide (pCer)-induced gel (L<sub>β</sub>) domains
in binary mixtures with either its sphingolipid relative palmitoylsphingomyelin
(pSM) or the glycerophospholipid dipalmitoylphosphorylcholine (DPPC)
and (ii) to evaluate effects of incorporating cholesterol (Chol) into
the previous mixtures in terms of Cer and Chol cooperation for the
generation of lamellar gel (L<sub>β</sub>) phases of ternary
composition. Binary phospholipid/pCer mixtures at <i>X</i><sub>pCer</sub> < 0.33 promote the generation of laterally segregated
micron-sized pCer-rich domains. Their analysis at different phospholipid/pCer
ratios, by means of domain thickness, roughness, and mechanical resistance
to tip piercing, reveals unvarying AFM-derived features over increasing
pCer concentrations. These results suggest that the domains grow in
size with increasing pCer concentrations while keeping a constant
phospholipid/pCer stoichiometry. Moreover, the data show important
differences between pCer interactions with pSM or DPPC. Gel domains
generated in pSM/pCer bilayers are thinner than the pSM-rich surrounding
phase, while the opposite is observed in DPPC/pCer mixtures. Furthermore,
a higher breakthrough force is observed for pSM/pCer as compared to
DPPC/pCer domains, which can be associated with the preferential pCer
interaction with its sphingolipid relative pSM. Cholesterol incorporation
into both binary mixtures at a high Chol and pCer ratio abolishes
any phospholipid/pCer binary domains. Bilayers with properties different
from any of the pure or binary samples are observed instead. The data
support no displacement of Chol by pCer or vice versa under these
conditions, but rather a preferential interaction between the two
hydrophobic lipids