OBSERVING THE NANOSCALE ORGANIZATION OF MODEL BIOLOGICAL MEMBRANES BY ATOMIC FORCE MICROSCOPY - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 8

been observed. 38 Additional problems of interpretation could come from the
fact that natural SM is able to form ripple phases or closely packed globular
structures using this binary lipid mixture ( Fig. 1.3a ) . Interestingly, this pattern
can be suppressed when divalent cations are added in the phosphate buffer
bathing the tip ( Fig. 1.3b ) , thereby strengthening again the importance of a
precise tuning of buffer composition for both SLBs formation and imaging. Chl
contents as well as the composition of fatty acid chains of the luid phase are
also critical parameters for the organization of membrane microdomains (see
“alkaline phosphatase” below), and we think that the phase of eukaryotic

cells should be preferentially mimicked using lipids with asymmetric fatty
acid chain, such as POPC, which are more representative of the PC species in
plasma membranes. 38
More recently, attention has been focused on ceramides (Cer), some
sphingolipids of eukaryotic membranes that have been lately postulated to be
important for membrane structure and function. This lipid can be produced by
de novo

ld

synthesis or through hydrolysis of the SM phosphocholine headgroup
by sphingomyelinase. In a DOPC/SM/Chl mixture, replacing SM by Cer induces
reorganization of the phase and formation of a gel phase enriched in Cer, 39,40

conirming that Chl and Cer could compete for SM association. Similarly, in
a ternary mixture of POPC, natural Cer and Chl, coexistence of gel-like and
lo

lo

domains was observed up to 20% Chl. 41 Taken together, these studies
demonstrate that Cer can induce gel-like domains within membranes (the
presence of gel phases in eukaryotic cell membranes is still a matter of debate)
and underline the notion that the behaviour of the lipid domain structure
as a function of the Chl content is different for membranes containing Cer
compared with those containing SM. This lateral organization is, therefore,
different from that proposed in the raft hypothesis. Interestingly, when Cer
is produced by SM hydrolysis, its effects on the DOPC/SM/Chl mixture's SLB
organization is much more pronounced compared with conditions where
Cer is directly incorporated in LUVs. It gives large clusters of domains that
are heterogeneous, with two distinct heights. 42 This more complex topology
could be explained by Cer lip-lop to the lower lealet, since the presence of
this component in model and cell membranes has been described to allow
rapid transverse diffusion between inner and outer lealets. 43 The fact that
SLBs are symmetric could inluence this phenomenon, compared with native
membranes.
Important insights in the distribution of proteins associated with raft have
also been obtained by AFM imaging of lipid phase-separated membranes. 44
One of these markers is alkaline phosphatase (AP), a protein expressed at the
membrane thanks to a glycosyl-phosphatidylinositol (GPI) anchor which, it
has been proposed, is involved in its partitioning in microdomains. In contrast