Membrane microdomains: Role of ceramides in the maintenance of their structure and functions

Abstract

Free-standing giant unilamellar vesicles were used to visualize the complex lateral heterogeneity, induced by ceramide in the membrane bilayer at micron scale using C(12)-NBD-PC probe partitioning under the fluorescence microscope. Ceramide gel domains exist as leaf-like structures in glycerophospholipid/ceramide mixtures. Cholesterol readily increases ceramide miscibility with glycerophospholipids but cholesterol-ceramide interactions are not involved in the organization of the liquid-ordered phase as exemplified by sphingomyelin/cholesterol mixtures. Sphingomyelin stabilizes the gel phase and thus decreases ceramide miscibility in the presence of cholesterol. Gel/liquid-ordered/liquid-disordered phase coexistence was visualized in quaternary phosphatidylcholine/sphingomyelin/ceramide/cholesterol mixtures as occurrence of dark leaf-like and circular domains within a bright liquid phase. Sphingomyelin initiates specific ceramide-sphingomyelin interactions to form a highly ordered gel phase appearing at temperatures higher than pure ceramide gel phase in phosphatidylcholine/ceramide mixtures. Less sphingomyelin is engaged in formation of liquid-ordered phase leading to a shift in its formation to lower temperatures. Sphingomyelinase activity on substrate vesicles destroys micron L(o) domains but induces the formation of a gel-like phase. The activation of phospholipase A(2) by ceramide on heterogeneous membranes was visualized. Changes in the phase state of the membrane bilayer initiates such morphological processes as membrane fragmentation, budding in and budding out was demonstrated.

Ceramide-enriched microdomains in planar membranes

“…Although the formation of three co-existing phases after Cer addition to ternary lipid mixtures was first visualized in supported bilayers [31], similar observations were subsequently reported for GUVs [39,40] and LUVs [41]. The formation of tightly-packed Cerenriched gel phase has been attributed to the strong intermolecular interactions between Cer molecules [42].…”

“…In two other cases, budding of small vesicles from the interior bilayer leaflet was observed after SMase application to the aqueous solution surrounding the GUVs [39,64], a result that is analogous to earlier observations by Holopainen in SM/PC GUVs [65]. Transformation of L o to gel phase was also observed at early times after enzyme addition [39,57]; co-existing gel, L o and L d phases were not observed, although they have been reported for GUVs with premixed Cer [39]. Thus, it appears that the use of planar membranes allows for observation of more of the intermediate steps in SMaseinduced membrane reorganization.…”

“…For example, an initial study of GUVs prepared from ternary lipid mixtures provided evidence for a SMase-induced transformation from a mixture of L o /L d phases to gel and liquid phases, followed by vesicle collapse [63]. In two other cases, budding of small vesicles from the interior bilayer leaflet was observed after SMase application to the aqueous solution surrounding the GUVs [39,64], a result that is analogous to earlier observations by Holopainen in SM/PC GUVs [65]. Transformation of L o to gel phase was also observed at early times after enzyme addition [39,57]; co-existing gel, L o and L d phases were not observed, although they have been reported for GUVs with premixed Cer [39].…”

Adenylyl cyclase AC8 directly controls its micro-environment by recruiting the actin cytoskeleton in a cholesterol-rich milieu

“…MbCD is a chelator of cholesterol, which alters cell morphology by extruding vesicles of cholesterol from the cell, which consequently reduces the cell surface area and rounds up the cells (Ilangumaran and Hoessli, 1998). The enzyme SMase, hydrolyses sphingomyelin into ceramide and phosphorylcholine to stimulate the internalization of cholesterol from the PM to the ER (Contreras et al, 2003;Staneva et al, 2009). Therefore, whereas MbCD entirely extrudes cholesterol from the cell, SMase internalizes the cholesterol.…”

Imaging the early stages of phospholipase C/sphingomyelinase activity on vesicles containing coexisting ordered-disordered and gel-fluid domains

“…Control vesicles, i.e., those containing PC:SM:PE:Chol (in equimolar proportions) become stained at a somewhat higher rate than those containing DAG and/or Cer in addition. Note that phase characterization in an identical system ( 22 ) and by the various important early ( 30,32,57 ) and contemporary ( 42,49,(58)(59)(60) studies of lipases and lipid domains that have been carried out using natural lipids. GUV are examined individually in most cases, without contact with other vesicles and under reduced mobility conditions; thus, vesicle aggregation or fusion cannot be observed under those conditions.…”