Shape Transitions and Lattice Structuring of Ceramide-Enriched Domains Generated by Sphingomyelinase in Lipid Monolayers

Abstract

Sphingomyelinases (SMases) hydrolyze the membrane constituent sphingomyelin (SM) to phosphocholine and ceramide (Cer). Growing evidence supports that SMase-induced SM-->Cer conversion leads to the formation of lateral Cer-enriched domains which drive structural reorganization in lipid membranes. We previously provided visual evidence in real-time for the formation of Cer-enriched domains in SM monolayers through the action of the neutral Bacillus cereus SMase. In this work, we disclose a succession of discrete morphologic transitions and lateral organization of Cer-enriched domains that underlay the SMase-generated surface topography. We further reveal how these structural parameters couple to the generation of two-dimensional electrostatic fields, based upon the specific orientation of the lipid dipole moments in the Cer-enriched domains. Advanced image processing routines in combination with time-resolved epifluorescence microscopy on Langmuir monolayers revealed: 1), spontaneous nucleation and circular growth of Cer-enriched domains after injection of SMase into the subphase of the SM monolayer; 2), domain-intrinsic discrete transitions from circular to periodically undulating shapes followed by a second transition toward increasingly branched morphologies; 3), lateral superstructure organization into predominantly hexagonal domain lattices; 4), formation of super-superstructures by the hexagonal lattices; and 5), rotationally and laterally coupled domain movement before domain border contact. All patterns proved to be specific for the SMase-driven system since they could not be observed with Cer-enriched domains generated by defined mixtures of SM/Cer in enzyme-free monolayers at the same surface pressure (pi = 10 mN/m). Following the theories of lateral shape transitions, dipolar electrostatic interactions of lipid domains, and direct determinations of the monolayer dipole potential, our data show that SMase induces a domain-specific packing and orientation of the molecular dipole moments perpendicular to the air/water interface. In consequence, protein-driven generation of specific out-of-equilibrium states, an accepted concept for maintenance of transmembrane lipid asymmetry, must also be considered on the lateral level. Lateral enzyme-specific out-of-equilibrium organization of lipid domains represents a new level of signal transduction from local (nm) to long-range (microm) scales. The cross-talk between lateral domain structures and dipolar electrostatic fields adds new perspectives to the mechanisms of SMase-mediated signal transduction in biological membranes.

Enzymatic generation of ceramide induces membrane restructuring: Correlated AFM and fluorescence imaging of supported bilayers

“…As a consequence, equilibration of enzyme between vesicles is slow compared to establishment of steady state kinetics (Yu et al, 2005a(Yu et al, , 2002. Similarly, studies of SMase activity for monolayers at the air-water interface have concluded that all interfacial-activated enzyme is associated irreversibly with the interface (Fanani et al, 2002;Hartel et al, 2005).…”

“…Thus, the increase in dye-excluded area cannot be directly related to the extent of SM hydrolysis. The situation is different for SM monolayers where changes in surface area due to enzymatic production of the smaller ceramide molecule are proportional to the extent of SM hydrolysis (Fanani et al, 2002;Hartel et al, 2005). Previous studies of PLA 2 have employed conditions where initial bilayer changes were attributable to single enzymes (Balashev et al, 2007).…”

“…For example, treating phosphatidylcholine/SM vesicles with SMase produced ceramide patches that coalesced into larger domains and then formed vesicles (Holopainen et al, 2000;Nurminen et al, 2002). Several other studies have provided evidence for ceramide-enriched domains in monolayers (Fanani et al, 2002;Hartel et al, 2005), in fluid phospholipid vesicles (Taniguchi et al, 2006) and in giant vesicles prepared from SM/ceramide or ceramide/POPC/cholesterol mixtures (Fidorra et al, 2006;Sot et al, 2006). These studies in monolayers and vesicles indicate that enzymatic generation of ceramide leads to more complex changes in membrane morphology than does direct ceramide incorporation in the same lipid membranes.…”

Recent advances in the immunobiology of ceramide

“…Furthermore, it has been indicated that lateral enzyme-specific out-of equilibrium organization of lipid domains represents a new level of signal transduction from local (nm) to long-range (μm) scales (Hartel et al, 2005). Cross-talk between lateral domain structures and dipolar electrostatic fields certainly adds new dimensions to the mechanisms of SMase-mediated signal transduction in biological membranes.…”

The many faces (and phases) of ceramide and sphingomyelin II – binary mixtures

“…As a consequence, such Cers form segregated solid-like condensed domains, in both bilayers and monolayers, that even exclude fluorescent probes and proteins (Carrer and Maggio 1999;Holopainen et al 2001;Härtel et al 2005;Castro et al 2007;Chiantia et al 2008;Busto et al 2009;Karttunen et al 2009;López-Montero et al 2010). Natural mixtures of Cer are mainly composed of long chain Cers.…”