Shape transitions and lattice structuring of ceramide-enriched domains generated by sphingomyelinase activity in lipid monolayers

STEFFEN HÄRTER, MARÍA L. FANANI, AND BRUNO MAGGIO

BIOPHYSICAL JOURNAL

Año: 2005 vol. 88 p. 287 - 304

0006-3495

  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 Cerenriched domains which drive structural reorganization in lipid membranes.Wepreviously 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 twodimensional electrostatic .elds, based upon the speci.c orientation of the lipid dipole moments in the Cer-enriched domains. Advanced image processing routines in combination with time-resolved epi.uorescence 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 speci.c for the SMase-driven system since they could not be observed with Cer-enriched domains generated by de.ned mixtures of SM/Cer in enzyme-free monolayers at the same surface pressure (P ¼ 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-speci.c packing and orientation of the molecular dipole moments perpendicular to the air/water interface. In  consequence, protein-driven generation of speci.c out-of-equilibrium states, an accepted concept for maintenance of transmembrane lipid asymmetry, must also be considered on the lateral level. Lateral enzyme-speci.c out-of-equilibrium organization of lipid domains represents a new level of signal transduction from local (nm) to long-range (mm) scales. The cross-talk between lateral domain structures and dipolar electrostatic .elds adds new perspectives to the mechanisms of SMase-mediated signal transduction in biological membranes.