Shape transitions and lattice structuring of ceramide-enriched domains generated by sphingomyelinase in lipid monolayers.
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.
Härtel
Steffen
S
Departamento de Química Biológica-CIQUIBIC, Facultad de Ciencias Químicas-CONICET, Universidad Nacional de Córdoba, Ciudad Universitaria, Córdoba, Argentina.
Fanani
María Laura
ML
Maggio
Bruno
B
D000818:Animals; D001409:Bacillus cereus / Q000201:enzymology; D055592:Biophysical Phenomena; D001703:Biophysics; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry; D002518:Ceramides / Q000737:chemistry*; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry*; D008055:Lipids / Q000737:chemistry; D008856:Microscopy, Fluorescence; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D012984:Software; D012997:Solvents / Q000737:chemistry; D013108:Sphingomyelin Phosphodiesterase / Q000737:chemistry*; D055672:Static Electricity; D013499:Surface Properties; D013696:Temperature; D013997:Time Factors
D002518:Ceramides; D008051:Lipid Bilayers; D008055:Lipids; D012997:Solvents; D013108:Sphingomyelin Phosphodiesterase