Myelin membrane assembly is driven by a phase transition of myelin basic proteins into a cohesive protein meshwork.

Rapid conduction of nerve impulses requires coating of axons by myelin. To function as an electrical insulator, myelin is generated as a tightly packed, lipid-rich multilayered membrane sheath. Knowledge about the mechanisms that govern myelin membrane biogenesis is required to understand myelin disassembly as it occurs in diseases such as multiple sclerosis. Here, we show that myelin basic protein drives myelin biogenesis using weak forces arising from its inherent capacity to phase separate. The association of myelin basic protein molecules to the inner leaflet of the membrane bilayer induces a phase transition into a cohesive mesh-like protein network. The formation of this protein network shares features with amyloid fibril formation. The process is driven by phenylalanine-mediated hydrophobic and amyloid-like interactions that provide the molecular basis for protein extrusion and myelin membrane zippering. These findings uncover a physicochemical mechanism of how a cytosolic protein regulates the morphology of a complex membrane architecture. These results provide a key mechanism in myelin membrane biogenesis with implications for disabling demyelinating diseases of the central nervous system.

Optical fusion assay based on membrane-coated spheres in a 2D assembly.

A major goal in neurophysiology and research on enveloped viruses is to understand and control the biology and physics of membrane fusion and its inhibition as a function of lipid and protein composition. This poses an experimental challenge in the realization of fast and reliable assays that allow us, with a minimal use of fluorescent or radioactive labels, to identify the different stages of membrane-membrane interaction ranging from docking to complete membrane merging. Here, an optical two-dimensional fusion assay based on monodisperse membrane-coated microspheres is introduced, allowing unequivocal assignment of docking and membrane fusion. The hard-sphere fluid captures and quantifies relevant stages of membrane fusion and its inhibition without interference from aggregation, liposome rupture, extensive fluorescence labeling, and light scattering. The feasibility of the approach is demonstrated by using an established model system based on coiled-coil heterodimers formed between two opposing membrane-coated microspheres.

Impact of peptide clustering on unbinding forces in the context of fusion mimetics.

Coiled-coil zipping and unzipping is a pivotal process in SNARE-regulated membrane fusion. In this study we examine this process mediated by a minimal model for coiled-coil formation employing force spectroscopy in the context of membrane-coated surfaces and probes. The interaction forces of several hundred pN are surprisingly low considering the proposed amount of molecular bonds in the contact zone. However, by means of high-resolution imaging employing atomic force microscopy and studying the lateral mobility of lipids and peptides as a function of coiled-coil formation, we are able to supply a detailed view on processes occurring on the membrane surfaces during force measurements. The interaction forces determined here are not only dependent on the peptide concentration on the surface, but also on the regional organization of lateral peptide clusters found prior to coiled-coil formation.

Coiled-coil formation on lipid bilayers--implications for docking and fusion efficiency.

Coiled-coil formation of four different oligopeptides was characterized in solution, on hydrogels, and on membranes by employing circular dichroism spectroscopy, surface plasmon resonance spectroscopy, attenuated total reflection infrared spectroscopy, and ellipsometry. Peptide sequences rich in either glutamic acid (E: E3Cys, i-E3Cys) or lysine (K: K3Cys, i-K3Cys) were used to represent minimal mimics of eukaryotic SNARE motifs. Half of the peptides were synthesized in reverse sequence, so that parallel and antiparallel heptad coiled-coil structures were formed. Either E-peptides or K-peptides were attached covalently to phospholipid anchors via maleimide chemistry, and served as receptors for the recognition of the corresponding binding partners added to solution. Attenuated total reflection infrared spectroscopy of single bilayers confirmed the formation of coiled-coil complexes at the membrane interface. Coiled-coil formation in solution, as compared with association at the membrane surface, displays considerably larger binding constants that are largely attributed to loss of translational entropy at the interface. Finally, the fusogenicity of the various coiled-coil motifs was explored, and the results provide clear evidence that hemifusion followed by full fusion requires a parallel orientation of α-helices, whereas antiparallel oriented coiled-coil motifs display only docking.

Hydrophobic mismatch sorts SNARE proteins into distinct membrane domains.

The clustering of proteins and lipids in distinct microdomains is emerging as an important principle for the spatial patterning of biological membranes. Such domain formation can be the result of hydrophobic and ionic interactions with membrane lipids as well as of specific protein-protein interactions. Here using plasma membrane-resident SNARE proteins as model, we show that hydrophobic mismatch between the length of transmembrane domains (TMDs) and the thickness of the lipid membrane suffices to induce clustering of proteins. Even when the TMDs differ in length by only a single residue, hydrophobic mismatch can segregate structurally closely homologous membrane proteins in distinct membrane domains. Domain formation is further fine-tuned by interactions with polyanionic phosphoinositides and homo and heterotypic protein interactions. Our findings demonstrate that hydrophobic mismatch contributes to the structural organization of membranes.

A size barrier limits protein diffusion at the cell surface to generate lipid-rich myelin-membrane sheets.

The insulating layers of myelin membrane wrapped around axons by oligodendrocytes are essential for the rapid conduction of nerve impulses in the central nervous system. To fulfill this function as an electrical insulator, myelin requires a unique lipid and protein composition. Here we show that oligodendrocytes employ a barrier that functions as a physical filter to generate the lipid-rich myelin-membrane sheets. Myelin basic protein (MBP) forms this molecular sieve and restricts the diffusion of proteins with large cytoplasmic domains into myelin. The barrier is generated from MBP molecules that line the entire sheet and is, thus, intimately intertwined with the biogenesis of the polarized cell surface. This system might have evolved in oligodendrocytes in order to generate an anisotropic membrane organization that facilitates the assembly of highly insulating lipid-rich membranes.