The invertebrate cytolysin lysenin is a member of the aerolysin family of pore-forming toxins that includes many representatives from pathogenic bacteria. Here we report the crystal structure of the lysenin pore and provide insights into its assembly mechanism. The lysenin pore is assembled from nine monomers via dramatic reorganization of almost half of the monomeric subunit structure leading to a β-barrel pore ∼10 nm long and 1.6–2.5 nm wide. The lysenin pore is devoid of additional luminal compartments as commonly found in other toxin pores. Mutagenic analysis and atomic force microscopy imaging, together with these structural insights, suggest a mechanism for pore assembly for lysenin. These insights are relevant to the understanding of pore formation by other aerolysin-like pore-forming toxins, which often represent crucial virulence factors in bacteria.

Sphingomyelin (SM) is a major sphingolipid in mammalian cells and is reported to form specific lipid domains together with cholesterol. However, methods to examine the membrane distribution of SM are limited. We demonstrated in model membranes that fluorescent protein conjugates of 2 specific SM-binding toxins, lysenin (Lys) and equinatoxin II (EqtII), recognize different membrane distributions of SM; Lys exclusively binds clustered SM, whereas EqtII preferentially binds dispersed SM. Freeze-fracture immunoelectron microscopy showed that clustered but not dispersed SM formed lipid domains on the cell surface. Glycolipids and the membrane concentration of SM affect the SM distribution pattern on the plasma membrane. Using derivatives of Lys and EqtII as SM distribution-sensitive probes, we revealed the exclusive accumulation of SM clusters in the midbody at the time of cytokinesis. Interestingly, apical membranes of differentiated epithelial cells exhibited dispersed SM distribution, whereas SM was clustered in basolateral membranes. Clustered but not dispersed SM was absent from the cell surface of acid sphingomyelinasedeficient Niemann-Pick type A cells. These data suggest that both the SM content and membrane distribution are crucial for pathophysiological events bringing therapeutic perspective in the role of SM membrane distribution.-Makino, A., Abe, M., Murate, M., Inaba, T.,

Pore-forming toxins (PFTs) are soluble proteins that can oligomerize on the cell membrane and induce cell death by membrane insertion. PFT oligomers sometimes form hexagonal close-packed (hcp) structures on the membrane. Here, we show the assembling of the sphingomyelin (SM)-binding PFT, lysenin, into an hcp structure after oligomerization on SM/cholesterol membrane. This process was monitored by high-speed atomic force microscopy. Hcp assembly was driven by reorganization of lysenin oligomers such as association/dissociation and rapid diffusion along the membrane. Besides rapid association/dissociation of oligomers, the height change for some oligomers, possibly resulting from conformational changes in lysenin, could also be visualized. After the entire membrane surface was covered with a well-ordered oligomer lattice, the lysenin molecules were firmly bound on the membrane and the oligomers neither dissociated nor diffused. Our results reveal the dynamic nature of the oligomers of a lipid-binding toxin during the formation of an hcp structure. Visualization of this dynamic process is essential for the elucidation of the assembling mechanism of some PFTs that can form ordered structures on the membrane.

We examined the effect of a sphingomyelin (SM)-binding pore-forming toxin (PFT), lysenin, on the dynamics of a phase-separated membrane of SM, where SM formed liquid-ordered (Lo) domains with cholesterol (Chol) within a phosphatidylcholine-rich liquid-disordered (Ld) phase. We visualized the lysenin-induced membrane reorganization using high-speed atomic force microscope (HS-AFM). Lysenin oligomerized on the SM-rich Lo domain and simultaneously its oligomers assembled into a hexagonal close-packed (hcp) structure. The phase boundary was stable during the assembling of lysenin on the SM-rich domain, indicating that lysenin did not affect the line tension between Lo and Ld phases. After the full coverage of the SM-rich domain by oligomers, their hcp assembly gradually expanded into the Ld phase and eventually covered the entire membrane. Our results suggest that pore formation, i.e., insertion of lysenin into the membrane in its oligomeric state, induced the exclusion of SM and Chol from the SM-rich domain, which was followed by further binding and oligomerization of lysenin.

Since the invention of scanning tunneling microscopy (STM), 2D supramolecular architectures have been observed under various experimental conditions. The construction of these architectures arises from the balance between interactions at the medium-solid interface. This review summarizes molecular motion observed in 2D-supramolecular structures on surfaces using nanospace resolution STM. The observation of molecular motion on surfaces provides a visual understanding of intermolecular interactions, which are the major driving force behind supramolecular arrangement.