Assembly of high molecular weight complexes of lipin on a supported lipid bilayer observed by atomic force microscopy

Lipins are phosphatidic acid phosphatases involved in the biosynthesis of triacylglycerols and phospholipids. They are associated with the endoplasmic reticulum but can also travel into the nucleus and alter gene expression. Previous studies indicate lipins in solution form high molecular weight complexes, possibly tetramers. This study was undertaken to determine if lipins form complexes on membranes as well. Murine lipin 1b was applied to a supported bilayer of phosphatidylcholine, phosphatidylserine, and cholesterol and examined by atomic force microscopy (AFM) over time. Lipin on bare mica appeared as a symmetric particle with a volume consistent with the size of a monomer. On the bilayer, lipin initially bound as asymmetric, curved particles that sometimes assembled into circular structures with an open center. Subsequently, lipin assemblies grew into large, symmetric particles with an average volume 12 times that of the monomer. Over time, some of the lipin assemblies were removed from the bilayer by the AFM probe leaving behind "footprints" composed of complex patterns that may reflect the substructure of the lipin assemblies. The lipin complexes appeared very flat, with a diameter 20 times their height. The footprints had a similar diameter, providing confirmation of the extensive deformation of the protein under the AFM probe. The ability of lipin to form large complexes on membranes may have significant implications for the local concentrations of the product, diacylglycerol, formed during hydrolysis of phosphatidic acid and for cooperative hormonal regulation of lipin activity through phosphorylation of one or more monomers in the complexes.