Morphological analysis on the distribution of membrane lipids and a membrane protein, NAP-22, during neuronal development in vitro

Specific localization of membrane proteins based on the interactions with membrane lipids at various microdomains (MDs) is under active investigation, since the elucidation of the molecular mechanism of the interactions could reveal a novel concept of cell organization. Due to the strong interactions not only between lipids but also between lipids and proteins, these MDs are considered to be recovered in a detergent-resistant low-density membrane fraction (DRM) after detergent extraction and density-gradient centrifugation. Neurons take well-developed membrane systems during maturation and specific localization of various membrane components, not only proteins but also lipids, is essential for the establishment of the nervous system. In previous studies, we showed that NAP-22 is a major protein of neuronal DRM and binds liposomes in a cholesterol-dependent manner. In this study, we analyzed the localization of membrane lipids during neuronal maturation in vitro and compared their distribution with that of NAP-22. In an attempt to detect DRM-associated lipids, we observed the staining patterns of neurons treated with Triton-X-100 at 4°C before fixation. Our results showed the less staining patterns of cholesterol and sphingomyelin at the axonal tips and a different staining pattern of two gangliosides, GM 1 and GD 3 . The enrichment of cholesterol at the NAP-22 localizing spots was observed after the treatment of the detergent. Since the application of maitotoxin, a calcium ion channel, caused the diminution of NAP-22 and cholesterol positive spots, the distribution of these molecules are considered under the calcium regulation.