Improved transfection methods for observation of cytoskeletal structures in primary cultured astrocytes

Astrocytes are the predominant type of glia in the central nervous system and have long, branched stem processes and perisynaptic/peripheral astrocyte processes (PAPs) contacting neurons and other glial cells. However, a common astrocyte culture method generates undesired fibroblast-like cells; thus, the roles of cytoskeletal proteins in astrocytes have not been well studied. Previously, we reported a method for culturing chicken astrocytes that results in cells with structures similar to stem processes and PAPs observed in vivo. In the current study, we improved methods for transfection of chicken astrocytes. The resulting transfected cells retained astrocyte morphology at low cell density, making them suitable for observing protein behaviors. Our cultured astrocytes had various actin-containing substructures such as filopodia, lamellipodia and microvilli in actively moving PAP-like structures. Moreover, LIM and SH3 protein 2 (lasp-2, highly expressed in cultured astrocytes) and the plasma membrane-actin-linking protein ezrin (a PAP marker in brain tissues) accumulated in different actin-containing substructures. Additionally, lasp-2 and F-actin colocalized as small elliptical structures at the base of lamellipodia and filopodia of process tips, which might be cell-substrate adhesions. Our developed methods offer significant advantages for analyzing the regulation of astrocyte morphology and motility.