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

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 generated undesired fibroblast-like cells; thus, the roles of cytoskeletal proteins in astrocytes have not been well studied. Previously, we reported a culture method of chicken astrocytes forming structures similar to stem processes and PAPs in vivo. In the current study, we improved transfection methods retaining astrocyte morphology at low cell density, 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, lasp-2 (LIM and SH3 protein 2, highly expressed in cultured astrocytes) and 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 may be cell-substrate adhesions. Our developed methods offer significant advantages for analyzing the regulation of astrocyte morphology and motility.