This chapter describes methods for forming tubulovesicular networks, separating the membranes into at least two fractions with little loss of network-forming activity, and extracting peripheral membrane proteins. It also presents various problems encountered in working with the tubulovesicular membrane networks. Several cell lines have been used to make networks, such as chick embryo fibroblasts (CEF), African green monkey kidney cells (CV-I), rat liver, and Xenopus egg extracts. The standard network mixture is placed between parallel lines of high-vacuum silicon grease that form a sample chamber. The mixture is incubated in a humidified chamber for 1–2 hours at room temperature or 30–60 minutes at 37°C. The microtubule motors kinesin and cytoplasmic dynein can be selectively removed from the high-speed supernatant. Standard differential interference contrast (DIC) microscopy can be used to a limited extent to visualize the tubulovesicular networks. Because the tubulovesicular membranes are about 0.1–0.2 μm in diameter and network formation is highly dependent on microtubule motility. The use of video-enhanced DIC microscopy is recommended.
ASJC Scopus subject areas
- Cell Biology