Microtubule Motor-Dependent Formation of Tubulovesicular Networks from Endoplasmic Reticulum and Golgi Membranes

James M. McIlvain, Carilee Lamb, Sandra Dabora, Michael Sheetz

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)227-236
Number of pages10
JournalMethods in Cell Biology
Volume39
Issue numberC
DOIs
StatePublished - Jan 1 1993
Externally publishedYes

Fingerprint

Microtubules
Endoplasmic Reticulum
Interference Microscopy
Membranes
Cytoplasmic Dyneins
Cercopithecus aethiops
Kinesin
Silicon
Chick Embryo
Vacuum
Xenopus
Ovum
Membrane Proteins
Fibroblasts
Kidney
Cell Line
Temperature
Liver

ASJC Scopus subject areas

  • Cell Biology

Cite this

Microtubule Motor-Dependent Formation of Tubulovesicular Networks from Endoplasmic Reticulum and Golgi Membranes. / McIlvain, James M.; Lamb, Carilee; Dabora, Sandra; Sheetz, Michael.

In: Methods in Cell Biology, Vol. 39, No. C, 01.01.1993, p. 227-236.

Research output: Contribution to journalArticle

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