Cdc42 and PI(4,5)P2-induced actin assembly in Xenopus egg extracts.
Xenopus egg cytoplasmic extracts have been used to study a variety of complex cellular processes. Given their amenability to biochemical manipulation and physiological balance of regulatory proteins, these extracts are an ideal system to dissect signal transduction pathways leading to actin assembly. We have developed methods to study Cdc42 and PI(4,5)P2-induced actin assembly in Xenopus egg extracts. In this chapter, we describe detailed procedures to prepare Xenopus egg extracts, Cdc42, and PI(4,5)P2 for use in actin assembly experiments. We also describe a fluorometric pyrene actin assay for quantitative kinetic analysis of actin polymerization and a microscopic rhodamine actin assay for quick measurement of actin rearrangements in extracts. Finally we provide a protocol for immunodepletion of proteins and discuss the use of immunodepletion and rescue experiments for functional analysis of components in the extracts.
Lebensohn
Andres M
AM
Harvard Medical School, Boston, MA, USA.
Ma
Le
L
Ho
Hsin-Yi Henry
HY
Kirschner
Marc W
MW
D000199:Actins / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000502:physiology; D002469:Cell Separation / Q000379:methods; D050556:Fatty Acid-Binding Proteins; D008081:Liposomes / Q000138:chemical synthesis; D010063:Ovum / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000502:physiology*; D017368:Protein Prenylation; D029867:Xenopus Proteins / Q000172:deficiency / Q000502:physiology; D014982:Xenopus laevis; D020764:cdc42 GTP-Binding Protein / Q000502:physiology*
D000199:Actins; D002352:Carrier Proteins; C488976:FNBP1L protein, Xenopus; D050556:Fatty Acid-Binding Proteins; D008081:Liposomes; D019269:Phosphatidylinositol 4,5-Diphosphate; D029867:Xenopus Proteins; D020764:cdc42 GTP-Binding Protein