In vitro reconstitution of cdc42-mediated actin assembly using purified components.
In the accompanying chapter, we describe an in vitro system that uses Xenopus egg extracts to study actin assembly induced by phosphatidylinositol (4,5)bisphosphate (PIP2) and Cdc42. Biochemical fractionation and candidate screening experiments conducted in the extract system have identified the Arp2/3 complex, the N-WASP-WIP (or N-WASP-CR16) complex, and the Cdc42-binding protein Toca-1 as important mediators of PIP2- and Cdc42-actin signaling. Toward our ultimate goal of reconstituting an in vitro system that recapitulates the signaling properties observed in vivo, we then developed a purified actin assembly assay system consisting of the regulatory components that we discovered from extracts. In these assays, the stereotypical sigmoidal kinetics of actin polymerization are monitored by pyrene-actin fluorescence in the presence of defined recombinant or purified proteins, enabling the detailed study of mechanism and protein function. In this chapter, we describe the preparation of the components used in these purified actin assembly reactions, as well as the assay conditions under which we monitor actin polymerization kinetics in vitro.
Division of Neuroscience, Children's Hospital, Boston, MA, USA.
Hsin-Yi Henry
HY
Ho
Rajat
R
Rohatgi
Andres M
AM
Lebensohn
Marc W
MW
Kirschner
D051376:Actin-Related Protein 2-3 Complex / Q000302:isolation & purification / Q000502:physiology*; D000199:Actins / Q000096:biosynthesis* / Q000378:metabolism; D000818:Animals; D001923:Brain Chemistry; D002352:Carrier Proteins / Q000302:isolation & purification / Q000502:physiology; D002417:Cattle; D003598:Cytoskeletal Proteins; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins / Q000502:physiology; D010063:Ovum / Q000737:chemistry; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000502:physiology; D010750:Phosphoproteins / Q000502:physiology; D011721:Pyrenes / Q000737:chemistry; D011994:Recombinant Proteins / Q000302:isolation & purification; D015398:Signal Transduction; D018411:Spodoptera; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal / Q000302:isolation & purification / Q000502:physiology*; D014982:Xenopus laevis; D020764:cdc42 GTP-Binding Protein / Q000502:physiology*
D051376:Actin-Related Protein 2-3 Complex; D000199:Actins; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; C482527:FNBP1L protein, human; D047908:Intracellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D010750:Phosphoproteins; D011721:Pyrenes; D011994:Recombinant Proteins; C109903:WIPF1 protein, human; C105364:Wipf3 protein, rat; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; C030984:pyrene; D020764:cdc42 GTP-Binding Protein