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Methods Cell Biol. 2015;128:125-47. doi: 10.1016/bs.mcb.2015.01.020. Epub 2015 Apr 8.

Triggering actin polymerization in Xenopus egg extracts from phosphoinositide-containing lipid bilayers.

Author information

1
Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge, UK.

Abstract

Xenopus egg extracts are a powerful tool to reconstitute complex cell biological processes using a cell-free strategy. When used in conjunction with liposomes and supported lipid bilayers, they can recapitulate the biochemical activities occurring at the cytosol/plasma membrane interface of the cell that underlie remodeling of the actin cytoskeleton. We use these in vitro systems to elucidate how membranes and proteins collaborate to make the appropriate actin structure at a given time and place. We have recently broadened the types of membrane substrate used, and also optimized protocols for preparation of Xenopus egg extracts for actin assembly assays from membranes. Tuning the lipid composition and curvature appropriately demands an appreciation of the native phospholipid and curvature environments that can form transiently in cells. Supported lipid bilayers on glass coverslips that contain phosphatidylserine and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) make actin bundles termed filopodia-like structures that contain fascin and have vasodilator-stimulated phosphoprotein (VASP) at their growing tips, indicating that these resemble filopodia growing from the plasma membrane. The combination of PI(4,5)P2 and phosphatidylinositol 3-phosphate in curved liposomes or supported bilayers on glass nanospheres uses Snx9, Cdc42, N-WASP (neuronal-Wiskott-Aldrich syndrome protein), and Arp2/3 complex for actin polymerization suggesting that this membrane may mimic the progression from plasma membrane to endosomes. Here we describe how to prepare high-speed supernatant frog egg extracts and phosphoinositide-containing liposomes and supported lipid bilayers that can assemble actin structures. We also describe the methods we use to assay actin polymerization using microscopy and spectrofluorometry and our protocol for immunodepleting specific proteins from extracts.

KEYWORDS:

Arp2/3 complex; Cdc42; Curvature; Cytoskeleton; Filopodia; Membrane; Microscopy; N-WASP; PI(4,5)P(2); Reconstitution

PMID:
25997346
DOI:
10.1016/bs.mcb.2015.01.020
[Indexed for MEDLINE]

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