Abstract
Membrane-associated actin polymerization is of considerable interest due to its role in cell migration and the motility of intracellular organelles. Intensive research efforts are underway to investigate the physiological role of membrane-associated actin as well as the regulation and mechanics of actin assembly. Branched actin polymerization on membranes is catalyzed by the Arp2/3 complex. Signaling events leading to the activation of the guanosine triphosphate (GTP)-binding protein Cdc42 stimulate Arp2/3-dependent actin polymerization. We have studied the role of Cdc42 at the Golgi apparatus in part by reconstituting actin polymerization on isolated Golgi membranes and on liposomes. In this manner, we showed that cytosolic proteins are sufficient for actin assembly on a phospholipid bilayer. Here we describe methods for the cell-free reconstitution of membrane-associated actin polymerization using liposomes and brain cytosol.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Cooke R (2004) The sliding filament model: 1972-2004. J Gen Physiol 123:643-656
Wu X, Jung G, Hammer JA III (2000) Functions of unconventional myosins. Curr Opin Cell Biol 12:42-51
Theriot JA (2000) The polymerization motor. Traffic 1:19-28
Ridley AJ (2006) Rho GTPases and actin dynamics in membrane protrusions and vesicle trafficking. Trends Cell Biol 16:522-529
Hehnly H, Stamnes M (2007) Regulating cytoskeleton-based vesicle motility. FEBS Lett 581:2112-2118
Fucini RV, Chen JL, Sharma C, Kessels MM, Stamnes M (2002) Golgi vesicle proteins are linked to the assembly of an actin complex defined by mAbp1. Mol Biol Cell 13:621-631
Chen JL, Fucini RV, Lacomis L, Erdjument-Bromage H, Tempst P, Stamnes M (2005) Coatomer-bound Cdc42 regulates dynein recruitment to COPI vesicles. J Cell Biol 169:383-389
Chen JL, Lacomis L, Erdjument-Bromage H, Tempst P, Stamnes M (2004) Cytosol-derived proteins are sufficient for Arp2/3 recruitment and ARF/coatomer-dependent actin polymerization on golgi membranes. FEBS Lett 566:281-286
Fucini RV, Navarrete A, Vadakkan C, Lacomis L, Erdjument-Bromage H, Tempst P, Stamnes M (2000) Activated ADP-ribosylation factor assembles distinct pools of actin on golgi membranes. J Biol Chem 275:18824-18829
Xu W, Stamnes M (2006) The actin-depolymerizing factor homology and charged/helical domains of drebrin and mAbp1 direct membrane binding and localization via distinct interactions with actin. J Biol Chem 281:11826-11833
Welch MD, Iwamatsu A, Mitchison TJ (1997) Actin polymerization is induced by Arp2/3 protein complex at the surface of Listeria monocytogenes. Nature 385:265-269
Heuvingh J, Franco M, Chavrier P, Sykes C (2007) ARF1-mediated actin polymerization produces movement of artificial vesicles. Proc Natl Acad Sci U S A 104:16928-16933
MacDonald RC, MacDonald RI, Menco BP, Takeshita K, Subbarao NK, Hu LR (1991) Small-volume extrusion apparatus for preparation of large, unilamellar vesicles. Biochim Biophys Acta 1061:297-303
Wattenberg BW, Rothman JE (1986) Multiple cytosolic components promote intra-Golgi protein transport. Resolution of a protein acting at a late stage, prior to membrane fusion. J Biol Chem 261:2208-2213
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Stamnes, M., Xu, W. (2010). The Reconstitution of Actin Polymerization on Liposomes. In: Weissig, V. (eds) Liposomes. Methods in Molecular Biology™, vol 606. Humana Press. https://doi.org/10.1007/978-1-60761-447-0_8
Download citation
DOI: https://doi.org/10.1007/978-1-60761-447-0_8
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60761-446-3
Online ISBN: 978-1-60761-447-0
eBook Packages: Springer Protocols