title: Dissecting principles governing actin assembly using yeast extracts.
abstract: In this chapter, we describe recent protocols that we have developed to trigger actin assembly and actin-based motility in yeast cell extracts. Our method allows for the fast preparation of yeast extracts that are competent in dynamic assembly of distinct actin filament structures of biologically appropriate protein composition. Compared to previous extract-based systems using other eukaryotic cell types, yeast provides a unique advantage for combining reconstituted assays with the preparation of extracts from genetically modified yeast strains. We present a global strategy for dissecting the functions of individual proteins, where the activities of the proteins are analyzed in systems of variable complexity, ranging from simple mixtures of pure proteins to the full complexity of a cell's cytoplasm.
authors.affiliation: Physics of the Cytoskeleton and Morphogenesis Group, institut de Recherches en Technologies et Sciences pour le Vivant, iRTSV, LPCV/CNRS/CEA/INRA/UJF, Grenoble, France. Electronic address: alphee.michelot@cea.fr.
authors.forename: Alphée
authors.initials: A
authors.lastname: Michelot
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, California, USA. Electronic address: drubin@berkeley.edu.
authors.forename: David G
authors.initials: DG
authors.lastname: Drubin
mesh_terms: D000199:Actins / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification / Q000378:metabolism*; D051316:Wiskott-Aldrich Syndrome Protein / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D000199:Actins; D029701:Saccharomyces cerevisiae Proteins; D051316:Wiskott-Aldrich Syndrome Protein
keywords: Actin; Arp2/3; Assay; Extract; Formin; Nucleation; WASP