title: Purification of recombinant human and Drosophila septin hexamers for TIRF assays of actin-septin filament assembly.
abstract: Septins are guanine nucleotide-binding proteins that are conserved from fungi to humans. Septins assemble into heterooligomeric complexes and higher-order structures with key roles in various cellular functions including cell migration and division. The mechanisms by which septins assemble and interact with other cytoskeletal elements like actin remain elusive. A powerful approach to address this question is by cell-free reconstitution of purified cytoskeletal proteins combined with fluorescence microscopy. Here, we describe procedures for the purification of recombinant Drosophila and human septin hexamers from Escherichia coli and reconstitution of actin-septin coassembly. These procedures can be used to compare assembly of Drosophila and human septins and their coassembly with the actin cytoskeleton by total internal reflection fluorescence microscopy.
authors.affiliation: Aix Marseille Université, CNRS, Centrale Marseille, Institut Fresnel, UMR 7249, Marseille, France.
authors.forename: M
authors.initials: M
authors.lastname: Mavrakis
authors.affiliation: FOM Institute AMOLF, Amsterdam, The Netherlands; Institut Curie, PSL Research University, CNRS UMR 168, Paris, France; Sorbonne Universités, UPMC Univ Paris 06, Paris, France.
authors.forename: F-C
authors.initials: FC
authors.lastname: Tsai
authors.affiliation: FOM Institute AMOLF, Amsterdam, The Netherlands.
authors.forename: G H
authors.initials: GH
authors.lastname: Koenderink
mesh_terms: D000199:Actins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D000818:Animals; D003599:Cytoskeleton / Q000737:chemistry / Q000235:genetics; D004331:Drosophila melanogaster / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D058112:Septins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D000199:Actins; D046912:Multiprotein Complexes; D011994:Recombinant Proteins; D058112:Septins
keywords: Actin; Cytoskeleton; Fluorescence microscopy; Reconstitution; Septins