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Article Dans Une Revue Methods in Cell Biology Année : 2016

Purification of recombinant human and Drosophila septin hexamers for TIRF assays of actin-septin filament assembly

Résumé

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 pu- rified 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.
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Dates et versions

hal-01363593 , version 1 (10-09-2016)

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Manos Mavrakis, Tsai Feng-Ching, Gijsje H. Koenderink. Purification of recombinant human and Drosophila septin hexamers for TIRF assays of actin-septin filament assembly. Methods in Cell Biology, 2016, Septins, 136, pp.199. ⟨10.1016/bs.mcb.2016.03.020⟩. ⟨hal-01363593⟩
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