Purification of recombinant human and Drosophila septin hexamers for   TIRF assays of actin-septin filament assembly
  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. 
Manos Mavrakis (MOSAIC), Tsai Feng-Ching (FOM Institute AMOLF), Gijsje   H. Koenderink (FOM Institute AMOLF)
q-bio.SC q-bio.CB