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

TL;DR

This paper presents methods for purifying recombinant human and Drosophila septin hexamers and reconstituting their coassembly with actin for TIRF microscopy studies, advancing understanding of cytoskeletal interactions.

Contribution

It introduces protocols for purifying septin complexes from E. coli and reconstituting actin-septin assembly for fluorescence microscopy analysis.

Findings

Successful purification of human and Drosophila septin hexamers.

Reconstitution of actin-septin coassembly observed via TIRF microscopy.

Protocols enable comparative studies of septin assembly and interaction with actin.

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 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.