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 hetero-oligomeric 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 2 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 co-assembly. These procedures can be used to compare assembly of Drosophila and human septins and their co-assembly with the actin cytoskeleton by total internal reflection fluorescence (TIRF) microscopy.

Membrane binding controls ordered self-assembly of animal septins

“…Recombinant Drosophila septin hexamers composed of DSep1 with an N-terminal His6 tag, DSep2, and Pnut with a C-terminal Strep tag II (WSHPQFEK, 1058 Da) were purified from Escherichia coli BL21(DE3) cells (Agilent Technologies) using a two-tag affinity purification scheme to aid the isolation of full-length complexes as explained in ref. [70]. Using the same cloning strategy as for wild type complexes [62], a coiled-coil truncation mutant of fly septin hexamers referred to as the ∆CC mutant was made with C-terminal truncations right after the end of the α6-helix (DSep1∆C56, DSep2∆C111, Pnut∆C123).…”

“…To test the ability of the recombinant fly septins to polymerize in bulk solution, we performed TIRF imaging of mEGFP-tagged septin hexamers ( Figure 1D) after rapid dilution from a high salt storage buffer containing 300 mM KCl to a low salt polymerization buffer containing 50 mM KCl. We expect fly septins to form bundles under these conditions [41,70]. To enable observation of septins in the thin (100 nm) evanescent TIRF field, we pushed them down onto a coverslip passivated with a neutral (PC) lipid bilayer with the crowding agent methylcellulose.…”

Septins Recognize and Entrap Dividing Bacterial Cells for Delivery to Lysosomes

“…Beads were centrifuged for 30 s and washed 4x in liposome buffer containing 50 mM MgCl 2 . SEPT2/6/7 was fluorescently labelled using Alexa Fluor 488 NHS Ester as previously described ( Mavrakis et al., 2016 ). After labelling, septins were dialysed against storage buffer (50 mM Tris, pH 8.0, 300 mM KCl, 5 mM MgCl 2 and 5 mM DTT).…”

CDC42EP5/BORG3 modulates SEPT9 to promote actomyosin function, migration, and invasion

“…pCSII-IRES2-blasti-GFP and pCSII-IRES2-blasti-mCherry were a kind gift from Erik Sahai (Francis Crick Institute, London, UK). pnCS-Strep-SEPT6/7 is a bicistronic, spectinomycin-resistant plasmid that expresses human SEPT6 and strep-SEPT7 in tandem; pET28-SEPT9 encodes a kanamycin-resistant His-tagged version of human SEPT9 (Mavrakis et al, 2016). Both plasmids were a kind gift from Elias Spiliotis (Drexel University, Philadelphia, PA).…”