In vitro reconstitution of cytoskeletal proteins is a vital tool to understand the basic functional properties of these proteins. The present paper describes a protocol to purify and assess the quality of recombinant septin complexes, which play a central role in cell division and migration.
Septins are a family of conserved eukaryotic GTP-binding proteins that can form cytoskeletal filaments and higher-order structures from hetero-oligomeric complexes. They interact with other cytoskeletal components and the cell membrane to participate in important cellular functions such as migration and cell division. Due to the complexity of septins’ many interactions, the large number of septin genes (13 in humans), and the ability of septins to form hetero-oligomeric complexes with different subunit compositions, cell-free reconstitution is a vital strategy to understand the basics of septin biology. The present paper first describes a method to purify recombinant septins in their hetero-oligomeric form using a two-step affinity chromatography approach. Then, the process of quality control used to check for the purity and integrity of the septin complexes is detailed. This process combines native and denaturing gel electrophoresis, negative stain electron microscopy, and interferometric scattering microscopy. Finally, a description of the process to check for the polymerization ability of septin complexes using negative stain electron microscopy and fluorescent microscopy is given. This demonstrates that it is possible to produce high-quality human septin hexamers and octamers containing different isoforms of septin_9, as well as Drosophila septin hexamers.
The cytoskeleton has been classically described as a three-component system consisting of actin filaments, microtubules, and intermediate filaments1, but recently, septins have been acknowledged as a fourth component of the cytoskeleton1. Septins are a family of GTP-binding proteins that are conserved in eukaryotes2. Septins are involved in many cellular functions such as cell division3, cell-cell adhesion4, cell motility5, morphogenesis6, cellular infection7, and the establishment and maintenance of cell polarity8. Despite their important functions, how septins are involved in such processes is poorly understood.
The septin family of proteins is subdivided into several subgroups (four or seven, depending on the classification) based on protein sequence similarity2. Members of different subfamilies can form palindromic hetero-oligomeric complexes, which are the building blocks of filaments and which, in turn, assemble into higher-order structures such as bundles, rings, and meshworks1,9,10,11,12. Further molecular complexity arises from the presence of different splice variants, an example being human SEPT9, where there is evidence for specific functions of different splice variants13,14,15. Additionally, the length of the hetero-oligomers depends on species and cell type. For instance, Caenorhabditis elegans septins form tetramers16, Drosophila melanogaster septins form hexamers17 (Figure 1A), Saccharomyces cerevisiae septins form octamers18, and human septins form both hexamers and octamers19 (Figure 1A). The ability of septin isoforms, splice variants, and post-translationally modified septins from the same subfamily to substitute each other in the complex and the (co-)existence of differently sized hetero-oligomers have made it difficult to delineate the cellular functions of different hetero-oligomeric complexes12.
Another interesting ability of septins is their ability to interact with many binding partners in the cell. Septins bind the plasma membrane and membranous organelles during interphase and cell division20,21,22. In dividing cells, septins cooperate with anillin23,24,25 and actin and myosin during cytokinesis26,27. At the late stages of cytokinesis, septins seem to regulate the endosomal sorting complexes required for the transport (ESCRT) system for midbody abscission28. Additionally, there is also evidence of septin located on the actin cortex and actin stress fibers of cells in interphase cells29,30,31. In specific cell types, septins also bind and regulate the microtubule cytoskeleton32,33.
All of these features make septins a very interesting protein system to study, but also a challenging one. The combination of the large number of septin subunits (13 genes in humans without counting splice variants2) with the potential of septin subunits from the same subfamily to substitute each other and form differently sized hetero-oligomers makes it difficult to draw a conclusion on the cellular function of a specific septin by genetic manipulation. Furthermore, the multiple interactions of septins make interpreting the effects of common research tools such as drugs34 directed at cytoskeletal or membrane components a hard task.
A way to overcome this situation is to complement research in cells with in vitro (cell-free) reconstitution of septins. In vitro reconstitution allows for the isolation of a single type of septin hetero-oligomers with a specific subunit composition and length18,35,36,37. This complex can then be studied in a controlled environment, either alone to discover the basic structural and physicochemical properties of septins38,39,40, or in combination with desired partners such as model biomembranes11,41,42, actin filaments10,27, or microtubules32,36 to decipher the nature of their interactions.
Therefore, a reliable method to purify different septin complexes efficiently is vital for septin research. However, even using the same protocol, different purifications can give proteins with different activity/functionality or even integrity. For commercially available proteins such as enzymes, the functionality and enzymatic activity are carefully validated43. Implementing careful quality control for cytoskeletal or structural proteins such as septins can be challenging, but it is essential to make experiments across labs comparable.
This paper describes a robust method to purify high-quality recombinant septins in their hetero-oligomeric form based on the simultaneous expression of two vectors containing mono- or bi-cistronic constructs (Table 1) in Escherichia coli cells. The method consists of a two-step affinity chromatography approach to capture septin hetero-oligomers containing both a his6-tagged septin and a Strep-II-tagged septin (Figure 1B,C). This protocol, first described in Iv et al.10, has been used to purify Drosophila septin hexamers11,27,35, human septin hexamers10, and several human septin octamers containing different native (isoform 1, 3, and 5)10,32 or mutated SEPT9 isoforms32. Furthermore, a description of a set of techniques to assess the quality of the purified septins is given. First, the integrity and correct stoichiometry of the septin subunits is checked using denaturing electrophoresis and transmission electron microscopy (TEM). Then, the presence of hetero-oligomers of the correct molecular mass and the presence of monomers or smaller oligomers indicative of complex instability are examined by native electrophoresis and mass photometry via interferometric scattering microscopy (iSCAT). Finally, the last step consists of the assessment of the polymerizing activity of the septins using fluorescence microscopy and TEM.
Figure 1: Purification strategy. (A) Schematics of the septin hetero-oligomers that exist in human (left) and Drosophila (right) cells. Numbers denote septin subunits from the indicated groups, and P denotes Peanut. Human SEPT9 can be any of its isoforms. The septin subunits have an asymmetric shape and are longitudinally associated with two distinct interfaces, the NC:NC and the G:G interface, as denoted by NC and G, respectively, on top of the human hexamer. (B,C) Schematic illustration of the two-step chromatography strategy, shown for (B) human septin hexamers and (C) octamers. H indicates the his-tags, while S indicates the Strep-II-tags. Please click here to view a larger version of this figure.
The method described here allows for the robust purification and quality control of pre-formed septin hetero-oligomers. Some of the key issues to consider for the correct application of the method are as follows. During the elution steps in the chromatographic separations, it is important to use the recommended (or lower) flow rate to minimize the dilution of the septin complexes. Additionally, to maximize the recovery of protein during the final concentration step, the concentrator column is oriented in such a way that the solution is not pushed against the filter (when there is only a filter on one side). If the solution goes directly to the filter, the protein sticks much more to it, substantially decreasing the final yield. It is also important to consider that the concentration step is not always necessary. Picking fractions only from a narrow range around the peak in the chromatogram usually gives a high enough stock concentration (>3,000 nM) for many reconstitution applications (which usually operate between 10-300 nM). Finally, for the quality control of the functionality of the septin complexes by fluorescence microscopy, it is important to correctly passivate the surface of the microscopy slides, since septin complexes avidly stick to glass. Passivation of the glass slides can be done either via PLL-PEG functionalization or by the formation of neutral (100% DOPC) supported lipid bilayers11,32.
Compared to the original purification protocol first described in Iv et al.10, there is a change in the buffer compositions (Table 2). The concentration of MgCl2 has been reduced from 5 mM to 2 mM, and the concentration and pH of Tris-HCl have been reduced from 50 mM to 20 mM and from 8.0 to 7.4, respectively. These changes were made to make the buffer conditions compatible with studies of the interactions of human septins with lipid bilayers, actin filaments, and microtubules10,11,32. This is because the authors formed supported lipid bilayers and polymerized actin in the F-buffer, whose composition is identical to that of darkSPB, apart from the presence of ATP in the F-buffer. The buffer change did not produce any changes in the quality or lifetime of the purified septins compared to the original buffers.
This method of purification still has several limitations. First, different purification attempts can vary in yield (0.5-1 mL of 2-5 μM septin complexes) and functional quality, as checked by the bundle formation ability of the purified septin complexes. That is why it is very important to consistently perform the quality checks described in this paper. Controlling very well the times of expression and the optical density of the bacterial culture can help mitigate the difference in yield. Second, this purification pipeline cannot distinguish between trimers and hexamers or between tetramers and octamers (Figure 1B). However, the quality control experiments can be used to prove that the majority of septin complexes are in their long oligomer form. In case an even narrower oligomer size distribution is required, size exclusion chromatography can be inserted in between step 1.6. and step 1.7. of the purification protocol. This optional step, however, dramatically decreases the yield, and it is not recommended unless it is strictly needed. A last, more fundamental, limitation comes from the use of E. coli as an expression system for recombinant septin complexes. Naturally, this system does not allow for post-translational modifications (PTMs), which have been reported in animal cells, such as phosphorylation, acetylation, and sumoylation6,51,52,53. These posttranslational modifications could be added by implementing a similar purification strategy in insect or human cells. Furthermore, this paper has only discussed the reconstitution of septins by themselves, but studies in cells indicate that regulatory proteins such as proteins from the Borg family54,55 and anillin24,25,56 can have substantial yet poorly understood effects on the assembly and functions of septins and are, therefore, important to eventually incorporate in in vitro studies. Protocols for the purification of Borg proteins and anillin have been reported54,57.
The septin purification protocol reported here offers a standardized way to purify septins in their oligomer form with the correct subunit stoichiometry, offering an important advance over many earlier in vitro studies relying on single septin subunits. Even though some septins in specific contexts can act as a single subunit2, the current body of literature strongly suggests that, in animal cells, septins mostly function in complexes9,58. Therefore, the use of pre-formed hetero-oligomers, such as the ones described in this paper and others10,11,18,32,35,36,37, is of great importance to study the structural and biophysical properties of septins via in vitro reconstitution to dissect their functions in the cell. Furthermore, septins are self-assembling proteins with many interaction partners, including the membrane and the cytoskeleton, which makes them of great interest for bottom-up synthetic biology59,60,61 and studies of protein-induced changes in membrane biophysical properties such as curvature42,62,63.
The authors have nothing to disclose.
We thank Cecilia de Agrela Pinto, Tomás de Garay, and Katharina Häußermann for their assistance with mass photometry (iSCAT) experiments; Arjen Jakobi and Wiel Evers for their help with TEM; Lucia Baldauf for her assistance with TIRF; Pascal Verdier-Pinard for his advice concerning native electrophoresis; Agata Szuba and Marjolein Vinkenoog for their help in setting up the Drosophila septin purification efforts, and the Cell and Tissue Imaging (PICT-IBiSA), Institut Curie, member of the French National Research Infrastructure France-BioImaging (ANR10-INBS-04). This research received funding from the Netherlands Organization for Scientific Research (NWO/OCW) through the 'BaSyC-Building a Synthetic Cell' Gravitation grant (024.003.019) and from the Agence Nationale pour la Recherche (ANR grants ANR-17-CE13-0014: “SEPTIMORF”; ANR-13-JSV8-0002-01: “SEPTIME”; and ANR-20-CE11-0014-01: “SEPTSCORT”).
488nm laser combiner iLAS2 | Gataca | TIRF microscope | |
488nm Sapphire laser lines | Coherent | Confocal microscope | |
4k X 4k F416 CMOS camera | TVIPS | For JEM-1400plus | |
4x sample buffer nativePAGE | Thermo Fisher scientific | BN2003 | |
6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (TROLOX) | Sigma-Aldrich | 238813 | To prevent blinking |
AKTA pure 25 M1 | GE healthcare | 1680311 | |
Ampicillin | Sigma-Aldrich | A9518-25G | |
Carbon Type-B, 300 mesh EM grid | Ted pella | 01813-F | |
Carbon Type-B, 300 mesh EM grid | Electron micoscopy sciences | CF300-Cu | |
Cover glass #1.5H | Thorslabs | CG15KH | |
CSU-X1-M1 confocal unit | Yokogawa | Confocal microscope | |
Desthiobiotin | Sigma-Aldrich | D1411-1G | |
Dithiothreitol (DTT) | Sigma-Aldrich | D9779 | |
DNAse | Sigma-Aldrich | 10104159001 | |
DOPC | Avanti Polar Lipids | 850375C | |
Eclipse Ti2-E | Nikon instruments | Confocal microscope | |
EDTA-free protease inhibtor cocktail | Roche | 481761 | |
HisTrap HP, 5 mL | GE healthcare | 29-0588-3 | |
iLAS2 azimuthal TIRF illumination system | Gataca | TIRF microscope | |
Imidazole | Sigma-Aldrich | 1202-1KG | |
InstantBlue Protein Gel Stain | Westburg Life Sciences | EP ab119211 | |
Isopropyl β-D-1-thiogalactopyranoside (IPTG) | Thermo Fisher scientific | 10849040 | |
iXon Ultra 888 EMCCD camera | Andor | Confocal microscope | |
iXon Ultra 897 EM-CCD | Andor | TIRF microscope | |
JEM-1400plus | JOEL | TEM microscope TUDelft | |
kappa-cassein | Sigma-Aldrich | C0406 | |
LB broth | Sigma-Aldrich | L3022-6X1KG | |
Lyzozyme | Sigma-Aldrich | 62971-10G-F | |
Magnesium Chloride | Sigma-Aldrich | M8266-100G | |
Magnesium sulfate | Sigma-Aldrich | 746452-1KG | |
Methylecllulose | Sigma-Aldrich | 8074844 | |
MilliQ system (Integral 10) | Merck-Millipore | I-water dispenser | |
Mini protean TGX gels | BIORAD | 4561086 | |
NativeMark unstained protein standard | Invitrogen | LC0725 | For iSCAT and Native gels |
NativePAGE 4-16% GELS | Thermo Fisher scientific | BN1002BOX | |
NativePAGE Running Buffer kit | Thermo Fisher scientific | BN2007 | |
Nikon Ti2-E | Nikon instruments | TIRF microscope | |
Nr. 1 Menzel coverslips | Thermo Fisher scientific | 11961988 | |
parafilm | Sigma-Aldrich | P7668 | |
Plan Apo ×100/1.45 NA oil immersion objective | Nikon instruments | Confocal microscope | |
PMSF | Sigma-Aldrich | 10837091001 | |
Poly(L-lysine)-graft-biotinylated PEG (PLL-PEG) | SuSoS | CHF560.00 | |
Poly-L-lysine solution 0.01% | Sigma-Aldrich | P4832 | For iSCAT glass slides |
Pottassium Chloride | Sigma-Aldrich | P9541-1KG | |
Power supply for native gels | CONSORT | S/N 71638 | |
POWERPAC UNIVERSAL | BIORAD | 042BR31206 | |
Protocatechuate 3,4-Dioxygenase (PCD) | Sigma-Aldrich | P8279-25UN | oxygen scavenger – enzyme |
Protocatechuic acid (PCA) | Sigma-Aldrich | 03930590-50MG | oxygen scavenger – reagent |
Q500 Sonicator | Qsonica | Q500-110 | |
Quemesa camera | Olympus | For Tecnai Spirit | |
Refeyn OneMP | Refeyn | ||
Sample buffer, laemmli 2x concentrate | Sigma-Aldrich | S3401-10vl | |
Silicon gaskets | Sigma-Aldrich | GBL103250-10EA | |
Slide-A-Lyzer Dialysis cassettes 30k MWCO 3mL | Thermo Fisher scientific | 66381 | |
Spectinomycin | Sigma-Aldrich | PHR1441-1G | |
StrepTrap HP, 1 mL | GE healthcare | 28-9075-46 | |
Tecnai Spirit microscope | Thermo Scientific, FEI | TEM microscope Institute Curie | |
Terrific broth | Sigma-Aldrich | T0918-1KG | |
Tris/Glyine/SDS buffer | BIORAD | 1610772 | |
Tris-HCl | Sigma-Aldrich | T5941-1KG | |
Ultrasonic cleaner | Branson | CPX2800H-E | |
Vivaspin 6, 30,000 MWCO PES | Sartorius | VS0622 |