Bottom-Up In Vitro Methods to Assay the Ultrastructural Organization, Membrane Reshaping, and Curvature Sensitivity Behavior of Septins

Summary

Septins are cytoskeletal proteins. They interact with lipid membranes and can sense but also generate membrane curvature at the micron scale. We describe in this protocol bottom-up in vitro methodologies for analyzing membrane deformations, curvature-sensitive septin binding, and septin filament ultrastructure.

Abstract

Membrane remodeling occurs constantly at the plasma membrane and within cellular organelles. To fully dissect the role of the environment (ionic conditions, protein and lipid compositions, membrane curvature) and the different partners associated with specific membrane reshaping processes, we undertake in vitro bottom-up approaches. In recent years, there has been keen interest in revealing the role of septin proteins associated with major diseases. Septins are essential and ubiquitous cytoskeletal proteins that interact with the plasma membrane. They are implicated in cell division, cell motility, neuro-morphogenesis, and spermiogenesis, among other functions. It is, therefore, important to understand how septins interact and organize at membranes to subsequently induce membrane deformations and how they can be sensitive to specific membrane curvatures. This article aims to decipher the interplay between the ultra-structure of septins at a molecular level and the membrane remodeling occurring at a micron scale. To this end, budding yeast, and mammalian septin complexes were recombinantly expressed and purified. A combination of in vitro assays was then used to analyze the self-assembly of septins at the membrane. Supported lipid bilayers (SLBs), giant unilamellar vesicles (GUVs), large unilamellar vesicles (LUVs), and wavy substrates were used to study the interplay between septin self-assembly, membrane reshaping, and membrane curvature.

Introduction

Septins are cytoskeletal filament-forming proteins that interact with lipid membranes. Septins are ubiquitous in eukaryotes and essential to numerous cellular functions. They have been identified as the main regulators of cell division in budding yeast and mammals^1,2. They are involved in membrane reshaping events, ciliogenesis³, and spermiogenesis⁴. Within mammalian cells, septins can also interact with actin and microtubules^5,6,7 in a binder of Rho GTPases (BORG)-depen....

Protocol

1. Determination of membrane reshaping using giant unilamellar vesicles (GUVs)

NOTE: In this section, GUVs are generated to mimic the membrane deformations possibly induced by septins in a cellular context. Indeed, in cells, septins are frequently found at sites with micrometer curvatures. GUVs have sizes ranging from a few to tens of micrometers and can be deformed. They are thus appropriate to assay any micrometer-scale septin-induced deformations. Fluorescent lipids, as well as fluorescently labeled septins (using Green Fluorescent Protein [GFP]), are used to follow the behavior of both lipids and proteins via flu....

Results

GUVs deformations
Typical confocal fluorescence images of GUVs reshaped after being incubated with septins are displayed in Figure 3, in conditions where septins polymerize. Bare GUVs (Figure 3A) were perfectly spherical. Upon incubation with more than 50 nM budding yeast septin filaments, the vesicles appeared deformed. Up to a concentration of 100 nM budding yeast septin octamers, the vesicles appeared facetted, and the deformations remain.......

Discussion

As stated above, a lipid mixture has been used that enhances PI(4,5)P₂ incorporation within the lipid bilayer and thus facilitates septin-membrane interactions. Indeed, we have shown elsewhere²⁵ that budding yeast septins interact with vesicles in a PI(4,5)P₂-specific fashion. This lipid composition was adjusted empirically from screening multiple compositions and is now widely used by the authors. PI(4,5)P₂ lipids have to be handled carefully. Stock solutions must.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine	Avanti Polar Lipids	850725
1,2-dioleoyl-sn-glycero-3-phospho-L-serine	Avanti Polar Lipids	840035
Bath sonicator	Elma		Elmasonic S10H
Bodipy-TR-Ceramide	invitrogen, Thermo Fischer scientific	11504726
Chemicals: NaCl, Tris-HCl, sucrose, KCl, MgCl2, B-casein, chloroform, sodium cacodylate, tannic acid, ethanol	Sigma Aldrich
Confocal microscope	nikon		spinning disk or confocal
Critical point dryer	Leica microsystems		CPD300
Deionized water generator	MilliQ	F1CA38083B	MilliQ integral 3
Egg L-α-phosphatidylcholine	Avanti Polar Lipids	840051
Field Emission Gun SEM (FESEM)	Carl Zeiss		Gemini SEM500
Glutaraldehyde 25 %, aqueous solution	Thermo Fischer scientific	50-262-19
High vacuum grease, Dow corning	VWR
IMOD software	https://bio3d.colorado.edu/imod/		software suite for tilted series image alignment and 3D reconstruction
Lacey Formvar/carbon electron microscopy grids	Eloise	01883-F
Lipids	Avanti Polar Lipids
L-α-phosphatidylinositol-4,5-bisphosphate	Avanti Polar Lipids	840046
Metal evaporator	Leica microsystems		EM ACE600
NOA (Norland Optical Adhesives), NOA 71 and NOA 81	Norland Products	NOA71, NOA81
Osmium tetraoxyde 4%	delta microscopies	19170
Osmometer	Löser		15 M
Plasma cleaner	Alcatel		pascal 2005 SD
Plasma generator	Electron Microscopy Science
Plunge freezing equipment	leica microsystems		EMGP
Transmission electron microscope	Thermofischer		Tecnai G2 200 kV, LaB6
Uranyl acetate	Electron Microscopy Science	22451	this product is not available for purchase any longer
Wax plates, Vitrex	VWR