Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions

Summary

Cell-free reconstitution has been a key tool to understand the cytoskeleton assembly, and work in the last decade has established approaches to study septin dynamics in minimal systems. Presented here are three complementary methods to observe septin assembly in different membrane contexts: planar bilayers, spherical supports, and rod supports.

Abstract

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component in coordinating shape changes like cytokinesis, polarized growth, and migration. Septins are filament-forming proteins that assemble to form diverse higher-order structures and, in many cases, are found in different areas of the plasma membrane, most notably in regions of micron-scale positive curvature. Monitoring the process of septin assembly in vivo is hindered by the limitations of light microscopy in cells, as well as the complexity of interactions with both membranes and cytoskeletal elements, making it difficult to quantify septin dynamics in living systems. Fortunately, there has been substantial progress in the past decade in reconstituting the septin cytoskeleton in a cell-free system to dissect the mechanisms controlling septin assembly at high spatial and temporal resolutions. The core steps of septin assembly include septin heterooligomer association and dissociation with the membrane, polymerization into filaments, and the formation of higher-order structures through interactions between filaments. Here, we present three methods to observe septin assembly in different contexts: planar bilayers, spherical supports, and rod supports. These methods can be used to determine the biophysical parameters of septins at different stages of assembly: as single octamers binding the membrane, as filaments, and as assemblies of filaments. We use these parameters paired with measurements of curvature sampling and preferential adsorption to understand how curvature sensing operates at a variety of length and time scales.

Introduction

The shapes of cells and many of their internal compartments are dependent on the lipid membranes that surround them. Membranes are viscoelastic structures that can be deformed through interactions with proteins, lipid sorting, and acting internal and external forces to generate a variety of shapes^1,2,3,4. These shapes are often described in terms of membrane curvature. Cells use a diverse suite of proteins capable of preferentially assembling onto, or "sensing", particular membrane curvatures to ensure defined spatio-temporal control....

Protocol

NOTE: Forming supported lipid bilayers requires the preparation of monodispersed small unilamellar vesicles (SUVs). Please refer to a previously published protocol²⁴ on SUV formation. Briefly, all SUVs are formed by probe sonication for 12 min in total at 70% amplitude via 4 min sonication periods followed by 2 min rest periods in ice-water. SUV solutions must be well clarified and monodispersed in size. Size distributions of SUVs can be measured, for example, by dynamic light scattering²⁵.

1. Planar lipid bilayers

1. Plasma cleaning of the slides
   NOTE: Plasma ....

Results

Following the preparation of each SLB, septins or the protein of interest may be incubated with the desired support and imaged via TIRFM, confocal microscopy, or SEM. The results shown here use septins recombinantly expressed and purified from E. coli¹⁷. Using TIRFM on planar SLBs, it is possible to determine the length of filaments and their flexibility, measure the diffusion coefficients and observe assembly over time^28,29. In o.......

Discussion

Cell membranes take on many different shapes, curvatures, and physicochemical properties. In order to study the nanometer-scale machinery through which cells build micrometer-scale assemblies, it is necessary to design minimal reconstitution systems of membrane mimetics. This protocol presents techniques that precisely control both membrane curvature and composition while allowing the user to easily take quantitative fluorescence measurements using widely available microscopy techniques.

The m.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.2 mL PCR Tubes with flat cap, Natural	Watson	137-211C(EX)
0.5 mL low adhesion tubes	USA Scientific	1405-2600
Beta mercaptoethanol (BME)	Sigma-Aldrich	M6250-100ML
Bovine Serum Albumin (BSA)	Sigma-Aldrich	A4612-25G
Coverglass for making PEGylated coverslips	Thermo Scientific	152450	Richard-Allan Scientific SLIP-RITE Cover Glass 24x50 #1.5
DOPC	Avanti Polar Lipids	850375
Egg Liss Rhodamine PE	Avanti Polar Lipids	810146
EMS Glutaraldehyde Aqueous 25%, EM Grade	VWR	16220
EMS Sodium Cacodylate Buffer	VWR	11652
Ethanol, 200 proof	Fisher Scientific	04-355-223EA
HEPES	Sigma Aldrich	H3375-1KG
Hexamethyldisilazane	Sigma-Aldrich	440191
Magnesium chloride	VWR	7791-18-6
Methyl cellulose 4000cp	Sigma-Aldrich	M052-100G
Microglass coverslips for planar bilayers	Matsunami	Discontinued	22x22
Mini centrifuge
Non-Functionalized Silica Microspheres	Bangs Laboratories, Inc.	Depends on size: SS0200*-SS0500*	Silica in aqueous suspension
Optical Adhesive	Norland Thorlabs	NOA 68	Flexible adhesive for glass or plastics
Osmium tetroxide	Millipore Sigma	20816-12-0
Parafilm	VWR	52858-000
Plasma Cleaner	Plasma Etch	PE-25	Voltage: 120V, 60Hz. Current: 15 AMPS
Potassium chloride	VWR	0395-1kg
Round coverglass, #1.5 12mm	VWR	64-0712
Sonicator bath	Branson	1510R-MT	Bransonic Ultrasonic cleaner. 50-60 Hz. Output: 70W
Soy PI	Avanti Polar Lipids	840044
Tabletop centrifuge	Eppendorf	22331
UV Lamp	Spectroline	ENF-260C	115 Volts, 60 Hz, 0.20 AMPS
WhatmanGlass Microfiber Filter Paper	VWR	28455-030	42.5 mm diameter, Grade GF/C