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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 shapes1,2,3,4. 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 scattering

Representative 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 time28,29. 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.......

Acknowledgements

This work was supported by the National Institutes of Health (NIH) Grant no. R01 GM-130934 and National Science Foundation (NSF) Grant MCB- 2016022. B.N.C, E.J.D.V., and K.S.C. were supported in part by a grant from the National Institute of General Medical Sciences under award T32 GM119999.

....

Materials

NameCompanyCatalog NumberComments
0.2 mL PCR Tubes with flat cap, NaturalWatson137-211C(EX)
0.5 mL low adhesion tubesUSA Scientific1405-2600
Beta mercaptoethanol (BME)Sigma-AldrichM6250-100ML
Bovine Serum Albumin (BSA)Sigma-AldrichA4612-25G
Coverglass for making PEGylated coverslipsThermo Scientific152450Richard-Allan Scientific SLIP-RITE Cover Glass 24x50 #1.5
DOPCAvanti Polar Lipids850375
Egg Liss Rhodamine PEAvanti Polar Lipids810146
EMS Glutaraldehyde Aqueous 25%, EM GradeVWR16220
EMS Sodium Cacodylate BufferVWR11652
Ethanol, 200 proofFisher Scientific04-355-223EA
HEPESSigma AldrichH3375-1KG
HexamethyldisilazaneSigma-Aldrich440191
Magnesium chlorideVWR7791-18-6
Methyl cellulose 4000cpSigma-AldrichM052-100G
Microglass coverslips for planar bilayersMatsunamiDiscontinued22x22
Mini centrifuge
Non-Functionalized Silica MicrospheresBangs Laboratories, Inc.Depends on size: SS0200*-SS0500*Silica in aqueous suspension
Optical AdhesiveNorland ThorlabsNOA 68Flexible adhesive for glass or plastics
Osmium tetroxideMillipore Sigma20816-12-0
ParafilmVWR52858-000
Plasma CleanerPlasma EtchPE-25Voltage: 120V, 60Hz. Current: 15 AMPS
Potassium chlorideVWR0395-1kg
Round coverglass, #1.5 12mm  VWR64-0712
Sonicator bathBranson1510R-MTBransonic Ultrasonic cleaner. 50-60 Hz. Output: 70W
Soy PIAvanti Polar Lipids840044
Tabletop centrifugeEppendorf22331
UV LampSpectrolineENF-260C115 Volts, 60 Hz, 0.20 AMPS
WhatmanGlass Microfiber Filter PaperVWR28455-03042.5 mm diameter, Grade GF/C

References

  1. Zimmerberg, J., Kozlov, M. M. How proteins produce cellular membrane curvature. Nature Reviews Molecular Cell Biology. 7 (1), 9-19 (2006).
  2. Parthasarathy, R., Groves, J. T. Curvature and spatial organization in biological membranes.

Explore More Articles

Membrane BindingMembrane ShapeProtein AssemblyLipid BilayerLiposomesPlasma CleaningUV activated AdhesiveSupported Lipid BilayerSeptinsTIRF MicroscopySilica MicrospheresSUVs

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