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

Using this protocol, we visualized the organization of cytoskeletal filamentous proteins on pattern substrates displaying various curvatures. We can test the curvature sensitivity. The resolution of scanning electron microscope images is high enough so that nanometer scale organizations are visualized.

The methods we use for fixation are mild enough to preserve the intra-structural organization of the protein. This remains in the scope of fundamental research to understand the behavior of cytoskeletal filaments. Begin by designing a wavy Norland Optical Adhesive, or NOA replica, in a clean room environment from wavy polydimethylsiloxane undulated patterns of 250 nanometer amplitude and two micrometer lateral periodicity.

To do so, deposit five microliters of liquid NOA on a circular glass cover slip of one centimeter diameter and then place the PDMS template on the drop. Treat the assembly with UV light at 320 nanometers for five minutes to photopolymerize the liquid NOA into a thin polymer film. Once done, gently peel off the PDMS template from the cover slip with the freshly polymerized NOA.

Treat the NOA film using an air plasma cleaner for five minutes to make the surface hydrophilic. Prepare a solution of small unilamellar vesicles, or SUVs, as described in the manuscript, and obtain the SUVs by resuspending a dried lipid film in the observation buffer under the sonication in a bath sonicator for five to 10 minutes until the solution is transparent. Later, insert the cover slips supporting the NOA patterns within the wells of cell culture boxes and incubate the freshly glow discharged NOA patterns with 100 microliters of one milligram per milliliter SUV solution for 30 minutes at room temperature to generate a supported lipid bilayer.

To remove the unfused SUV, rinse the sides thoroughly six times with the septin low salt buffer without letting the sample completely dry between two washes. Dilute the octomeric septin stock solution in a septin low salt buffer to final concentrations, ranging from 10 to 100 nanomolar and volumes of one milliliter. Then, incubate the protein solution on the slides for one hour at room temperature.

Wash the NOA slides containing fused supported lipid bilayers and incubate protein with septin low salt buffer. Replace septin low salt buffer with 2%glutaraldehyde fixative solution in 0.1 molar sodium cacodylate buffer, prewarmed at 37 degrees Celsius, and let the reaction proceed for 15 minutes, and wash the fixed sample thrice, five minutes each with 0.1 molar sodium cacodylate. After washing, incubate the sample in the fixative solution of osmium tetroxide, then the filtered tannic acid, and finally the filtered uranyl acetate for 10 minutes each.

Wash the sample thrice in distilled water after every solution. Incubate the sample serially in ethanol solutions starting from 50%to 100%for two to three minutes each. Transfer the glass slides inside the critical point dryer prefilled with ethanol and follow the manufacturer's instructions for drying.

As dried samples are highly hydroscopic, coat them immediately by attaching the cover slip to the stubs. Then add a strip of silver paint to the upper face of the cover slip without creating paint deglomerates. Make sure that the connection with the stub is satisfactory.

When the sample evaporates completely, use an apparatus equipped with a plasma magnetron sputtering head and a rotary planetary stage, and follow standard protocols provided by the manufacturer. Perform pre-sputtering at 120 milliamperes for 60 seconds to remove the oxide layer at the surface. Then deposit 1.5 nanometers of tungsten with a film thickness monitor at 90 milliamperes and a working distance of 50 milliliters.

Later, store the samples under a vacuum to protect them from the ambient air until and throughout the SEM analyses. To achieve high solution imaging by detecting the secondary electrons with the inland detector, set the accelerating voltage to three kilovolts and the beam current to 20 micrometer aperture. For observations, use resolutions ranging from 21.25 nanometers per pixel to 1.224 nanometers per pixel.

Use a resolution of 5.58 nanometers per pixel for data analysis. Set the working distance between one to two millimeters for high resolution observation and around three millimeters if an increased depth of field is required. Adjust the scan speed and line integration continuously to ensure a constant signal to noise ratio with an acquisition time of around 30 to 45 seconds per image.

The representative analysis illustrates the effect of the material deposited on the septin filaments on wavy PDMS patterns with 1.5 nanometers of platinum and 1.5 nanometers of tungsten. It was observed that bare giant unilamellar vesicles, or GUVs, were perfectly spherical. After the septin-induced deformation, the vesicles appeared faceted and the deformations remained static, and thus did not fluctuate.

At higher concentrations of the septins, periodic deformations in the vesicles were observed. Self-assembly of the septin filaments bound to large unilamellar vesicles, or LUVs, was examined with a cryo electron microscopy image and 3D reconstruction was obtained from a tilted series. The curvature-dependent arrangement of the septin filaments was visualized by scanning electron microscopy.

At low magnification, a wavy pattern with the periodicity of negative and positive curvatures was visible. The ultra-structural organization of the septin filaments was viewed at higher magnification. The NOA have to be gently peeled off to prevent degradation.

After imaging, some image analysis can be performed to quantify the ultra-structure features visualized by SCM, such as filament spacing, and orientation of the filament. The present method has been applied to septins, but it could also be used for other cytoskeletal proteins or proteins that organize as long filaments and thus be sensitive to curvature.