Polylysine

Fresh fractions from the Superose 6 Increase column containing DARP14-3G124Mut5 bound with sfGFP V206A were pooled and concentrated to 2 mg/mL. The sample was diluted to 1 mg/mL and final buffer composition of 10 mM Tris pH 7.5, 500 mM NaCl, 1 mM DTT, 0.5% glycerol immediately prior to freezing. Quantifoil 200 mesh 1.2/1.3 copper grids (Electron Microscopy Sciences) was treated with 0.1% poly-lysine (Sigma-Aldrich) for 4–6 h prior to freezing and cleaned of excess poly-lysine by washing with filtered water three times. 2.5 μL of sample was applied to the grids without glow discharging and frozen using a Vitrobot Mark IV (FEI). 1,929 movies were collected on a FEI titan Krios microscope (Thermo Fisher) with a Gatan K2 Summit direct electron detector in counting mode with image shift at a pixel size of 1.07 Å, and defocus values around −2.5 μm. Movies with 40 frames were collected over 8 s with ~7.00 e⁻* A⁻²* s⁻¹ dose rate.

The HEK293T cells (CRL-11268, ATCC) were grown at 37 °C and 5% CO₂ in a humidified atmosphere incubator (Thermo). The culture medium contained Dulbecco’s modified Eagle’s medium (Gibco), 10% fetal bovine serum, and penicillin-streptomycin (50 μ/ml and 50 μg/ml). The HEK293T cells were seeded at the density of 100,000 per 8-mm × 8-mm, and the cover slip was coated with poly-lysine (Sigma) in a 24-well plate before transduction.
The dissociated hippocampal neurons were prepared from P0 pups, as described previously14 (link). The neurons were plated at the density of 80,000 per 8-mm × 8-mm cover slip coated with poly-lysine (Sigma). The infected neuronal cultures were fixed with 4% paraformaldehyde/4% sucrose in phosphate buffered saline, and fluorescent images were collected with a laser confocal microscope (Olympus) using a 20× objective.

We imaged 3 freshly sacrificed zebrafish using a non-destructive volume visualization for each condition and at 17 dpf and 30 dpf. This method enables the visualization of soft tissues without need for chemical fixation or staining [32 (link)]. The fish were euthanized with MS222. We coated the surface of a plastic sheet 3x1x0.2 cm in size with a drop of polylysine (Sigma-Aldrich) to allow the interaction between the polyanionic surfaces of the fish and the polycationic layer of adsorbed polylysine. The sample was placed in a custom-made sample holder that allowed maintenance of high humidity around the sample and was observed using an Xradia Micro-CT-400 (Zeiss X-Ray Microscopy, Pleasanton, CA, USA), with an X-ray source of 30 kV, current 150 μA and magnification 10X. The pixel size for the 17 dpf specimen and for the tail was 0.6x0.6x0.6 microns and for the 30 dpf specimen was 1.3x1.3x1.3 microns. 1,200 projection images were recorded with 20 sec exposure time. In order to compare all the intensities of all the acquisitions, a scale was designed using a standard phantom. In addition, a hydroxyapatite CT phantom (QRM, Möhrendorf, Germany) was used as a calibration standard for the quantification of the density of zebrafish bones. Obtained data are given as mean ± standard deviation.