ds3  (Digitimer North America LLC)

Journal: bioRxiv

Article Title: Integrating GlycoSHIELD Modeling and DNA-PAINT SMLM to Map the Glycosylation-Dependent Distribution of the Na,K-ATPase

doi: 10.64898/2026.03.27.714919

Figure Lengend Snippet: DNA-PAINT SMLM shows that Na,K-ATPase β1 3NQ mutants cluster less frequently and form smaller clusters compared to the WT protein. a) Schematic representation of the SMLM workflow, starting with focusing on the suitable region of the cell in TIRF mode using the GFP signal of the expressed protein (either WT β1-GFP or 3NQ β1-GFP), followed by recording 10, 000 frames of the blinking sequence of ATTO 655 imagers hybridizing to a docking strand on the anti-GFP nanobody, which allows for indirect detection of Na,K-ATPase β1 WT or 3NQ mutant, scale bars 10 µm. b) Ripley’s H curves for selected ROIs for Na,K-ATPase B1 WT (blue) and 3NQ (orange), show that the peak clustering occurs in the mean radius of 61.2 nm. This value was used as epsilon for the DBSCAN analysis. c) (i) 3NQ mutants show approximately two-fold lower number of localizations per ROI as compared to the WT protein, median 1680 vs 3031 points per ROI. DBSCAN analysis (min points= 5, ε = 61.2) shows that 3NQ mutants form a fewer number of clusters median 73 vs 101 for WT (ii) and clusters of a smaller diameter mean 109 nm vs 144 nm for WT (iii). All p < 0.05.

Article Snippet: Samples were washed three times with 1X washing buffer (Massive Photonics), and prior to imaging, immersed in a 1 nM solution of ATTO 655 DS3 imager strands diluted in imaging buffer (Massive Photonics).

Techniques: Sequencing, Mutagenesis