Fig. 7: Detecting individual protein status from their dynamics.

a Confocal images of a COS-7 cell stably expressing the ER marker mEmerald^ER, an ER-targeted anti-alfatag intrabody fused to an HaloTag (stained with Halo-JFX650), an Amyloid Precursor Protein containing the Alfatag peptide and fused to a SnapTag (stained with Snap-TMR) and the merged image showing the near perfect correlation of the three signals. b Principle of the intrabody status detection method at the single-molecule level. Unbound intrabodies have a relatively fast luminal dynamic while bound intrabodies acquire the slower membrane dynamics of their APP target (Created with Biorender⁶⁰). c TIRF images of ER structures (top) and associated FidlTrack reconstructed single-particle trajectories (colour-coded by the average trajectory displacement length of each trajectory) for cells expressing: the intrabody together with the ER-retained Snap-tagged APP (stained with Snap-PA-JF646, left), the ER-retained intrabody alone (stained with Halo-PA-JF646) (middle), or the intrabody (stained with Halo-PA-JF646) together with the APP construct (right). d Distributions of the pooled averaged trajectory displacement lengths for the unbound intrabody (red), APP (blue) and intrabody + APP (purple) recordings. The dashed lines correspond to fits of the distributions to single-components Gaussian models. The pooling is over 5 recordings for each condition. e Distributions of the averaged trajectory displacement lengths for 5 individual recordings of the intrabody in the presence of APP. The dashed lines correspond to fits of the distributions to two-component Gaussian models. f Average trajectory displacement length parameter (fitted value ± 95% CI) of each component extracted from the fits of the different distributions presented in (e). The dashed lines correspond to the average trajectory displacement length parameters of the single-component Gaussian fits of the APP (blue) and intrabody-only (red) recordings from (d). g Associated proportion of bound and unbound intrabody populations for the fits presented in (e) (see Methods section “Binding status extraction from intrabody trajectories”). h Principle of the procedure for unmixing APP-bound and unbound intrabody trajectories. i Unmixing of APP vs unbound intrabodies on a recording, from left to right: ER structure of a cell expressing both the intrabody and APP, recorded trajectories, APP trajectories and unbound trajectories, colour coded by individual trajectory. Source data are provided as a Source Data file.