Fig. 2: Quantification of CTR1 oligomeric states in endocytic vesicles using TIRF imaging and single-molecule localization microscopy.

a Schematic representation of wild-type CTR1^mEos (WT) and mutant CTR1(M150L)^mEos constructs. b Illustration of Rab5 signal under TIRF illumination: Rab5 marks early/sorting endosomes, with higher intensity observed in smaller, newly formed vesicles near the plasma membrane and decreasing signal as vesicles mature and move deeper into the cell. c Rab5 intensity versus punta area in COS7 cells expressing WT (blue) or M150L (red) under basal (dash) and Cu-treated (solid) conditions. All conditions display a similar rise to 0.4 μm² followed by a decline, indicating comparable endocytic progression across conditions. d Representative images of COS7 cells showing the transmission (top), CTR1^mEos TIRF (middle), and Rab5-mCherry TIRF (bottom) signals. e Workflow for the smND assay, demonstrated by using images enlarged from d (yellow square). Neighboring CTR1 molecules within a 40 nm radius were counted to generate an ND histogram. Experimental ND distributions (PND_exp) were fitted with theoretical distributions (PND_theo) to obtain CTR1 oligomer populations and \(\bar{N}\). f Two dissociation models used to estimate the lower (Model 1) and upper (Model 2) bounds of CTR1 trimer populations from smND data. Data of (c) were collected from three biologically independent experiments. n = 26 (WT-Basal), 31 (WT-Cu), 29 (M150L-Basal) and 37 (M150L-Cu). Graph represents the mean ± SEM of normalized ROI intensities segregated in groups by punta size with bin width of 0.1 µm².