U2OS cells (50x103) were grown on autoclaved coverslips and were placed individually in 12-well plates. Transient transfection was performed by pre-mixing 1 µg DNA and 3 µg PEI (polyethylenimine, MW 25 kDa) in 100 µl OptiMEM (without serum and antibiotics). The transfection mix was incubated for 15 min at RT and 30µl were added dropwise to the wells. Transfection incubation time was 16 h and then media was replaced by DMEM high glucose media and incubated further for 48 h. Prior fixation, the U2OS cells were incubated with 100 nM Lysotracker (Red DND-99) for 30 min at 37°C for co-staining for Lysosomes, or 2µg/ml WGA Texas Red-X for 10 min at 4°C for co-staining of plasma membrane. Cells were washed three times with PBS, fixed in 4% formaldehyde (v/v) in PBS for 10 min and incubated 5 min with DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride, 0.02 μg/μl in PBS) at RT, then washed 3 times with PBS. Coverslips were mounted in Permafluor mounting medium using glass slides and dried at RT overnight. All samples were stored at 4°C in the dark until further analysis by microscopy. SIM imaging (structured illumination microscopy) was performed using a Zeiss Elyra 7 lattice SIM system (Zeiss, Germany) equipped with a 63x/1.4NA oil objective and an additional 1.6x Optovar magnification, resulting in a final optical pixel size of 62 nm. Multicolor z-stacks were acquired with a physical step size of 110 nm according to Nyquist sampling. Each 3D SIM volume was recorded using Zeiss’s lattice SIM mode, with each optical plane reconstructed from 13 raw phase images acquired with an exposure time of 250 ms each. A quad-band dichroic mirror and emission filter (LBF 405/488/561/642) enabled detection of multiple fluorophores. SIM reconstruction was carried out using Zeiss Zen Black software (v3.0) with default settings and the ‘precise’ reconstruction mode and a resulting pixel size of 32.24 nm. No baseline cut was applied. Subsequent post-processing, contrast adjustment, and image export were performed in ImageJ/Fiji. SIM datasets were preprocessed using a customized Fiji macro (ImageJ v1.53) to generate binary masks suitable for quantitative analysis. Raw .CZI (optional .tif) files were imported using the Bio-Formats Importer in hyperstack mode with default autoscaling. Subsequently, channels were split and processed individually.To extract object-specific binary masks, the Otsu's automatic thresholding method (script included here) was applied to each 2D slice independently using the Otsu dark mode, followed by manual outlier compensation and conversion to binary masks with black background enforced. All resulting masks were saved in TIFF format. Object-based analysis was used to assess the spatial association between GFP-positive structures and LysoTracker-labeled lysosomes. Binary images were analyzed using a custom Python script (Python 3.10) to quantify proximity-based colocalization between GFP-positive objects and lysosomal structures. Briefly, binary masks for GFP and Lysotracker channels were loaded as 2D arrays. Each image pair was analyzed independently. GFP-positive objects were identified by connected-component labeling, and computing their centroids. Similarly, lysosomal structures were segmented, and a cKDTree (SciPy) spatial index was constructed from their centroid coordinates. A GFP object was classified as "associated" if its centroid was within a 500 nm euclidean distance to the nearest lysosome centroid (based on a calibrated pixel size of 32.24 nm). The area-based association was calculated for each image individually. Aggregated results were summarized as boxplots per group (WT EHRPLL, signal mutant AHRPAA). Quantification pipeline is included in the colocalization analysis script.

Microscopy raw data related to Figure S4 Figure_S4_Overview_Microscopy Representative_Images_SLC45A1_Lysotracker (S4J) Signal_Mutant_SLC45A1 (TIF File and PNGs) WT_SLC45A1 (TIF File and PNGs) Quantification_SLC45A1_Lysotracker Related to Figure S4L Signal_Mutant_SLC45A1 (TIF Files) WT_SLC45A1 (TIF Files) Quantification Table (gfp_lyso_association_results_SLC45A1) Franke_Heiby_Otsu_preprocessing Franke_Heiby_colocalization_analysis Representative_Images_SLC45A1_Plasma_Membrane (S4K) Signal_Mutant_SLC45A1 (TIF Files and PNGs) WT_SLC45A1 (TIF Files and PNGs) Quantification_SLC45A1_Plasma_Membrane Related to Figure S4M (number of PM+ cells) Signal_Mutant_SLC45A1 (czi) WT_SLC45A1 (czi) Quantification Table

Microscopy raw data related to Figure S4 and Quantification analysis script (suitable for Fiji/ImageJ)