Fig 2: Live-cell imaging of Miro1 with selected Miro1-Cbs. (A) Representative fluorescence images of living HeLa cells transiently expressing GFP-Miro1 (left column) in combination with red fluorescently labelled (TagRFP) M41-, M85- or M114-Cb (middle column). Scale bars 20 µm. (B) Time-lapse microscopy of U2OS cells transiently expressing GFP-Miro1 in combination with either M114-Cb or mito-Mkate2. To visually track morphological mitochondrial changes, cells were treated with either DMSO as a control (top two rows) or 10 µM Sorafenib (bottom two rows) followed by time-lapse imaging over a 2 h period. Shown are representative images of three biological replicates. Scale bar 25 µm. Squares at the bottom right represent enlargements of the selected image section.

Fig 3: Biochemical characterization of Miro1 specific Nbs. (A) Amino acid sequence alignment of the complementary determining region (CDR) 3 of seven unique Miro1-Nbs positively identified by phage ELISA (complete amino acid sequences of all Miro1-Nbs are shown in Supplementary Table S2). (B) Recombinant expression and purification of Nbs using immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC). (C) For biolayer interferometry based affinity measurements, Miro1-Nbs were biotinylated and immobilized on streptavidin sensors. Kinetic measurements were performed by using four concentrations of purified Miro1 As an example, the sensogram of Miro1 at concentrations 7.8, 15.6, 31.2, and 62.5 nM on immobilized M41-Nb (illustrated with increasingly darker shades from low to high concentration) is shown and global 1:1 fits are illustrated as dashed line (upper panel).

Fig 4: Domain mapping of Miro1-Nbs. (A) Schematic illustration of GFP-labelled Miro1 deletion constructs and domains used for domain specific binding studies. (B) Soluble protein fractions of HEK293 cells transiently expressing indicated Miro1 deletion constructs or GFP (as control) were subjected to immunoprecipitation with selected Miro1 nanotraps followed by western blot analysis of input (IN) and bound fractions with an anti-GFP antibody.

Fig 5: Targeted intracellular degradation of endogenous Miro1 by Fbox-Nb-based degrons. (A) Schematic illustration of the targeted degradation of Miro1 mediated by Miro1-Nb-Fbox fusions (illustration created with Biorender.com). (B) Representative confocal images of HeLa cells transiently expressing indicated Miro1-specific Fbox-Nbs (Fbox-M41 and Fbox-M114) or a non-related Fbox-Nb (Fbox-NR) construct. For quantitative IF analysis, cells were fixed and permeabilized 24 h after transfection followed by detection of endogenous Miro1 with Miro1 antibody (shown in green). Fbox-Nb expressing cells were identified by a nuclear TagRFP signal (white arrows) and subjected to automated image analysis and quantification as described in Supplementary Figure S13 and Material and Methods. Scale bar 20 µm. (C) Mean Miro1 fluorescence intensity from HeLa cells expressing Fbox-Nbs determined by quantitative fluorescence imaging. Mean Miro1 fluorescence was calculated from four samples (N = 4; n > 500 cells) and normalized to untransfected cells, UT (set to 1). As control, a non-related Fbox-Nb construct (Fbox-NR) was used. Data are represented as mean ± SEM. For statistical analysis, student’s t-test was performed, **p < 0.01, ***p < 0.001.