Supplementary material Legends to Supplementary Figures Figure S1. Figure S2. Figure S3.
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1 Supplementary material Legends to Supplementary Figures. Figure S1. Expression of BICD-N-MTS fusion does not affect the distribution of the Golgi and endosomes. HeLa cells were transfected with GFP-BICD-N-MTS (A,D) and stained for the Golgi marker GM130 (B) and endosomal marker transferrin receptor (E). GFP signals are shown in the left panel, stained organelles in the middle panels, and an overlay of the portion of the figure, indicated by a white rectangle (with GFP signal in green, organelles in red), in the right panels. Figure S2. Overexpression of dynein subunits, fused to the MTS, does not induce relocalization of mitochondria. A. Schematic representation of the fusion constructs. B-G. HeLa cells were transfected with GFP-fusion constructs and stained for mitochondria. GFP signals are shown in the left panel, mitochondria in the middle panels, and an overlay of the portion of the figure, indicated by a white rectangle (with GFP signal in green, mitochondria in red), in the right panels. Figure S3. Inducible relocalization of mitochondria, using regulated heterodimerization system. HeLa cells were cotransfected with GFP-BICD-N-FRB and HA-FKBP2-MTS, stained with MitoTracker, fixed, and stained for the HA tag. HA-specific signal is shown in (A), GFP signals in (C,E,G and I), while the MitoTracker signals are shown in all right panels. Cells were treated with 125 nm AP21967 for 5 min (E,F), 10 min (G,H) or 20 min (I,J) prior to fixation.
2 Figure S4. Organization of the microtubule network in cells, co-expressing HA-BICD-N- MTS and GFP-dynamitin. Cells were transfected with HA-BICD-N-MTS (left panels) and GFP-dynamitin (right panels) and stained for β-tubulin (middle panels). Cells with normal nuclear appearance and a tight mitochondrial cluster, displayed comparatively normal microtubule network (A-C). Cells with multilobular nuclei (which strongly correlated with partially dispersed mitochondrial aggregate) displayed disorganized and entangled microtubule network (D- F). Figure S5. Example of an image used to quantify the expression levels of the BICD2- fusion proteins. HeLa cells were transfected with GFP-BICD-N-MTS (A) and stained for BICD2 with the antibody #2293 (B). An example of a cell with the expression level defined as medium is shown. 2
3 Legends to Supplementary Movies. Movie S1. Induced movement of mitochondria in HeLa cells HeLa cells, transfected with GFP-BICD-N-FRB and HA-FKBP2-MTS, were analyzed 1 day after transfection by confocal microscopy. Cells were stained for mitochondria with MitoTracker Red CMXRos for 30 minutes, washed and maintained in normal culture medium at 37 C. A total of 20 images were collected every minute. After 2 frames (2 minutes) 125 nm AP21967 was added to the cells (indicated by a dark frame). Notice the movement of the mitochondria to the perinuclear area in the GFP-BICD-N-FRB and HA- FKBP2-MTS transfected cell (the cell with the green cytoplasmic signal in Fig.3 C,D) after adding the FRB/FKBP dimerizer AP Movies S2A, S2B, S3A, S3B. Induced movement of mitochondria in CAR fish fibroblasts. CAR fish fibroblasts were transfected with HA-BICD-N-FRB and GFP-FKBP2-MTS. 48 hours after transfection cells were microinjected with Cy3-tubulin. Videos S2A and S3A were taken before AP21967 was added. Videos S2B and S3B were taken immediately after the addition of 250 nm AP Time between frames was 15 sec. Organelles are labelled green, and the microtubule network is red. Movie S4. Dispersion of the mitochondrial aggregate, induced by GFP-BICD-N-MTS, by microinjection of anti-dynein antibodies CAR fish fibroblast was transfected with GFP-BICD-N-MTS and injected with the M-74-2 anti dynein antibody (5mg/ml). The sequence shows the time point just before injection (first frame) and after injection (subsequent frames). Times between frames after injection were 30 sec. Note dispersal of GFP-tagged organelles towards cell periphery in response to the dynein inhibitory antibody. 3
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