Supplemental Material

This article contains the following supporting material:

• Movie 1 - Podosomes are contacted by microtubule plus-ends. Primary human macrophage expressing mRFP-β-actin (red), labeling podosomes, and GFP-CLIP170 (green), labeling microtubule plus-ends. Confocal time lapse series of substrate-attached part of the cell (exposure time: 488 nm: 1500ms, 568 nm: 3000 ms, frame rate: 10 f/s; sequence: 394 s).

• Movie 2 - Podosome dissolution after repeated contact with microtubule plus-ends. ROI (region of interest) from video-1 (exposure time: 488 nm: 1500 ms, 568 nm: 3000 ms, frame rate: 10 f/s; sequence: 394 s)

• Movie 3 - A podosome is repeatedly contacted by microtubule plus-ends but remains stable. ROI from video-1 (exposure time: 488 nm: 1500ms, 568 nm: 3000 ms, frame rate: 10 f/s; sequence: 394 s

• Movie 4 - Fission of a podosome precursor cluster after repeated contact by microtubule plus-ends. ROI from video-1 (exposure time: 488 nm: 1500ms, 568 nm: 3000ms, frame rate: 10 f/s; sequence: 394 s)

• Movie 5 - KIF1C is localized at sites of high podosome turnover. Primary human macrophage expressing KIF1C-GFP (green), and mRFP-β-actin (red), labeling podosomes. Confocal time lapse series of substrate-attached part of the cell (exposure time: 488 nm: 2000ms, 568 nm: 12000 ms, frame rate: 10 f/s; sequence: 506 s)

• Movie 6 - KIF1C-GFP accumulations target a region of high podosome turnover. ROI from video-5 (exposure time: 488 nm: 2000ms, 568 nm: 12000 ms, frame rate: 10 f/s; sequence: 506 s)

• Movie 7 - KIF1C-GFP can localize to microtubule plus-ends. Primary human macrophage expressing KIF1C-GFP (green), and DsRED-EB1 (red). Confocal time lapse series of substrate-attached part of the cell (exposure time: 488 nm: 1000 ms, 568 nm: 3500 ms, frame rate: 10 f/s; sequence: 650 s)

• Movie 8 - KIF1C-GFP can localize to microtubule plus-ends. ROI from video-7 (exposure time: 488 nm: 2000ms, 568 nm: 12000 ms, frame rate: 10 f/s; sequence: 506 s)

• Figure 1 - A KIF5B-inhibitory antibody blocks mitochondria plus-end directed transport in macrophages. (A,B) Fluorescence micrographs of primary macrophages microinjected with anti-KIF5B inhibitiory antibody (4 μg/μl), fixed (A) 0h or (B) 1h post injection. Mitochondria are visualized by staining subunit I of cytochrome C oxidase complex IV (OxPhos), with specific primary antibody (red), rat IgG (green) stained as injection marker. Cell circumference is depicted by dashed white line. Scale bar 10 μm. Note accumulation of mitochondria at cell center 1h after injection of antibody. (C) Evaluation of mitochondria localization in microinjected macrophages. For each value, 3x 30 cells were counted. Values are given as mean percentage ± SD of total counts. Cells with mitochondria predominantly at cell center: 17.8% ± 6.5 % for uninjected cells, 18.9% ± 5.3 % for injected cells at 0 h, 83.3% ± 7.7% for injected cells at 1 h.

• Figure 2 - Dynein inhibition blocks minus-end directed transport of TRITC-dextran-loaded vesicles in macrophages. (A,B) Fluorescence micrographs of primary macrophages microinjected with (A) rat IgG (1 μg/μl) or (B) anti-dynein inhibitiory antibody (4 μg/μl), fixed 1h post injection. Dextran containing vesicles visible by TRITC signal (red), rat IgG stained as injection marker (green). Cell circumference is depicted by dashed white line. Scale bar 10 μm. Note that normal juxtanuclear accumulation of dextran vesicles is disrupted after anti-dynein antibody injection. (C) Evaluation of localization of TRITC-dextran containing vesicles in macrophages. For each value, 3x 30 cells were counted. Values are given as mean percentage ± SD of total counts. Uninjected cells: 90.0% ± 2.6% for cells with central, and 10.0% ± 2.6% for cells with peripheral TRITC-dextran vesicles; mock-injected cells: 88.9% ± 3.9% for cells with central, and 11.1% ± 3.9% for cells with peripheral TRITC-dextran vesicles; anti-dynein antibody injected: 3.3% ± 2.6% for cells with central, and 96.7% ± 2.6% for cells with peripheral TRITC-dextran vesicles; dynein inhibitor erythro-9-3-(2-hydroxynonyl) adenine (EHNA) injected (1 mM): 27.8% ± 5.3% for cells with central, 3.3% ± 0.0% for cells with peripheral, and 68.9% ± 5.3% for cells with diffusely localized TRITC-dextran vesicles.

• Figure 3 - (A) Gradual podosome loss following KIF1C shRNA expression. Podosome formation was evaluated in cells expressing KIF1C-specific shRNA or scrambled shRNA, 8h, 16h, 20h and 24h post transfection. For each value, 3 x 30 cells were evaluated. Values are given as mean percentage ± SD of total counts. KIF1C shRNA: 8h: 68.9% ± 1.9%, 16h: 61.1% ± 6.9%, 20h: 60.0% ± 8.8%, 24h: 52.2% ± 3.8%; eGFP-N1: 8h: 80.0% ± 0.0%, 16h: 90.0% ± 5.8%, 20h: 82.2% ± 1.9%, 24h: 85.6% ± 7.7%. For differences between values for KIF1C shRNA and values for scrambled controls or for differences between the 8h value and subsequent time points for KIF1C shRNA, a P value <0.02 was considered significant, (indicated by asterisk). (B) KIF1C overexpression does not lead to alterations in podosome content. Evaluation of podosome numbers in cells expressing KIF1C-GFP or eGFP. For each value, 3x 30 cells were counted. Values are given as mean percentage ± SD of total counts in supplementary table S2. A P value <0.05 was considered significant, but was not reached in any of the compared datasets.

• Figure 4 - (A) KIF1C-GFP localizes preferentially to microtubule ends in the cell periphery. Still image from confocal time lapse video of a primary macrophage expressing KIF1C-GFP (green) and DsRed-EB1 (red), note EB1-positive but KIF1C-GFP-negative microtubule plus ends in the central part of the cell. (B-D) KIF1C-GFP localizes to microtubule ends. Confocal laser scanning micrograph of substrate-attached part of primary macrophage expressing KIF1C-GFP (green; B), and stained for β-tubulin (red; C); (D) overlay of (B) and (C), white arrows indicate KIF1C-GFP localizing at microtubule ends. Scale bars 10 μm.

• Figure 5 - KIF1C-GFP-decorated microtubule plus-ends preferentially target podosomes at the cell periphery. Confocal laser scanning micrographs (image of time lapse series) of primary macrophages expressing mRFP-β-actin and (A) GFP-CLIP170 or (B) KIF1C-GFP. Podosomes were analyzed for contact by the respectively labeled microtubule plus-ends, and squares of overlaid grid were colored according to the ratio of contacted podosomes therein: 0%-25% (blue), 26%-50% (green), 51%-75% (orange), 76%-100% (red). Note that CLIP-170-decorated microtubule plus-ends evenly contact podosomes over the whole ventral surface, while KIF1C-decorated microtuble plus-ends preferentially contact podosomes in the cell periphery. Scale bars 10 μm.

• Table 1
• Table 2