JCS03913 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Depletion of microtubule plus-end proteins in B16F1 cells and mouse dermal fibroblasts (MDF). (A) B16F1 cells transfected with an RNA interference construct directed against both CLIP170 and 115 (CLIP KD), and mouse dermal fibroblasts (MDF) deficient in CLIP170 and 115 (CLIP KO) were immunostained with anti-CLIP head domain antibodies and anti-tubulin antibodies. Combined images shown CLIP staining in green and microtubules in red. (C) B16F1 cells were transfected with single RNA interference constructs directed against the end-binding proteins EB1, EB2, EB3 or cotransfected with the EB1 and EB3 constructs (EB1+EB3). Cells were immunostained with the respective anti-EB antibodies and anti-tubulin antibodies. The combined images show EB staining in green and microtubules in red. Boxed regions indicate microtubule ends. Nontransfected B16F1 cells in the same field serve as controls. Bars, 2 μm. (B) Quantification of CLIP immunofluorescence at the ends of microtubules in CLIP KD and CLIP KO cells. (D) Immunofluorescence of EB proteins at the ends of microtubules in B16F1 knockdown cells. Numbers expressed as percent control levels. Error bars indicate s.e.

• Supplemental Figure 2 -

  Fig. S2. Microtubule targeting is independent of microtubule plus-end proteins. Combined images of microtubules (green) and actin (red) in (A) B16F1 knock down (CLIP KD) cells and MDF dual knock out (CLIP KO) cells, or (B) B16F1 knock down cells deficient in EB proteins (B16F1 EB KD). Arrowheads indicate targeting of microtubules to filopodia. Bar, 2 μm. (C) Percent filopodia targeting in B16F1 CLIP KD cells and MDF CLIP KO cells. (D) Percent filopodia targeting in B16F1 EB KD cells. Nontransfected B16F1 cells and wild-type MDF cells served as controls. Error bars indicate s.e. Bars, 2μm.

• Movie 1 -

  Movie 1. Turning of targeted filopodia. A filopodium is targeted by a microtubule at 25 seconds and then targeted again by another microtubule at 60 seconds. The targeted filopodium turns toward the lamellipodium midline.

• Movie 2 -

  Movie 2. Merging of targeted filopodia. A filopodium is targeted by a microtubule at 35 seconds. The targeted filopodium turns toward the lamellipodium midline and merges with a nearby filopodium.