Figure 1.
Figure 1. MCAK limits MT growth to regulate EC branching morphogenesis and directional migration. (A) A HUVEC at the edge of a monolayer wound overexpressing GFP-MCAK and immunolabeled for α-tubulin displays few MTs compared with untransfected cells. (B and C) A wound-edge HUVEC expressing low levels of GFP-MCAK (green). GFP-MCAK tracks with growing, mApple-EB3–labeled MT plus ends (red) in HUVECs (see also Video 1). (C) Zoom of red-boxed region in B highlights GFP-MCAK and mApple-EB3 colocalization. In A–C, the wound edge faces up. (D) MT tracks shown in E–H are color coded according to their growth speed and lifetime. (E–H), MT growth tracks from 2-min time-lapse videos of mApple-EB3 (2-s intervals) from plusTipTracker software. Whole cells (left) and zoom of boxed regions (right). (E) HUVEC expressing control shRNA. (F) HUVEC expressing shRNA targeting MCAK. (G) HUVEC expressing GFP-MCAK. (H) HUVEC expressing MCAK shRNA and GFP-MCAK (Rescue). (I) Western blot of HUVEC lysates for MCAK in untreated (control) or MCAK shRNA-, GFP-MCAK-, or Rescue-treated HUVECs. (J and K) Comparison of mean MT growth speeds (J) and mean MT growth excursion lifetimes (K) from mApple EB3 tracks for control, MCAK shRNA-, GFP-MCAK–, or Rescue-treated HUVECs. (L) DIC images of HUVECs plated on 0.7 kPa fibronectin-coupled polyacrylamide substrates to induce cell branching. Cells were expressing a fluorescent volume marker (mApple-C1) and either BFP-control shRNA, BFP-MCAK shRNA, GFP-MCAK, or Rescue. (M) Phase-contrast images of BFP-control shRNA, BFP-MCAK shRNA, or GFP-MCAK–expressing HUVECs migrating in an experimentally induced wound. Fluorescence (green) and red arrows point to wound-edge GFP-expressing cells at the start point (left) and end point (right) of the migration assay. (N–P) Quantification of HUVEC branch number per cell (N) and branch length (O). (P) Quantification of directional migration in a wound-edge migration assay. Bars: (A and L) 25 µm; (L, inset) 10 µm; (B–H, main) 10 µm; (B–H, zoomed) 2 µm; (M) 50 µm. *, P < 0.001; **, P < 0.05. Error bars show ± standard error in J and K and ± standard deviation in N–P.

MCAK limits MT growth to regulate EC branching morphogenesis and directional migration. (A) A HUVEC at the edge of a monolayer wound overexpressing GFP-MCAK and immunolabeled for α-tubulin displays few MTs compared with untransfected cells. (B and C) A wound-edge HUVEC expressing low levels of GFP-MCAK (green). GFP-MCAK tracks with growing, mApple-EB3–labeled MT plus ends (red) in HUVECs (see also Video 1). (C) Zoom of red-boxed region in B highlights GFP-MCAK and mApple-EB3 colocalization. In A–C, the wound edge faces up. (D) MT tracks shown in E–H are color coded according to their growth speed and lifetime. (E–H), MT growth tracks from 2-min time-lapse videos of mApple-EB3 (2-s intervals) from plusTipTracker software. Whole cells (left) and zoom of boxed regions (right). (E) HUVEC expressing control shRNA. (F) HUVEC expressing shRNA targeting MCAK. (G) HUVEC expressing GFP-MCAK. (H) HUVEC expressing MCAK shRNA and GFP-MCAK (Rescue). (I) Western blot of HUVEC lysates for MCAK in untreated (control) or MCAK shRNA-, GFP-MCAK-, or Rescue-treated HUVECs. (J and K) Comparison of mean MT growth speeds (J) and mean MT growth excursion lifetimes (K) from mApple EB3 tracks for control, MCAK shRNA-, GFP-MCAK–, or Rescue-treated HUVECs. (L) DIC images of HUVECs plated on 0.7 kPa fibronectin-coupled polyacrylamide substrates to induce cell branching. Cells were expressing a fluorescent volume marker (mApple-C1) and either BFP-control shRNA, BFP-MCAK shRNA, GFP-MCAK, or Rescue. (M) Phase-contrast images of BFP-control shRNA, BFP-MCAK shRNA, or GFP-MCAK–expressing HUVECs migrating in an experimentally induced wound. Fluorescence (green) and red arrows point to wound-edge GFP-expressing cells at the start point (left) and end point (right) of the migration assay. (N–P) Quantification of HUVEC branch number per cell (N) and branch length (O). (P) Quantification of directional migration in a wound-edge migration assay. Bars: (A and L) 25 µm; (L, inset) 10 µm; (B–H, main) 10 µm; (B–H, zoomed) 2 µm; (M) 50 µm. *, P < 0.001; **, P < 0.05. Error bars show ± standard error in J and K and ± standard deviation in N–P.

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