Centrosome overamplification affects 3D migration and blood vessel sprouting. (A) HUVECs on microcarrier beads in sprouting angiogenesis assay infected with the indicated viral constructs (green) and stained with phalloidin to delineate sprout structures. Bars, 100 µm. (B and C) Mean of sprouts/bead (B) and branches/bead (C) in the indicated groups in a sprouting angiogenesis assay (empty vector [EV], n = 20 beads; Cdc14B KD, n = 20 beads; Plk4 OE, n = 8 beads). Error bars show SDs. Statistical comparisons versus empty vector. *, P < 0.05. (D) Live-imaging micrographs with fluorescence (centrin::GFP) overlaid on phase (see also Video 10). Arrowheads, nucleus position of a given cell at the indicated time points; blue ovals, nucleus. Bar, 20 µm. (insets) Fluorescence of centrosomes at higher magnification. Bar, 2 µm. White dotted lines, sprout borders. (E and F) Scatter plot with mean (middle bars) and 95% confidence intervals of total distance traveled (E) and persistence (F) of individual nuclei tracked within sprouts (1–2 centrosomes, n = 15 cells; >2 centrosomes, n = 14 cells for both graphs). *, P < 0.05; ***, P < 0.0001. (G) Model depicting mechanism of supernumerary centrosome effects on Golgi organization, migratory polarity, and blood vessel formation. Endothelial cells with supernumerary centrosomes may have reduced Plk1/centrosome, leading to reduced levels of γ-tubulin and fewer MT nucleations/centrosome. Reduced MT nucleations compromise centrosome clustering at the MTOC, which leads to disorganized Golgi that randomizes vesicle trafficking and directional migration. In blood vessels, this abnormal migration perturbs vessel morphogenesis and integrity.