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Among the three nonmuscle myosin 2 (NM2) paralogs, NM 2A and 2B, but not 2C, are detected in endothelial cells. To study the role of NM2 in vascular formation we ablate NM2 in endothelial cells in mice. Ablating NM2A, but not NM2B, results in reduced blood vessel coverage and increased vascular branching in the developing mouse skin and coronary vasculature. NM2B becomes essential for vascular formation when NM2A expression is limited. Mice ablated for NM2B and one allele of NM2A develop vascular abnormalities similar to NM2A ablated mice. Using the embryoid body angiogenic sprouting assay in collagen gels reveals that NM2A is required for persistent angiogenic sprouting by stabilizing the endothelial cell cortex, thereby preventing excessive branching and ensuring persistent migration of the endothelial sprouts. Mechanistically, NM2 promotes focal adhesion formation and cortical protrusion retraction during angiogenic sprouting. Further studies demonstrate the critical role of Rho kinase activated NM2 signaling in the regulation of angiogenic sprouting in vitro and in vivo.Movie S1Movie S1aEffect of NM2A and 2B ablation on EB angiogenic sprouts over 6 hrs. Phase contrast time lapse videos of EB sprouts at 1min/frame with 10X objective. a. wild-type sprout, b. A−/A−sprouts, c. B−/B−sprouts. In general the wild-type and B−/B−sprouts develop one dominant protrusion resulting in a persistent growth of sprouts (a,c). A−/A−sprouts often develop multiple protrusions at the tip causing frequent splitting and/or turning of the knockout sprouts (b). 1min/frame (capture rate), 100fps (playback).Movie S1Movie S1bEffect of NM2A and 2B ablation on EB angiogenic sprouts over 6 hrs. Phase contrast time lapse videos of EB sprouts at 1min/frame with 10X objective. a. wild-type sprout, b. A−/A−sprouts, c. B−/B−sprouts. In general the wild-type and B−/B−sprouts develop one dominant protrusion resulting in a persistent growth of sprouts (a,c). A−/A−sprouts often develop multiple protrusions at the tip causing frequent splitting and/or turning of the knockout sprouts (b). 1min/frame (capture rate), 100fps (playback).Movie S1Movie S1cEffect of NM2A and 2B ablation on EB angiogenic sprouts over 6 hrs. Phase contrast time lapse videos of EB sprouts at 1min/frame with 10X objective. a. wild-type sprout, b. A−/A−sprouts, c. B−/B−sprouts. In general the wild-type and B−/B−sprouts develop one dominant protrusion resulting in a persistent growth of sprouts (a,c). A−/A−sprouts often develop multiple protrusions at the tip causing frequent splitting and/or turning of the knockout sprouts (b). 1min/frame (capture rate), 100fps (playback).Movie S2Movie S2aEffect of NM2A and 2B ablation on tip cell dynamics in EB sprouts. Phase contrast time lapse videos of EB sprouts at 1sec/frame with 40X objective. (a) wild-type sprout, (b) A−/A−sprout, (c) B−/B−sprouts. Wild-type and B−/B−tips cells generate numerous cortical blebs (a,c, arrow). A−/A−tip cells develop cortical ruffles (b, arrow), but no blebbing. 1sec/frame (capture rate), 100fps (playback).Movie S2Movie S2bEffect of NM2A and 2B ablation on tip cell dynamics in EB sprouts. Phase contrast time lapse videos of EB sprouts at 1sec/frame with 40X objective. (a) wild-type sprout, (b) A−/A−sprout, (c) B−/B−sprouts. Wild-type and B−/B−tips cells generate numerous cortical blebs (a,c, arrow). A−/A−tip cells develop cortical ruffles (b, arrow), but no blebbing. 1sec/frame (capture rate), 100fps (playback).Movie S2Movie S2cEffect of NM2A and 2B ablation on tip cell dynamics in EB sprouts. Phase contrast time lapse videos of EB sprouts at 1sec/frame with 40X objective. (a) wild-type sprout, (b) A−/A−sprout, (c) B−/B−sprouts. Wild-type and B−/B−tips cells generate numerous cortical blebs (a,c, arrow). A−/A−tip cells develop cortical ruffles (b, arrow), but no blebbing. 1sec/frame (capture rate), 100fps (playback).Movie S3Movie S3NM2A dynamics in EB sprouting and cortical ruffling. A time lapse video of EGFP-NM2A in an EB sprout. NM2A accumulates during the retraction of the cortical protrusion in angiogenic sprouts.Movie S4Movie S4aEffect of NM2 inhibition on tip cell dynamics in EB Sprouts. Phase contrast time lapse videos of EB sprouts at 1sec/frame with 40X objective. (a) 10μM blebbistatin, (b) 10μM Y27632, (c) 20μM ML-7. Blebbistatin inhibition of NM2 activity (a) and Y27632 inhibition of Rho kinase (b) result in abnormal cortical protrusion (arrows) and blocked tip cell blebbing. ML-7 inhibition of MLCK has no effect on blebbing (c, arrow) in EB sprouting angiogenesis. 1sec/frame (capture rate), 100fps (playback).Movie S4Movie S4bEffect of NM2 inhibition on tip cell dynamics in EB Sprouts. Phase contrast time lapse videos of EB sprouts at 1sec/frame with 40X objective. (a) 10μM blebbistatin, (b) 10μM Y27632, (c) 20μM ML-7. Blebbistatin inhibition of NM2 activity (a) and Y27632 inhibition of Rho kinase (b) result in abnormal cortical protrusion (arrows) and blocked tip cell blebbing. ML-7 inhibition of MLCK has no effect on blebbing (c, arrow) in EB sprouting angiogenesis. 1sec/frame (capture rate), 100fps (playback).Movie S4Movie S4cEffect of NM2 inhibition on tip cell dynamics in EB Sprouts. Phase contrast time lapse videos of EB sprouts at 1sec/frame with 40X objective. (a) 10μM blebbistatin, (b) 10μM Y27632, (c) 20μM ML-7. Blebbistatin inhibition of NM2 activity (a) and Y27632 inhibition of Rho kinase (b) result in abnormal cortical protrusion (arrows) and blocked tip cell blebbing. ML-7 inhibition of MLCK has no effect on blebbing (c, arrow) in EB sprouting angiogenesis. 1sec/frame (capture rate), 100fps (playback).