title: Automated screening of microtubule growth dynamics identifies MARK2 as a regulator of leading edge microtubules downstream of Rac1 in migrating cells.
abstract: Polarized microtubule (MT) growth in the leading edge is critical to directed cell migration, and is mediated by Rac1 GTPase. To find downstream targets of Rac1 that affect MT assembly dynamics, we performed an RNAi screen of 23 MT binding and regulatory factors and identified RNAi treatments that suppressed changes in MT dynamics induced by constitutively activated Rac1. By analyzing fluorescent EB3 dynamics with automated tracking, we found that RNAi treatments targeting p150(glued), APC2, spastin, EB1, Op18, or MARK2 blocked Rac1-mediated MT growth in lamellipodia. MARK2 was the only protein whose RNAi targeting additionally suppressed Rac1 effects on MT orientation in lamellipodia, and thus became the focus of further study. We show that GFP-MARK2 rescued effects of MARK2 depletion on MT growth lifetime and orientation, and GFP-MARK2 localized in lamellipodia in a Rac1-activity-dependent manner. In a wound-edge motility assay, MARK2-depleted cells failed to polarize their centrosomes or exhibit oriented MT growth in the leading edge, and displayed defects in directional cell migration. Thus, automated image analysis of MT assembly dynamics identified MARK2 as a target regulated downstream of Rac1 that promotes oriented MT growth in the leading edge to mediate directed cell migration.
authors: Cell Biology and Physiology Center, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
authors: Yukako
authors: Y
authors: Nishimura
authors: Kathryn
authors: K
authors: Applegate
authors: Michael W
authors: MW
authors: Davidson
authors: Gaudenz
authors: G
authors: Danuser
authors: Clare M
authors: CM
authors: Waterman
mesh_terms: D045744:Cell Line, Tumor; D002465:Cell Movement / Q000502:physiology*; D018385:Centrosome / Q000378:metabolism; D006801:Humans; D008870:Microtubules / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011554:Pseudopodia / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: C107953:RAC1 protein, human; C094670:MARK2 protein, human; D017346:Protein Serine-Threonine Kinases; D020830:rac1 GTP-Binding Protein