title: Knockdown of kinesin KIF11 abrogates directed migration in response to epidermal growth factor-mediated chemotaxis.
abstract: Establishment of microtubule polarity is critical for directional cell migration involved in morphogenesis, differentiation, cell division, and metastasis. Current models, involving iterative microtubule capture and inactivation of microtubule depolymerizing mechanisms at the leading edge, cannot account for the biased migration exhibited by cells in culture in the absence of directional cues, suggesting central mechanisms governing microtubule polarity remain unknown. We engineered two human MDA-MB-231/IMP1 breast carcinoma cell lines, denoted kdKIF11-1 and kdKIF11-2, in which the kinesin KIF11 (also known as Eg5) was stably knocked down by two different shRNAs. Western blot analysis showed knockdown by each shRNA decreased KIF11 expression by 58% and 79% for kdKIF11-1 and kdKIF11-2, respectively, whereas Rac1 expression was unaffected. All cell lines retained a well-defined microtubule structure. Compared to cells infected with the control viral vector, both KIF11 knockdown cell lines displayed a 14-45% increase in cell motility in a scratch wound healing assay. In contrast, KIF11 knockdown decreased invasion by 70%, compared to the control, as measured by invasion through Matrigel-coated transwells. To determine whether the reduction in invasion was due to reduced chemotaxis, we substituted collagen for Matrigel in the transwell assay and similarly observed a 44-54% reduction in migration, using EGF as the chemoattractant. However, when including EGF in both the upper and lower chambers of the transwell to stimulate migration but eliminate chemotaxis, transwell migration decreased for the control cell line only, indicating that KIF11 knockdown did not impair migration, but severely impaired chemotaxis. We conclude KIF11 is a key downstream molecule that responds to directional cues in chemotaxis to govern the direction of migration.
authors.affiliation: Department of Pathophysiology, Key Immunology Laboratory of Guangdong Province, Shantou University College of Medicine, 22 Xinling Road, Shantou, Guangdong 515041, China; Center for Neuroscience, Shantou University College of Medicine, 22 Xinling Road, Shantou, Guangdong 515041, China.
authors.forename: Fang
authors.initials: F
authors.lastname: Wang
authors.affiliation: Department of Pathophysiology, Key Immunology Laboratory of Guangdong Province, Shantou University College of Medicine, 22 Xinling Road, Shantou, Guangdong 515041, China; Center for Neuroscience, Shantou University College of Medicine, 22 Xinling Road, Shantou, Guangdong 515041, China. Electronic address: stanleylin@stu.edu.cn.
authors.forename: Stanley Li
authors.initials: SL
authors.lastname: Lin
mesh_terms: D001681:Biological Assay; D060846:Cell Engineering; D045744:Cell Line, Tumor; D002633:Chemotaxis / Q000187:drug effects*; D003094:Collagen / Q000737:chemistry; D015194:Diffusion Chambers, Culture; D004338:Drug Combinations; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D005260:Female; D055785:Gene Knockdown Techniques; D006801:Humans; D016547:Kinesins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D007797:Laminin / Q000737:chemistry; D008870:Microtubules / Q000187:drug effects / Q000648:ultrastructure; D011509:Proteoglycans / Q000737:chemistry; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D014945:Wound Healing / Q000187:drug effects; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D004338:Drug Combinations; C470243:KIF11 protein, human; D007797:Laminin; D011509:Proteoglycans; C107953:RAC1 protein, human; D034741:RNA, Small Interfering; C059485:matrigel; D004815:Epidermal Growth Factor; D003094:Collagen; D016547:Kinesins; D020830:rac1 GTP-Binding Protein
keywords: Chemotaxis; Epidermal growth factor; Invasion; KIF11; Migration