Automated screening of microtubule growth dynamics identifies MARK2 as a regulator of leading edge microtubules downstream of Rac1 in migrating cells
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 s...
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| Veröffentlicht in: | PloS one 2012-07, Vol.7 (7), p.e41413-e41413 |
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| creator | Nishimura, Yukako Applegate, Kathryn Davidson, Michael W Danuser, Gaudenz Waterman, Clare M |
| description | 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. |
| doi_str_mv | 10.1371/journal.pone.0041413 |
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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.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0041413</identifier><identifier>PMID: 22848487</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Assembly ; Automation ; Biochemistry ; Biology ; Bone cancer ; Cell adhesion & migration ; Cell Line, Tumor ; Cell migration ; Cell Movement - physiology ; Centrosome - metabolism ; Centrosomes ; Cytoskeleton ; Downstream ; Drosophila ; Dynamics ; Fluorescence ; Guanosine triphosphatases ; Humans ; Image analysis ; Image processing ; Insects ; Kinases ; Lamellipodia ; Leukocyte migration ; Medical research ; Medical screening ; Microtubules ; Microtubules - genetics ; Microtubules - metabolism ; Morphology ; Neurosciences ; Physiology ; Polymerization ; Protein-Serine-Threonine Kinases - genetics ; Protein-Serine-Threonine Kinases - metabolism ; Protein-serine/threonine kinase ; Proteins ; Pseudopodia ; Pseudopodia - genetics ; Pseudopodia - metabolism ; rac1 GTP-Binding Protein - genetics ; rac1 GTP-Binding Protein - metabolism ; Rac1 protein ; RNA Interference ; RNA-mediated interference ; Target recognition ; Wounds</subject><ispartof>PloS one, 2012-07, Vol.7 (7), p.e41413-e41413</ispartof><rights>COPYRIGHT 2012 Public Library of Science</rights><rights>2012. This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. 2012</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c758t-b202d17e7b56a06d5ff606174c8dafee0613c262eb96ea890b799f7cc8d224eb3</citedby><cites>FETCH-LOGICAL-c758t-b202d17e7b56a06d5ff606174c8dafee0613c262eb96ea890b799f7cc8d224eb3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3404095/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3404095/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,27901,27902,53766,53768,79342,79343</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/22848487$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Nishimura, Yukako</creatorcontrib><creatorcontrib>Applegate, Kathryn</creatorcontrib><creatorcontrib>Davidson, Michael W</creatorcontrib><creatorcontrib>Danuser, Gaudenz</creatorcontrib><creatorcontrib>Waterman, Clare M</creatorcontrib><title>Automated screening of microtubule growth dynamics identifies MARK2 as a regulator of leading edge microtubules downstream of Rac1 in migrating cells</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>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.</description><subject>Assembly</subject><subject>Automation</subject><subject>Biochemistry</subject><subject>Biology</subject><subject>Bone cancer</subject><subject>Cell adhesion & migration</subject><subject>Cell Line, Tumor</subject><subject>Cell migration</subject><subject>Cell Movement - physiology</subject><subject>Centrosome - metabolism</subject><subject>Centrosomes</subject><subject>Cytoskeleton</subject><subject>Downstream</subject><subject>Drosophila</subject><subject>Dynamics</subject><subject>Fluorescence</subject><subject>Guanosine triphosphatases</subject><subject>Humans</subject><subject>Image analysis</subject><subject>Image processing</subject><subject>Insects</subject><subject>Kinases</subject><subject>Lamellipodia</subject><subject>Leukocyte migration</subject><subject>Medical research</subject><subject>Medical screening</subject><subject>Microtubules</subject><subject>Microtubules - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Nishimura, Yukako</au><au>Applegate, Kathryn</au><au>Davidson, Michael W</au><au>Danuser, Gaudenz</au><au>Waterman, Clare M</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Automated screening of microtubule growth dynamics identifies MARK2 as a regulator of leading edge microtubules downstream of Rac1 in migrating cells</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2012-07-24</date><risdate>2012</risdate><volume>7</volume><issue>7</issue><spage>e41413</spage><epage>e41413</epage><pages>e41413-e41413</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><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.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>22848487</pmid><doi>10.1371/journal.pone.0041413</doi><tpages>e41413</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Assembly Automation Biochemistry Biology Bone cancer Cell adhesion & migration Cell Line, Tumor Cell migration Cell Movement - physiology Centrosome - metabolism Centrosomes Cytoskeleton Downstream Drosophila Dynamics Fluorescence Guanosine triphosphatases Humans Image analysis Image processing Insects Kinases Lamellipodia Leukocyte migration Medical research Medical screening Microtubules Microtubules - genetics Microtubules - metabolism Morphology Neurosciences Physiology Polymerization Protein-Serine-Threonine Kinases - genetics Protein-Serine-Threonine Kinases - metabolism Protein-serine/threonine kinase Proteins Pseudopodia Pseudopodia - genetics Pseudopodia - metabolism rac1 GTP-Binding Protein - genetics rac1 GTP-Binding Protein - metabolism Rac1 protein RNA Interference RNA-mediated interference Target recognition Wounds |
| title | Automated screening of microtubule growth dynamics identifies MARK2 as a regulator of leading edge microtubules downstream of Rac1 in migrating cells |
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