A Fluorescent Resonant Energy Transfer-Based Biosensor Reveals Transient and Regional Myosin Light Chain Kinase Activation in Lamella and Cleavage Furrows
Approaches with high spatial and temporal resolution are required to understand the regulation of nonmuscle myosin II in vivo. Using fluorescence resonance energy transfer we have produced a novel biosensor allowing simultaneous determination of myosin light chain kinase (MLCK) localization and its...
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| Veröffentlicht in: | The Journal of cell biology 2002-02, Vol.156 (3), p.543-553 |
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| container_title | The Journal of cell biology |
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| creator | Chew, Teng-Leong Wolf, Wendy A. Gallagher, Patricia J. Matsumura, Fumio Chisholm, Rex L. |
| description | Approaches with high spatial and temporal resolution are required to understand the regulation of nonmuscle myosin II in vivo. Using fluorescence resonance energy transfer we have produced a novel biosensor allowing simultaneous determination of myosin light chain kinase (MLCK) localization and its [ Ca2+]4/calmodulin-binding state in living cells. We observe transient recruitment of diffuse MLCK to stress fibers and its in situ activation before contraction. MLCK is highly active in the lamella of migrating cells, but not at the retracting tail. This unexpected result highlights a potential role for MLCK-mediated myosin contractility in the lamella as a driving force for migration. During cytokinesis, MLCK was enriched at the spindle equator during late metaphase, and was maximally activated just before cleavage furrow constriction. As furrow contraction was completed, active MLCK was redistributed to the poles of the daughter cells. These results show MLCK is a myosin regulator in the lamella and contractile ring, and pinpoints sites where myosin function may be mediated by other kinases. |
| doi_str_mv | 10.1083/jcb.200110161 |
| format | Article |
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Using fluorescence resonance energy transfer we have produced a novel biosensor allowing simultaneous determination of myosin light chain kinase (MLCK) localization and its [ Ca2+]4/calmodulin-binding state in living cells. We observe transient recruitment of diffuse MLCK to stress fibers and its in situ activation before contraction. MLCK is highly active in the lamella of migrating cells, but not at the retracting tail. This unexpected result highlights a potential role for MLCK-mediated myosin contractility in the lamella as a driving force for migration. During cytokinesis, MLCK was enriched at the spindle equator during late metaphase, and was maximally activated just before cleavage furrow constriction. As furrow contraction was completed, active MLCK was redistributed to the poles of the daughter cells. These results show MLCK is a myosin regulator in the lamella and contractile ring, and pinpoints sites where myosin function may be mediated by other kinases.</description><identifier>ISSN: 0021-9525</identifier><identifier>EISSN: 1540-8140</identifier><identifier>DOI: 10.1083/jcb.200110161</identifier><identifier>PMID: 11815633</identifier><language>eng</language><publisher>United States: Rockefeller University Press</publisher><subject>Animals ; Biosensing Techniques - methods ; Calcium - metabolism ; Calcium Signaling - physiology ; Calmodulin - metabolism ; Cell Division - physiology ; Cell lines ; Cell motility ; Cell Movement - physiology ; Cell Polarity - physiology ; Cells ; COS cells ; Cytokinesis ; Cytoskeleton - metabolism ; Dipodomys ; Endothelial cells ; HeLa cells ; Intracellular Membranes - metabolism ; Microscopy, Fluorescence - methods ; Myosin-Light-Chain Kinase - metabolism ; Myosins - metabolism ; Phosphorylation ; Pseudopodia - metabolism ; Smooth muscle ; Stress fibers</subject><ispartof>The Journal of cell biology, 2002-02, Vol.156 (3), p.543-553</ispartof><rights>Copyright 2002 The Rockefeller University Press</rights><rights>Copyright © 2002, The Rockefeller University Press</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c470t-a5c3cfd3f546d4bfdc8f7651fac52930834d30fd106d817d42adea1b311c97ee3</citedby><cites>FETCH-LOGICAL-c470t-a5c3cfd3f546d4bfdc8f7651fac52930834d30fd106d817d42adea1b311c97ee3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>230,314,780,784,885,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/11815633$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Chew, Teng-Leong</creatorcontrib><creatorcontrib>Wolf, Wendy A.</creatorcontrib><creatorcontrib>Gallagher, Patricia J.</creatorcontrib><creatorcontrib>Matsumura, Fumio</creatorcontrib><creatorcontrib>Chisholm, Rex L.</creatorcontrib><title>A Fluorescent Resonant Energy Transfer-Based Biosensor Reveals Transient and Regional Myosin Light Chain Kinase Activation in Lamella and Cleavage Furrows</title><title>The Journal of cell biology</title><addtitle>J Cell Biol</addtitle><description>Approaches with high spatial and temporal resolution are required to understand the regulation of nonmuscle myosin II in vivo. Using fluorescence resonance energy transfer we have produced a novel biosensor allowing simultaneous determination of myosin light chain kinase (MLCK) localization and its [ Ca2+]4/calmodulin-binding state in living cells. We observe transient recruitment of diffuse MLCK to stress fibers and its in situ activation before contraction. MLCK is highly active in the lamella of migrating cells, but not at the retracting tail. This unexpected result highlights a potential role for MLCK-mediated myosin contractility in the lamella as a driving force for migration. During cytokinesis, MLCK was enriched at the spindle equator during late metaphase, and was maximally activated just before cleavage furrow constriction. As furrow contraction was completed, active MLCK was redistributed to the poles of the daughter cells. These results show MLCK is a myosin regulator in the lamella and contractile ring, and pinpoints sites where myosin function may be mediated by other kinases.</description><subject>Animals</subject><subject>Biosensing Techniques - methods</subject><subject>Calcium - metabolism</subject><subject>Calcium Signaling - physiology</subject><subject>Calmodulin - metabolism</subject><subject>Cell Division - physiology</subject><subject>Cell lines</subject><subject>Cell motility</subject><subject>Cell Movement - physiology</subject><subject>Cell Polarity - physiology</subject><subject>Cells</subject><subject>COS cells</subject><subject>Cytokinesis</subject><subject>Cytoskeleton - metabolism</subject><subject>Dipodomys</subject><subject>Endothelial cells</subject><subject>HeLa cells</subject><subject>Intracellular Membranes - metabolism</subject><subject>Microscopy, Fluorescence - methods</subject><subject>Myosin-Light-Chain Kinase - metabolism</subject><subject>Myosins - metabolism</subject><subject>Phosphorylation</subject><subject>Pseudopodia - metabolism</subject><subject>Smooth muscle</subject><subject>Stress fibers</subject><issn>0021-9525</issn><issn>1540-8140</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2002</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpVkU1vEzEQhi1ERdPCkRtCPnHb4ll7P3JBSqOmVAQhoXK2HHu8cbSxi70blL_Cr8UhUT9OHnmeeefjJeQ9sCtgLf-80aurkjEABjW8IhOoBCtaEOw1mTBWQjGtyuqcXKS0YYyJRvA35BygharmfEL-zuiiH0PEpNEP9Cem4FUObjzGbk_vo_LJYiyuVUJDr11I6FOIGdyh6tMRcIdS5U3-7Vyu7-n3fUjO06Xr1gOdr1WOvzmfNehMD26nhozRA6C22Pfqf_G8R7VTHdLFGGP4k96SM5tb4LvTe0l-LW7u51-L5Y_bu_lsWWjRsKFQlebaGm4rURuxska3tqkrsEpX5ZTnGwnDmTXAatNCY0SpDCpYcQA9bRD5Jfly1H0YV1s0hztE1cuH6LYq7mVQTr7MeLeWXdjJEhrOyzYLfDoJxPB7xDTIrcvnzHt5DGOSDQiRsTqDxRHUMaQU0T42ASYPbsrspnx0M_Mfn0_2RJ_sy8CHI7BJQ4hP-bpkeVX-DyoGqFY</recordid><startdate>20020204</startdate><enddate>20020204</enddate><creator>Chew, Teng-Leong</creator><creator>Wolf, Wendy A.</creator><creator>Gallagher, Patricia J.</creator><creator>Matsumura, Fumio</creator><creator>Chisholm, Rex L.</creator><general>Rockefeller University Press</general><general>The Rockefeller University Press</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20020204</creationdate><title>A Fluorescent Resonant Energy Transfer-Based Biosensor Reveals Transient and Regional Myosin Light Chain Kinase Activation in Lamella and Cleavage Furrows</title><author>Chew, Teng-Leong ; Wolf, Wendy A. ; Gallagher, Patricia J. ; Matsumura, Fumio ; Chisholm, Rex L.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c470t-a5c3cfd3f546d4bfdc8f7651fac52930834d30fd106d817d42adea1b311c97ee3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2002</creationdate><topic>Animals</topic><topic>Biosensing Techniques - methods</topic><topic>Calcium - metabolism</topic><topic>Calcium Signaling - physiology</topic><topic>Calmodulin - metabolism</topic><topic>Cell Division - physiology</topic><topic>Cell lines</topic><topic>Cell motility</topic><topic>Cell Movement - physiology</topic><topic>Cell Polarity - physiology</topic><topic>Cells</topic><topic>COS cells</topic><topic>Cytokinesis</topic><topic>Cytoskeleton - metabolism</topic><topic>Dipodomys</topic><topic>Endothelial cells</topic><topic>HeLa cells</topic><topic>Intracellular Membranes - metabolism</topic><topic>Microscopy, Fluorescence - methods</topic><topic>Myosin-Light-Chain Kinase - metabolism</topic><topic>Myosins - metabolism</topic><topic>Phosphorylation</topic><topic>Pseudopodia - metabolism</topic><topic>Smooth muscle</topic><topic>Stress fibers</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Chew, Teng-Leong</creatorcontrib><creatorcontrib>Wolf, Wendy A.</creatorcontrib><creatorcontrib>Gallagher, Patricia J.</creatorcontrib><creatorcontrib>Matsumura, Fumio</creatorcontrib><creatorcontrib>Chisholm, Rex L.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>The Journal of cell biology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Chew, Teng-Leong</au><au>Wolf, Wendy A.</au><au>Gallagher, Patricia J.</au><au>Matsumura, Fumio</au><au>Chisholm, Rex L.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A Fluorescent Resonant Energy Transfer-Based Biosensor Reveals Transient and Regional Myosin Light Chain Kinase Activation in Lamella and Cleavage Furrows</atitle><jtitle>The Journal of cell biology</jtitle><addtitle>J Cell Biol</addtitle><date>2002-02-04</date><risdate>2002</risdate><volume>156</volume><issue>3</issue><spage>543</spage><epage>553</epage><pages>543-553</pages><issn>0021-9525</issn><eissn>1540-8140</eissn><abstract>Approaches with high spatial and temporal resolution are required to understand the regulation of nonmuscle myosin II in vivo. Using fluorescence resonance energy transfer we have produced a novel biosensor allowing simultaneous determination of myosin light chain kinase (MLCK) localization and its [ Ca2+]4/calmodulin-binding state in living cells. We observe transient recruitment of diffuse MLCK to stress fibers and its in situ activation before contraction. MLCK is highly active in the lamella of migrating cells, but not at the retracting tail. This unexpected result highlights a potential role for MLCK-mediated myosin contractility in the lamella as a driving force for migration. During cytokinesis, MLCK was enriched at the spindle equator during late metaphase, and was maximally activated just before cleavage furrow constriction. As furrow contraction was completed, active MLCK was redistributed to the poles of the daughter cells. These results show MLCK is a myosin regulator in the lamella and contractile ring, and pinpoints sites where myosin function may be mediated by other kinases.</abstract><cop>United States</cop><pub>Rockefeller University Press</pub><pmid>11815633</pmid><doi>10.1083/jcb.200110161</doi><tpages>11</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Animals Biosensing Techniques - methods Calcium - metabolism Calcium Signaling - physiology Calmodulin - metabolism Cell Division - physiology Cell lines Cell motility Cell Movement - physiology Cell Polarity - physiology Cells COS cells Cytokinesis Cytoskeleton - metabolism Dipodomys Endothelial cells HeLa cells Intracellular Membranes - metabolism Microscopy, Fluorescence - methods Myosin-Light-Chain Kinase - metabolism Myosins - metabolism Phosphorylation Pseudopodia - metabolism Smooth muscle Stress fibers |
| title | A Fluorescent Resonant Energy Transfer-Based Biosensor Reveals Transient and Regional Myosin Light Chain Kinase Activation in Lamella and Cleavage Furrows |
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