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 [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.
Chew
Teng-Leong
TL
Department of Cell and Molecular Biology, R.H. Lurie Comprehensive Cancer Center and Center for Genetic Medicine, Northwestern University Medical School, Chicago, IL 60611, USA.
Wolf
Wendy A
WA
Gallagher
Patricia J
PJ
Matsumura
Fumio
F
Chisholm
Rex L
RL
D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology; D002147:Calmodulin / Q000378:metabolism; D002455:Cell Division / Q000502:physiology; D002465:Cell Movement / Q000502:physiology*; D016764:Cell Polarity / Q000502:physiology; D003599:Cytoskeleton / Q000378:metabolism*; D004173:Dipodomys; D007425:Intracellular Membranes / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D011554:Pseudopodia / Q000378:metabolism*
D002147:Calmodulin; D009219:Myosin-Light-Chain Kinase; D009218:Myosins; D002118:Calcium