Ca2+-dependent myosin II activation is required for uropod retraction during neutrophil migration.
Buffering of intracellular Ca2+ transients in human neutrophils leads to reduced motility due to defective uropod detachment on fibronectin and vitronectin-coated surfaces. Since one potential target of a rise in [Ca2+]i is the activation of myosin II, we characterized the role of myosin II during motility. Treatment of neutrophils with a myosin inhibitor (2,3-butanedione monoxime), or myosin light chain kinase inhibitors (ML-7, ML-9, or KT5926) resulted in impaired uropod retraction and a dose-dependent decrease in chemokinesis following stimulation with N-formyl-Met-Leu-Phe (fMLP). Treatment with ML-9 resulted in a redistribution of F-actin and talin to the non-retracted uropods, mimicking the redistribution observed during [Ca2+]i buffering. Impairment of uropod retraction and redistribution of F-actin and talin by myosin II inhibition was only observed on adhesive substrates such as fibronectin and not on poorly adhesive substrates such as human serum-coated glass. At higher concentrations of ML-9, cell polarization was inhibited and pseudopod extension occurred radially. Using an antibody specific for serine 19-phosphorylated regulatory light chain of myosin II, regions of activated myosin II were found at the leading edge as well as the uropod in motile fMLP-stimulated cells. [Ca2+]i depletion caused a 50% decrease in the level of serine 19-phosphorylated myosin II suggesting that activation of myosin II by intracellular Ca2+ transients may be an essential step in establishing a polarized pseudopod and providing the force required for uropod retraction during PMN motility on adhesive surfaces.
Eddy
R J
RJ
Department of Biochemistry, Weill Medical College of Cornell University, New York, NY, USA.
Pierini
L M
LM
Matsumura
F
F
Maxfield
F R
FR
D000199:Actins / Q000378:metabolism; D001381:Azepines / Q000494:pharmacology; D002021:Buffers; D002118:Calcium / Q000502:physiology*; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology*; D016764:Cell Polarity / Q000187:drug effects; D002634:Chemotaxis, Leukocyte / Q000187:drug effects / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors; D009218:Myosins / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D011554:Pseudopodia / Q000187:drug effects / Q000502:physiology*; D016608:Talin / Q000378:metabolism
D000199:Actins; D001381:Azepines; D002021:Buffers; D004791:Enzyme Inhibitors; D016608:Talin; C056218:ML 9; D009219:Myosin-Light-Chain Kinase; D009218:Myosins; D002118:Calcium