Nonmuscle myosin II molecules carry out a wide variety of functions within cells. There are three nonmuscle myosin II genes. We are using optical trapping nanometry to study the interaction of nonmuscle myosin IIB with actin. When phosphorylated this myosin has long attachment times as would be expected from such a slow myosin, but that it does not move processively in the optical trap and shows only attachments and detachments without stepping in a single molecule motility assay in which actin is bound to the surface and the interaction with fluorescently-labeled myosin is observed. We have expressed full length nonmuscle myosins IIA and IIB and have characterized their steady state MgATPase properties. We have examined the filament structure of these myosins using negative staining electron microscopy and find that both form short bipolar filaments of similar length and thickness. These short filaments move processively in the above mentioned motility assay. We will use a combination of solution studies and electron microscopy to study the assembly mechanism for these myosins and observe their interaction with actin. We have also expressed mutant forms of nonmuscle myosin IIA corresponding to naturally occuring, disease causing mutations that give rise to giant platelet disorders and will characterize the effect of these mutations on myosin function and filament assembly.

Project Start
Project End
Budget Start
Budget End
Support Year
32
Fiscal Year
2011
Total Cost
$478,064
Indirect Cost
Name
National Heart, Lung, and Blood Institute
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Type
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Ecsédi, Péter; Billington, Neil; Pálfy, Gyula et al. (2018) Multiple S100 protein isoforms and C-terminal phosphorylation contribute to the paralog-selective regulation of non-muscle myosin 2 filaments. J Biol Chem :
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Lopata, Anna; Hughes, Ruth; Tiede, Christian et al. (2018) Affimer proteins for F-actin: novel affinity reagents that label F-actin in live and fixed cells. Sci Rep 8:6572
Melli, Luca; Billington, Neil; Sun, Sara A et al. (2018) Bipolar filaments of human nonmuscle myosin 2-A and 2-B have distinct motile and mechanical properties. Elife 7:
Liu, Xiong; Billington, Neil; Shu, Shi et al. (2017) Effect of ATP and regulatory light-chain phosphorylation on the polymerization of mammalian nonmuscle myosin II. Proc Natl Acad Sci U S A 114:E6516-E6525
Chinthalapudi, Krishna; Heissler, Sarah M; Preller, Matthias et al. (2017) Mechanistic insights into the active site and allosteric communication pathways in human nonmuscle myosin-2C. Elife 6:
Bruun, Kyle; Beach, Jordan R; Heissler, Sarah M et al. (2017) Re-evaluating the roles of myosin 18A? and F-actin in determining Golgi morphology. Cytoskeleton (Hoboken) 74:205-218
Baird, Michelle A; Billington, Neil; Wang, Aibing et al. (2017) Local pulsatile contractions are an intrinsic property of the myosin 2A motor in the cortical cytoskeleton of adherent cells. Mol Biol Cell 28:240-251
Liu, Xiong; Shu, Shi; Billington, Neil et al. (2016) Mammalian Nonmuscle Myosin II Binds to Anionic Phospholipids with Concomitant Dissociation of the Regulatory Light Chain. J Biol Chem 291:24828-24837
Heissler, Sarah M; Sellers, James R (2016) Various Themes of Myosin Regulation. J Mol Biol 428:1927-46

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