Publications by authors named "Neil Billington"

31 Publications

Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays.

J Vis Exp 2021 02 4(168). Epub 2021 Feb 4.

Laboratory of Molecular Physiology, Cell and Developmental Biology Center, National Heart, Lung and Blood Institute, National Institutes of Health;

Myosin proteins bind and interact with filamentous actin (F-actin) and are found in organisms across the phylogenetic tree. Their structure and enzymatic properties are adapted for the particular function they execute in cells. Myosin 5a processively walks on F-actin to transport melanosomes and vesicles in cells. Conversely, nonmuscle myosin 2b operates as a bipolar filament containing approximately 30 molecules. It moves F-actin of opposite polarity toward the center of the filament, where the myosin molecules work asynchronously to bind actin, impart a power stroke, and dissociate before repeating the cycle. Nonmuscle myosin 2b, along with its other nonmuscle myosin 2 isoforms, has roles that include cell adhesion, cytokinesis, and tension maintenance. The mechanochemistry of myosins can be studied by performing in vitro motility assays using purified proteins. In the gliding actin filament assay, the myosins are bound to a microscope coverslip surface and translocate fluorescently labeled F-actin, which can be tracked. In the single molecule/ensemble motility assay, however, F-actin is bound to a coverslip and the movement of fluorescently labeled myosin molecules on the F-actin is observed. In this report, the purification of recombinant myosin 5a from Sf9 cells using affinity chromatography is outlined. Following this, we outline two fluorescence microscopy-based assays: the gliding actin filament assay and the inverted motility assay. From these assays, parameters such as actin translocation velocities and single molecule run lengths and velocities can be extracted using the image analysis software. These techniques can also be applied to study the movement of single filaments of the nonmuscle myosin 2 isoforms, discussed herein in the context of nonmuscle myosin 2b. This workflow represents a protocol and a set of quantitative tools that can be used to study the single molecule and ensemble dynamics of nonmuscle myosins.
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http://dx.doi.org/10.3791/62180DOI Listing
February 2021

A binding protein regulates myosin-7a dimerization and actin bundle assembly.

Nat Commun 2021 01 25;12(1):563. Epub 2021 Jan 25.

Laboratory of Molecular Physiology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, 20892, USA.

Myosin-7a, despite being monomeric in isolation, plays roles in organizing actin-based cell protrusions such as filopodia, microvilli and stereocilia, as well as transporting cargoes within them. Here, we identify a binding protein for Drosophila myosin-7a termed M7BP, and describe how M7BP assembles myosin-7a into a motile complex that enables cargo translocation and actin cytoskeletal remodeling. M7BP binds to the autoinhibitory tail of myosin-7a, extending the molecule and activating its ATPase activity. Single-molecule reconstitution show that M7BP enables robust motility by complexing with myosin-7a as 2:2 translocation dimers in an actin-regulated manner. Meanwhile, M7BP tethers actin, enhancing complex's processivity and driving actin-filament alignment during processive runs. Finally, we show that myosin-7a-M7BP complex assembles actin bundles and filopodia-like protrusions while migrating along them in living cells. Together, these findings provide insights into the mechanisms by which myosin-7a functions in actin protrusions.
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http://dx.doi.org/10.1038/s41467-020-20864-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7835385PMC
January 2021

The BAR domain of the Arf GTPase-activating protein ASAP1 directly binds actin filaments.

J Biol Chem 2020 08 22;295(32):11303-11315. Epub 2020 May 22.

Department of Physiology and Cell Biology, Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University Wexner Medical Center, Columbus, Ohio, USA

The Arf GTPase-activating protein (Arf GAP) with SH3 domain, ankyrin repeat and PH domain 1 (ASAP1) establishes a connection between the cell membrane and the cortical actin cytoskeleton. The formation, maintenance, and turnover of actin filaments and bundles in the actin cortex are important for cell adhesion, invasion, and migration. Here, using actin cosedimentation, polymerization, and depolymerization assays, along with total internal reflection fluorescence (TIRF), confocal, and EM analyses, we show that the N-terminal N-BAR domain of ASAP1 directly binds to F-actin. We found that ASAP1 homodimerization aligns F-actin in predominantly unipolar bundles and stabilizes them against depolymerization. Furthermore, the ASAP1 N-BAR domain moderately reduced the spontaneous polymerization of G-actin. The overexpression of the ASAP1 BAR-PH tandem domain in fibroblasts induced the formation of actin-filled projections more effectively than did full-length ASAP1. An ASAP1 construct that lacked the N-BAR domain failed to induce cellular projections. Our results suggest that ASAP1 regulates the dynamics and the formation of higher-order actin structures, possibly through direct binding to F-actin via its N-BAR domain. We propose that ASAP1 is a hub protein for dynamic protein-protein interactions in mechanosensitive structures, such as focal adhesions, invadopodia, and podosomes, that are directly implicated in oncogenic events. The effect of ASAP1 on actin dynamics puts a spotlight on its function as a central signaling molecule that regulates the dynamics of the actin cytoskeleton by transmitting signals from the plasma membrane.
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http://dx.doi.org/10.1074/jbc.RA119.009903DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7415992PMC
August 2020

Megakaryocyte migration defects due to nonmuscle myosin IIA mutations underlie thrombocytopenia in MYH9-related disease.

Blood 2020 05;135(21):1887-1898

Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA.

Megakaryocytes (MKs), the precursor cells for platelets, migrate from the endosteal niche of the bone marrow (BM) toward the vasculature, extending proplatelets into sinusoids, where circulating blood progressively fragments them into platelets. Nonmuscle myosin IIA (NMIIA) heavy chain gene (MYH9) mutations cause macrothrombocytopenia characterized by fewer platelets with larger sizes leading to clotting disorders termed myosin-9-related disorders (MYH9-RDs). MYH9-RD patient MKs have proplatelets with thicker and fewer branches that produce fewer and larger proplatelets, which is phenocopied in mouse Myh9-RD models. Defective proplatelet formation is considered to be the principal mechanism underlying the macrothrombocytopenia phenotype. However, MYH9-RD patient MKs may have other defects, as NMII interactions with actin filaments regulate physiological processes such as chemotaxis, cell migration, and adhesion. How MYH9-RD mutations affect MK migration and adhesion in BM or NMIIA activity and assembly prior to proplatelet production remain unanswered. NMIIA is the only NMII isoform expressed in mature MKs, permitting exploration of these questions without complicating effects of other NMII isoforms. Using mouse models of MYH9-RD (NMIIAR702C+/-GFP+/-, NMIIAD1424N+/-, and NMIIAE1841K+/-) and in vitro assays, we investigated MK distribution in BM, chemotaxis toward stromal-derived factor 1, NMIIA activity, and bipolar filament assembly. Results indicate that different MYH9-RD mutations suppressed MK migration in the BM without compromising bipolar filament formation but led to divergent adhesion phenotypes and NMIIA contractile activities depending on the mutation. We conclude that MYH9-RD mutations impair MK chemotaxis by multiple mechanisms to disrupt migration toward the vasculature, impairing proplatelet release and causing macrothrombocytopenia.
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http://dx.doi.org/10.1182/blood.2019003064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7243143PMC
May 2020

Competition between kinesin-1 and myosin-V defines posterior determination.

Elife 2020 02 14;9. Epub 2020 Feb 14.

Department of Cell and Developmental Biology, Feinberg School of Medicine, Northwestern University, Chicago, United States.

Local accumulation of ) mRNA in the oocyte determines the posterior pole of the future embryo. Two major cytoskeletal components, microtubules and actin filaments, together with a microtubule motor, kinesin-1, and an actin motor, myosin-V, are essential for mRNA posterior localization. In this study, we use Staufen, an RNA-binding protein that colocalizes with mRNA, as a proxy for mRNA. We demonstrate that posterior localization of /Staufen is determined by competition between kinesin-1 and myosin-V. While kinesin-1 removes /Staufen from the cortex along microtubules, myosin-V anchors /Staufen at the cortex. Myosin-V wins over kinesin-1 at the posterior pole due to low microtubule density at this site, while kinesin-1 wins at anterior and lateral positions because they have high density of cortically-anchored microtubules. As a result, posterior determinants are removed from the anterior and lateral cortex but retained at the posterior pole. Thus, posterior determination of oocytes is defined by kinesin-myosin competition, whose outcome is primarily determined by cortical microtubule density.
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http://dx.doi.org/10.7554/eLife.54216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7112953PMC
February 2020

Multiple S100 protein isoforms and C-terminal phosphorylation contribute to the paralog-selective regulation of nonmuscle myosin 2 filaments.

J Biol Chem 2018 09 7;293(38):14850-14867. Epub 2018 Aug 7.

From the Department of Biochemistry,

Nonmuscle myosin 2 (NM2) has three paralogs in mammals, NM2A, NM2B, and NM2C, which have both unique and overlapping functions in cell migration, formation of cell-cell adhesions, and cell polarity. Their assembly into homo- and heterotypic bipolar filaments in living cells is primarily regulated by phosphorylation of the N-terminally bound regulatory light chain. Here, we present evidence that the equilibrium between these filaments and single NM2A and NM2B molecules can be controlled via S100 calcium-binding protein interactions and phosphorylation at the C-terminal end of the heavy chains. Furthermore, we show that in addition to S100A4, other members of the S100 family can also mediate disassembly of homotypic NM2A filaments. Importantly, these proteins can selectively remove NM2A molecules from heterotypic filaments. We also found that tail phosphorylation (at Ser-1956 and Ser-1975) of NM2B by casein kinase 2, as well as phosphomimetic substitutions at sites targeted by protein kinase C (PKC) and transient receptor potential cation channel subfamily M member 7 (TRPM7), down-regulates filament assembly in an additive fashion. Tail phosphorylation of NM2A had a comparatively minor effect on filament stability. S100 binding and tail phosphorylation therefore preferentially disassemble NM2A and NM2B, respectively. These two distinct mechanisms are likely to contribute to the temporal and spatial sorting of the two NM2 paralogs within heterotypic filaments. The existence of multiple NM2A-depolymerizing S100 paralogs offers the potential for diverse regulatory inputs modulating NM2A filament disassembly in cells and provides functional redundancy under both physiological and pathological conditions.
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http://dx.doi.org/10.1074/jbc.RA118.004277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6153290PMC
September 2018

Recruitment of two dyneins to an mRNA-dependent Bicaudal D transport complex.

Elife 2018 06 26;7. Epub 2018 Jun 26.

Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, United States.

We investigated the role of full-length Bicaudal D (BicD) binding partners in dynein-dynactin activation for mRNA transport on microtubules. Full-length BicD robustly activated dynein-dynactin motility only when both the mRNA binding protein Egalitarian (Egl) and mRNA cargo were present, and electron microscopy showed that both Egl and mRNA were needed to disrupt a looped, auto-inhibited BicD conformation. BicD can recruit two dimeric dyneins, resulting in faster speeds and longer runs than with one dynein. Moving complexes predominantly contained two Egl molecules and one mRNA. This mRNA-bound configuration makes Egl bivalent, likely enhancing its avidity for BicD and thus its ability to disrupt BicD auto-inhibition. Consistent with this idea, artificially dimerized Egl activates dynein-dynactin-BicD in the absence of mRNA. The ability of mRNA cargo to orchestrate the activation of the mRNP (messenger ribonucleotide protein) complex is an elegant way to ensure that only cargo-bound motors are motile.
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http://dx.doi.org/10.7554/eLife.36306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6056235PMC
June 2018

Quantitative mass imaging of single biological macromolecules.

Science 2018 04;360(6387):423-427

Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QZ, UK.

The cellular processes underpinning life are orchestrated by proteins and their interactions. The associated structural and dynamic heterogeneity, despite being key to function, poses a fundamental challenge to existing analytical and structural methodologies. We used interferometric scattering microscopy to quantify the mass of single biomolecules in solution with 2% sequence mass accuracy, up to 19-kilodalton resolution, and 1-kilodalton precision. We resolved oligomeric distributions at high dynamic range, detected small-molecule binding, and mass-imaged proteins with associated lipids and sugars. These capabilities enabled us to characterize the molecular dynamics of processes as diverse as glycoprotein cross-linking, amyloidogenic protein aggregation, and actin polymerization. Interferometric scattering mass spectrometry allows spatiotemporally resolved measurement of a broad range of biomolecular interactions, one molecule at a time.
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http://dx.doi.org/10.1126/science.aar5839DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6103225PMC
April 2018

Bipolar filaments of human nonmuscle myosin 2-A and 2-B have distinct motile and mechanical properties.

Elife 2018 02 8;7. Epub 2018 Feb 8.

Cell Biology and Physiology Center, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, United States.

Nonmusclemyosin 2 (NM-2) powers cell motility and tissue morphogenesis by assembling into bipolar filaments that interact with actin. Although the enzymatic properties of purified NM-2 motor fragments have been determined, the emergent properties of filament ensembles are unknown. Using single myosin filament in vitro motility assays, we report fundamental differences in filaments formed of different NM-2 motors. Filaments consisting of NM2-B moved processively along actin, while under identical conditions, NM2-A filaments did not. By more closely mimicking the physiological milieu, either by increasing solution viscosity or by co-polymerization with NM2-B, NM2-A containing filaments moved processively. Our data demonstrate that both the kinetic and mechanical properties of these two myosins, in addition to the stochiometry of NM-2 subunits, can tune filament mechanical output. We propose altering NM-2 filament composition is a general cellular strategy for tailoring force production of filaments to specific functions, such as maintaining tension or remodeling actin.
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http://dx.doi.org/10.7554/eLife.32871DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5829915PMC
February 2018

Effect of ATP and regulatory light-chain phosphorylation on the polymerization of mammalian nonmuscle myosin II.

Proc Natl Acad Sci U S A 2017 08 24;114(32):E6516-E6525. Epub 2017 Jul 24.

Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892;

Addition of 1 mM ATP substantially reduces the light scattering of solutions of polymerized unphosphorylated nonmuscle myosin IIs (NM2s), and this is reversed by phosphorylation of the regulatory light chain (RLC). It has been proposed that these changes result from substantial depolymerization of unphosphorylated NM2 filaments to monomers upon addition of ATP, and filament repolymerization upon RLC-phosphorylation. We now show that the differences in myosin monomer concentration of RLC-unphosphorylated and -phosphorylated recombinant mammalian NM2A, NM2B, and NM2C polymerized in the presence of ATP are much too small to explain their substantial differences in light scattering. Rather, we find that the decrease in light scattering upon addition of ATP to polymerized unphosphorylated NM2s correlates with the formation of dimers, tetramers, and hexamers, in addition to monomers, an increase in length, and decrease in width of the bare zones of RLC-unphosphorylated filaments. Both effects of ATP addition are reversed by phosphorylation of the RLC. Our data also suggest that, contrary to previous models, assembly of RLC-phosphorylated NM2s at physiological ionic strength proceeds from folded monomers to folded antiparallel dimers, tetramers, and hexamers that unfold and polymerize into antiparallel filaments. This model could explain the dynamic relocalization of NM2 filaments in vivo by dephosphorylation of RLC-phosphorylated filaments, disassembly of the dephosphorylated filaments to folded monomers, dimers, and small oligomers, followed by diffusion of these species, and reassembly of filaments at the new location following rephosphorylation of the RLC.
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http://dx.doi.org/10.1073/pnas.1702375114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5559010PMC
August 2017

non-muscle myosin II motor activity determines the rate of tissue folding.

Elife 2016 12 30;5. Epub 2016 Dec 30.

Department of Biology, Massachusetts Institute of Technology, Cambridge, United States.

Non-muscle cell contractility is critical for tissues to adopt shape changes. Although, the non-muscle myosin II holoenzyme (myosin) is a molecular motor that powers contraction of actin cytoskeleton networks, recent studies have questioned the importance of myosin motor activity cell and tissue shape changes. Here, combining the biochemical analysis of enzymatic and motile properties for purified myosin mutants with in vivo measurements of apical constriction for the same mutants, we show that in vivo constriction rate scales with myosin motor activity. We show that so-called phosphomimetic mutants of the regulatory light chain (RLC) do not mimic the phosphorylated RLC state in vitro. The defect in the myosin motor activity in these mutants is evident in developing embryos where tissue recoil following laser ablation is decreased compared to wild-type tissue. Overall, our data highlights that myosin activity is required for rapid cell contraction and tissue folding in developing embryos.
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http://dx.doi.org/10.7554/eLife.20828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5201417PMC
December 2016

Local pulsatile contractions are an intrinsic property of the myosin 2A motor in the cortical cytoskeleton of adherent cells.

Mol Biol Cell 2017 01 23;28(2):240-251. Epub 2016 Nov 23.

Cell Biology and Physiology Center, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892

The role of nonmuscle myosin 2 (NM2) pulsatile dynamics in generating contractile forces required for developmental morphogenesis has been characterized, but whether these pulsatile contractions are an intrinsic property of all actomyosin networks is not known. Here we used live-cell fluorescence imaging to show that transient, local assembly of NM2A "pulses" occurs in the cortical cytoskeleton of single adherent cells of mesenchymal, epithelial, and sarcoma origin, independent of developmental signaling cues and cell-cell or cell-ECM interactions. We show that pulses in the cortical cytoskeleton require Rho-associated kinase- or myosin light chain kinase (MLCK) activity, increases in cytosolic calcium, and NM2 ATPase activity. Surprisingly, we find that cortical cytoskeleton pulses specifically require the head domain of NM2A, as they do not occur with either NM2B or a 2B-head-2A-tail chimera. Our results thus suggest that pulsatile contractions in the cortical cytoskeleton are an intrinsic property of the NM2A motor that may mediate its role in homeostatic maintenance of tension in the cortical cytoskeleton of adherent cells.
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http://dx.doi.org/10.1091/mbc.E16-05-0335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5231893PMC
January 2017

Mammalian Nonmuscle Myosin II Binds to Anionic Phospholipids with Concomitant Dissociation of the Regulatory Light Chain.

J Biol Chem 2016 Nov 3;291(48):24828-24837. Epub 2016 Oct 3.

From the Laboratories of Cell Biology and

Mammalian cells express three Class II nonmuscle myosins (NM): NM2A, NM2B, and NM2C. The three NM2s have well established essential roles in cell motility, adhesion, and cytokinesis and less well defined roles in vesicle transport and other processes that would require association of NM2s with cell membranes. Previous evidence for the mechanism of NM2-membrane association includes direct interaction of NM2s with membrane lipids and indirect interaction by association of NM2s with membrane-bound F-actin or peripheral membrane proteins. Direct binding of NM2s to phosphatidylserine-liposomes, but not to phosphatidylcholine-liposomes, has been reported, but the molecular basis of the interaction between NM2s and acidic phospholipids has not been previously investigated. We now show that filamentous, full-length NM2A, NM2B, and NM2C and monomeric, non-filamentous heavy meromyosin bind to liposomes containing one or more acidic phospholipids (phosphatidylserine, phosphatidylinositol 4,5-diphosphate, and phosphatidylinositol 3,4,5-triphosphate) but do not bind to 100% phosphatidylcholine-liposomes. Binding of NM2s to acidic liposomes occurs predominantly through interaction of the liposomes with the regulatory light chain (RLC) binding site in the myosin heavy chain with concomitant dissociation of the RLC. Phosphorylation of myosin-bound RLC by myosin light chain kinase substantially inhibits binding to liposomes of both filamentous NM2 and non-filamentous heavy meromyosin; the addition of excess unbound RLC, but not excess unbound essential light chain, competes with liposome binding. Consistent with the in vitro data, we show that endogenous and expressed NM2A associates with the plasma membrane of HeLa cells and fibrosarcoma cells independently of F-actin.
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http://dx.doi.org/10.1074/jbc.M116.739185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5122755PMC
November 2016

A single-headed fission yeast myosin V transports actin in a tropomyosin-dependent manner.

J Cell Biol 2016 07 11;214(2):167-79. Epub 2016 Jul 11.

Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, VT 05405

Myo51, a class V myosin in fission yeast, localizes to and assists in the assembly of the contractile ring, a conserved eukaryotic actomyosin structure that facilitates cytokinesis. Rng8 and Rng9 are binding partners that dictate the cellular localization and function of Myo51. Myo51 was expressed in insect cells in the presence or absence of Rng8/9. Surprisingly, electron microscopy of negatively stained images and hydrodynamic measurements showed that Myo51 is single headed, unlike most class V myosins. When Myo51-Rng8/9 was bound to actin-tropomyosin, two attachment sites were observed: the typical ATP-dependent motor domain attachment and a novel ATP-independent binding of the tail mediated by Rng8/9. A modified motility assay showed that this additional binding site anchors Myo51-Rng8/9 so that it can cross-link and slide actin-tropomyosin filaments relative to one another, functions that may explain the role of this motor in contractile ring assembly.
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http://dx.doi.org/10.1083/jcb.201511102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4949448PMC
July 2016

Myosin 18A coassembles with nonmuscle myosin 2 to form mixed bipolar filaments.

Curr Biol 2015 Mar 5;25(7):942-8. Epub 2015 Mar 5.

Laboratory of Molecular Physiology, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD 20892-8015, USA. Electronic address:

Class-18 myosins are most closely related to conventional class-2 nonmuscle myosins (NM2). Surprisingly, the purified head domains of Drosophila, mouse, and human myosin 18A (M18A) lack actin-activated ATPase activity and the ability to translocate actin filaments, suggesting that the functions of M18A in vivo do not depend on intrinsic motor activity. M18A has the longest coiled coil of any myosin outside of the class-2 myosins, suggesting that it might form bipolar filaments similar to conventional myosins. To address this possibility, we expressed and purified full-length mouse M18A using the baculovirus/Sf9 system. M18A did not form large bipolar filaments under any of the conditions tested. Instead, M18A formed an ∼ 65-nm-long bipolar structure with two heads at each end. Importantly, when NM2 was polymerized in the presence of M18A, the two myosins formed mixed bipolar filaments, as evidenced by cosedimentation, electron microscopy, and single-molecule imaging. Moreover, super-resolution imaging of NM2 and M18A using fluorescently tagged proteins and immunostaining of endogenous proteins showed that NM2 and M18A are present together within individual filaments inside living cells. Together, our in vitro and live-cell imaging data argue strongly that M18A coassembles with NM2 into mixed bipolar filaments. M18A could regulate the biophysical properties of these filaments and, by virtue of its extra N- and C-terminal domains, determine the localization and/or molecular interactions of the filaments. Given the numerous, fundamental cellular and developmental roles attributed to NM2, our results have far-reaching biological implications.
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http://dx.doi.org/10.1016/j.cub.2015.02.012DOI Listing
March 2015

Chaperone-enhanced purification of unconventional myosin 15, a molecular motor specialized for stereocilia protein trafficking.

Proc Natl Acad Sci U S A 2014 Aug 11;111(34):12390-5. Epub 2014 Aug 11.

Laboratory of Molecular Genetics, National Institute on Deafness and Other Communication Disorders, and

Unconventional myosin 15 is a molecular motor expressed in inner ear hair cells that transports protein cargos within developing mechanosensory stereocilia. Mutations of myosin 15 cause profound hearing loss in humans and mice; however, the properties of this motor and its regulation within the stereocilia organelle are unknown. To address these questions, we expressed a subfragment 1-like (S1) truncation of mouse myosin 15, comprising the predicted motor domain plus three light-chain binding sites. Following unsuccessful attempts to express functional myosin 15-S1 using the Spodoptera frugiperda (Sf9)-baculovirus system, we discovered that coexpression of the muscle-myosin-specific chaperone UNC45B, in addition to the chaperone heat-shock protein 90 (HSP90) significantly increased the yield of functional protein. Surprisingly, myosin 15-S1 did not bind calmodulin with high affinity. Instead, the IQ domains bound essential and regulatory light chains that are normally associated with class II myosins. We show that myosin 15-S1 is a barbed-end-directed motor that moves actin filaments in a gliding assay (∼ 430 nm · s(-1) at 30 °C), using a power stroke of 7.9 nm. The maximum ATPase rate (k(cat) ∼ 6 s(-1)) was similar to the actin-detachment rate (k(det) = 6.2 s(-1)) determined in single molecule optical trapping experiments, indicating that myosin 15-S1 was rate limited by transit through strongly actin-bound states, similar to other processive myosin motors. Our data further indicate that in addition to folding muscle myosin, UNC45B facilitates maturation of an unconventional myosin. We speculate that chaperone coexpression may be a simple method to optimize the purification of other myosin motors from Sf9 insect cells.
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http://dx.doi.org/10.1073/pnas.1409459111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4151768PMC
August 2014

Myosin-10 produces its power-stroke in two phases and moves processively along a single actin filament under low load.

Proc Natl Acad Sci U S A 2014 May 21;111(18):E1833-42. Epub 2014 Apr 21.

Laboratory of Molecular Physiology, National Heart, Lung and Blood, Institute, National Institutes of Health, Bethesda, MD 20892.

Myosin-10 is an actin-based molecular motor that participates in essential intracellular processes such as filopodia formation/extension, phagocytosis, cell migration, and mitotic spindle maintenance. To study this motor protein's mechano-chemical properties, we used a recombinant, truncated form of myosin-10 consisting of the first 936 amino acids, followed by a GCN4 leucine zipper motif, to force dimerization. Negative-stain electron microscopy reveals that the majority of molecules are dimeric with a head-to-head contour distance of ∼50 nm. In vitro motility assays show that myosin-10 moves actin filaments smoothly with a velocity of ∼310 nm/s. Steady-state and transient kinetic analysis of the ATPase cycle shows that the ADP release rate (∼13 s(-1)) is similar to the maximum ATPase activity (∼12-14 s(-1)) and therefore contributes to rate limitation of the enzymatic cycle. Single molecule optical tweezers experiments show that under intermediate load (∼0.5 pN), myosin-10 interacts intermittently with actin and produces a power stroke of ∼17 nm, composed of an initial 15-nm and subsequent 2-nm movement. At low optical trap loads, we observed staircase-like processive movements of myosin-10 interacting with the actin filament, consisting of up to six ∼35-nm steps per binding interaction. We discuss the implications of this load-dependent processivity of myosin-10 as a filopodial transport motor.
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http://dx.doi.org/10.1073/pnas.1320122111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4020102PMC
May 2014

Mutations in TBC1D24, a gene associated with epilepsy, also cause nonsyndromic deafness DFNB86.

Am J Hum Genet 2014 Jan;94(1):144-52

Laboratory of Molecular Genetics, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Rockville, MD 20850, USA. Electronic address:

Inherited deafness is clinically and genetically heterogeneous. We recently mapped DFNB86, a locus associated with nonsyndromic deafness, to chromosome 16p. In this study, whole-exome sequencing was performed with genomic DNA from affected individuals from three large consanguineous families in which markers linked to DFNB86 segregate with profound deafness. Analyses of these data revealed homozygous mutation c.208G>T (p.Asp70Tyr) or c.878G>C (p.Arg293Pro) in TBC1D24 as the underlying cause of deafness in the three families. Sanger sequence analysis of TBC1D24 in an additional large family in which deafness segregates with DFNB86 identified the c.208G>T (p.Asp70Tyr) substitution. These mutations affect TBC1D24 amino acid residues that are conserved in orthologs ranging from fruit fly to human. Neither variant was observed in databases of single-nucleotide variants or in 634 chromosomes from ethnically matched control subjects. TBC1D24 in the mouse inner ear was immunolocalized predominantly to spiral ganglion neurons, indicating that DFNB86 deafness might be an auditory neuropathy spectrum disorder. Previously, six recessive mutations in TBC1D24 were reported to cause seizures (hearing loss was not reported) ranging in severity from epilepsy with otherwise normal development to epileptic encephalopathy resulting in childhood death. Two of our four families in which deafness segregates with mutant alleles of TBC1D24 were available for neurological examination. Cosegregation of epilepsy and deafness was not observed in these two families. Although the causal relationship between genotype and phenotype is not presently understood, our findings, combined with published data, indicate that recessive alleles of TBC1D24 can cause either epilepsy or nonsyndromic deafness.
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http://dx.doi.org/10.1016/j.ajhg.2013.12.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3882911PMC
January 2014

Flexibility within the heads of muscle myosin-2 molecules.

J Mol Biol 2014 Feb 9;426(4):894-907. Epub 2013 Dec 9.

School of Molecular and Cellular Biology and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK. Electronic address:

We show that negative-stain electron microscopy and image processing of nucleotide-free (apo) striated muscle myosin-2 subfragment-1 (S1), possessing one light chain or both light chains, is capable of resolving significant amounts of structural detail. The overall appearance of the motor and the lever is similar in rabbit, scallop and chicken S1. Projection matching of class averages of the different S1 types to projection views of two different crystal structures of apo S1 shows that all types most commonly closely resemble the appearance of the scallop S1 structure rather than the methylated chicken S1 structure. Methylation of chicken S1 has no effect on the structure of the molecule at this resolution: it too resembles the scallop S1 crystal structure. The lever is found to vary in its angle of attachment to the motor domain, with a hinge point located in the so-called pliant region between the converter and the essential light chain. The chicken S1 crystal structure lies near one end of the range of flexion observed. The Gaussian spread of angles of flexion suggests that flexibility is driven thermally, from which a torsional spring constant of ~23 pN·nm/rad² is estimated on average for all S1 types, similar to myosin-5. This translates to apparent cantilever-type stiffness at the tip of the lever of 0.37 pN/nm. Because this stiffness is lower than recent estimates from myosin-2 heads attached to actin, we suggest that binding to actin leads to an allosteric stiffening of the motor-lever junction.
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http://dx.doi.org/10.1016/j.jmb.2013.11.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3919154PMC
February 2014

Characterization of three full-length human nonmuscle myosin II paralogs.

J Biol Chem 2013 Nov 26;288(46):33398-410. Epub 2013 Sep 26.

From the Laboratories of Molecular Physiology and.

Nonmuscle myosin IIs (NM IIs) are a group of molecular motors involved in a wide variety of cellular processes including cytokinesis, migration, and control of cell morphology. There are three paralogs of the NM II heavy chain in humans (IIA, IIB, and IIC), each encoded by a separate gene. These paralogs are expressed at different levels according to cell type and have different roles and intracellular distributions in vivo. Most previous studies on NM II used tissue-purified protein or expressed fragments of the molecule, which presents potential drawbacks for characterizing individual paralogs of the intact protein in vitro. To circumvent current limitations and approach their native properties, we have successfully expressed and purified the three full-length human NM II proteins with their light chains, using the baculovirus/Sf9 system. The enzymatic and structural properties of the three paralogs were characterized. Although each NM II is capable of forming bipolar filaments, those formed by IIC tend to contain fewer constituent molecules than those of IIA and IIB. All paralogs adopt the compact conformation in the presence of ATP. Phosphorylation of the regulatory light chain leads to assembly into filaments, which bind to actin in the presence of ATP. The nature of interactions with actin filaments is shown with different paralogs exhibiting different actin binding behaviors under equivalent conditions. The data show that although NM IIA and IIB form filaments with similar properties, NM IIC forms filaments that are less well suited to roles such as tension maintenance within the cell.
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http://dx.doi.org/10.1074/jbc.M113.499848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3829186PMC
November 2013

Visualization of human Bloom's syndrome helicase molecules bound to homologous recombination intermediates.

FASEB J 2013 Dec 4;27(12):4954-64. Epub 2013 Sep 4.

3Department of Biochemistry, Eötvös University, Pázmány P. s. 1/c, H-1117 Budapest, Hungary.

Homologous recombination (HR) is a key process in the repair of double-stranded DNA breaks (DSBs) that can initiate cancer or cell death. Human Bloom's syndrome RecQ-family DNA helicase (BLM) exerts complex activities to promote DSB repair while avoiding illegitimate HR. The oligomeric assembly state of BLM has been a key unresolved aspect of its activities. In this study we assessed the structure and oligomeric state of BLM, in the absence and presence of key HR-intermediate DNA structures, by using single-molecule visualization (electron microscopic and atomic force microscopic single-particle analysis) and solution biophysical (dynamic light scattering, kinetic and equilibrium binding) techniques. Besides full-length BLM, we used a previously characterized truncated construct (BLM(642-1290)) as a monomeric control. Contrary to previous models proposing a ring-forming oligomer, we found the majority of BLM molecules to be monomeric in all examined conditions. However, BLM showed a tendency to form dimers when bound to branched HR intermediates. Our results suggest that HR activities requiring single-stranded DNA translocation are performed by monomeric BLM, while complex DNA structures encountered and dissolved by BLM in later stages of HR induce partial oligomerization of the helicase.
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http://dx.doi.org/10.1096/fj.13-234088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3834772PMC
December 2013

Silica encapsulation of fluorescent nanodiamonds for colloidal stability and facile surface functionalization.

J Am Chem Soc 2013 May 22;135(21):7815-8. Epub 2013 Apr 22.

Laboratory of Molecular Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, 50 South Drive, Building 50, Room 3517, Bethesda, Maryland 20892, USA.

Fluorescent nanodiamonds (FNDs) emit in the near-IR and do not photobleach or photoblink. These properties make FNDs better suited for numerous imaging applications compared with commonly used fluorescence agents such as organic dyes and quantum dots. However, nanodiamonds do not form stable suspensions in aqueous buffer, are prone to aggregation, and are difficult to functionalize. Here we present a method for encapsulating nanodiamonds with silica using an innovative liposome-based encapsulation process that renders the particle surface biocompatible, stable, and readily functionalized through routine linking chemistries. Furthermore, the method selects for a desired particle size and produces a monodisperse agent. We attached biotin to the silica-coated FNDs and tracked the three-dimensional motion of a biotinylated FND tethered by a single DNA molecule with high spatial and temporal resolution.
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http://dx.doi.org/10.1021/ja4016815DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3689586PMC
May 2013

Perrault syndrome is caused by recessive mutations in CLPP, encoding a mitochondrial ATP-dependent chambered protease.

Am J Hum Genet 2013 Apr 28;92(4):605-13. Epub 2013 Mar 28.

Centre for Genetic Medicine, Institute of Human Development, Faculty of Medical and Human Sciences, University of Manchester and Central Manchester University Hospitals NHS Foundation Trust as part of Manchester Academic Health Science Centre, Manchester M13 9WL, UK.

Perrault syndrome is a genetically and clinically heterogeneous autosomal-recessive condition characterized by sensorineural hearing loss and ovarian failure. By a combination of linkage analysis, homozygosity mapping, and exome sequencing in three families, we identified mutations in CLPP as the likely cause of this phenotype. In each family, affected individuals were homozygous for a different pathogenic CLPP allele: c.433A>C (p.Thr145Pro), c.440G>C (p.Cys147Ser), or an experimentally demonstrated splice-donor-site mutation, c.270+4A>G. CLPP, a component of a mitochondrial ATP-dependent proteolytic complex, is a highly conserved endopeptidase encoded by CLPP and forms an element of the evolutionarily ancient mitochondrial unfolded-protein response (UPR(mt)) stress signaling pathway. Crystal-structure modeling suggests that both substitutions would alter the structure of the CLPP barrel chamber that captures unfolded proteins and exposes them to proteolysis. Together with the previous identification of mutations in HARS2, encoding mitochondrial histidyl-tRNA synthetase, mutations in CLPP expose dysfunction of mitochondrial protein homeostasis as a cause of Perrault syndrome.
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http://dx.doi.org/10.1016/j.ajhg.2013.02.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3617381PMC
April 2013

Mammalian myosin-18A, a highly divergent myosin.

J Biol Chem 2013 Mar 4;288(13):9532-48. Epub 2013 Feb 4.

Laboratory of Molecular Physiology, NHLBI, National Institutes of Health, Bethesda, MD 20892-8015, USA.

The Mus musculus myosin-18A gene is expressed as two alternatively spliced isoforms, α and β, with reported roles in Golgi localization, in maintenance of cytoskeleton, and as receptors for immunological surfactant proteins. Both myosin-18A isoforms feature a myosin motor domain, a single predicted IQ motif, and a long coiled-coil reminiscent of myosin-2. The myosin-18Aα isoform, additionally, has an N-terminal PDZ domain. Recombinant heavy meromyosin- and subfragment-1 (S1)-like constructs for both myosin-18Aα and -18β species were purified from the baculovirus/Sf9 cell expression system. These constructs bound both essential and regulatory light chains, indicating an additional noncanonical light chain binding site in the neck. Myosin-18Aα-S1 and -18Aβ-S1 molecules bound actin weakly with Kd values of 4.9 and 54 μm, respectively. The actin binding data could be modeled by assuming an equilibrium between two myosin conformations, a competent and an incompetent form to bind actin. Actin binding was unchanged by presence of nucleotide. Both myosin-18A isoforms bound N-methylanthraniloyl-nucleotides, but the rate of ATP hydrolysis was very slow (<0.002 s(-1)) and not significantly enhanced by actin. Phosphorylation of the regulatory light chain had no effect on ATP hydrolysis, and neither did the addition of tropomyosin or of GOLPH3, a myosin-18A binding partner. Electron microscopy of myosin-18A-S1 showed that the lever is strongly angled with respect to the long axis of the motor domain, suggesting a pre-power stroke conformation regardless of the presence of ATP. These data lead us to conclude that myosin-18A does not operate as a traditional molecular motor in cells.
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http://dx.doi.org/10.1074/jbc.M112.441238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3611021PMC
March 2013

Kinetic characterization of nonmuscle myosin IIb at the single molecule level.

J Biol Chem 2013 Jan 12;288(1):709-22. Epub 2012 Nov 12.

Laboratory of Molecular Physiology, HLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.

Nonmuscle myosin IIB (NMIIB) is a cytoplasmic myosin, which plays an important role in cell motility by maintaining cortical tension. It forms bipolar thick filaments with ~14 myosin molecule dimers on each side of the bare zone. Our previous studies showed that the NMIIB is a moderately high duty ratio (~20-25%) motor. The ADP release step (~0.35 s(-1)) of NMIIB is only ~3 times faster than the rate-limiting phosphate release (0.13 ± 0.01 s(-1)). The aim of this study was to relate the known in vitro kinetic parameters to the results of single molecule experiments and to compare the kinetic and mechanical properties of single- and double-headed myosin fragments and nonmuscle IIB thick filaments. Examination of the kinetics of NMIIB interaction with actin at the single molecule level was accomplished using total internal reflection fluorescence (TIRF) with fluorescence imaging with 1-nm accuracy (FIONA) and dual-beam optical trapping. At a physiological ATP concentration (1 mm), the rate of detachment of the single-headed and double-headed molecules was similar (~0.4 s(-1)). Using optical tweezers we found that the power stroke sizes of single- and double-headed heavy meromyosin (HMM) were each ~6 nm. No signs of processive stepping at the single molecule level were observed in the case of NMIIB-HMM in optical tweezers or TIRF/in vitro motility experiments. In contrast, robust motility of individual fluorescently labeled thick filaments of full-length NMIIB was observed on actin filaments. Our results are in good agreement with the previous steady-state and transient kinetic studies and show that the individual nonprocessive nonmuscle myosin IIB molecules form a highly processive unit when polymerized into filaments.
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http://dx.doi.org/10.1074/jbc.M112.424671DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3537070PMC
January 2013

Dynein struts its stuff.

Nat Struct Mol Biol 2011 Jun;18(6):635-6

National Heart, Lung, and Blood Institute, U.S. National Institutes of Health, Bethesda, Maryland, USA.

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http://dx.doi.org/10.1038/nsmb.2082DOI Listing
June 2011

Role of the tail in the regulated state of myosin 2.

J Mol Biol 2011 May 23;408(5):863-78. Epub 2011 Mar 23.

Institute of Molecular and Cellular Biology and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.

Myosin 2 from vertebrate smooth muscle or non-muscle sources is in equilibrium between compact, inactive monomers and thick filaments under physiological conditions. In the inactive monomer, the two heads pack compactly together, and the long tail is folded into three closely packed segments that are associated chiefly with one of the heads. The molecular basis of the folding of the tail remains unexplained. By using electron microscopy, we show that compact monomers of smooth muscle myosin 2 have the same structure in both the native state and following specific, intramolecular photo-cross-linking between Cys109 of the regulatory light chain (RLC) and segment 3 of the tail. Nonspecific cross-linking between lysine residues of the folded monomer by glutaraldehyde also does not perturb the compact conformation and stabilizes it against unfolding at high ionic strength. Sequence comparisons across phyla and myosin 2 isoforms suggest that the folding of the tail is stabilized by ionic interactions between the positively charged N-terminal sequence of the RLC and a negatively charged region near the start of tail segment 3 and that phosphorylation of the RLC could perturb these interactions. Our results support the view that interactions between the heads and the distal tail perform a critical role in regulating activity of myosin 2 molecules through stabilizing the compact monomer conformation.
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http://dx.doi.org/10.1016/j.jmb.2011.03.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3776433PMC
May 2011

The kinetic mechanism of mouse myosin VIIA.

J Biol Chem 2011 Mar 6;286(11):8819-28. Epub 2011 Jan 6.

Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, Virginia 23507, USA.

Myosin VIIa is crucial in hearing and visual processes. We examined the kinetic and association properties of the baculovirus expressed, truncated mouse myosin VIIa construct containing the head, all 5IQ motifs and the putative coiled coil domain (myosin VIIa-5IQ). The construct appears to be monomeric as determined by analytical ultracentrifugation experiments, and only single headed molecules were detected by negative stain electron microscopy. The relatively high basal steady-state rate of 0.18 s(-1) is activated by actin only by ∼3.5-fold resulting in a V(max) of 0.7 s(-1) and a K(ATPase) of 11.5 μM. There is no single rate-limiting step of the ATP hydrolysis cycle. The ATP hydrolysis step (M·T M·D·P) is slow (12 s(-1)) and the equilibrium constant (K(H)) of 1 suggests significant reversal of hydrolysis. In the presence of actin ADP dissociates with a rate constant of 1.2 s(-1). Phosphate dissociation is relatively fast (>12 s(-1)), but the maximal rate could not be experimentally obtained at actin concentrations ≤ 50 μM because of the weak binding of the myosin VIIa-ADP-P(i) complex to actin. At higher actin concentrations the rate of attached hydrolysis (0.4 s(-1)) becomes significant and partially rate-limiting. Our findings suggest that the myosin VIIa is a "slow", monomeric molecular motor with a duty ratio of 0.6.
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http://dx.doi.org/10.1074/jbc.M110.163592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3059067PMC
March 2011

Myosin complexed with ADP and blebbistatin reversibly adopts a conformation resembling the start point of the working stroke.

Proc Natl Acad Sci U S A 2010 Apr 29;107(15):6799-804. Epub 2010 Mar 29.

Department of Biochemistry, Eötvös University, H-1117 Budapest, Pázmány Peter Setany 1/C, Hungary.

The powerstroke of the myosin motor is the basis of cell division and bodily movement, but has eluded empirical description due to the short lifetime and low abundance of intermediates during force generation. To gain insight into this process, we used well-established single-tryptophan and pyrene fluorescent sensors and electron microscopy to characterize the structural and kinetic properties of myosin complexed with ADP and blebbistatin, a widely used inhibitor. We found that blebbistatin does not weaken the tight actin binding of myosin.ADP, but unexpectedly it induces lever priming, a process for which the gamma-phosphate of ATP (or its analog) had been thought necessary. The results indicate that a significant fraction of the myosin.ADP.blebbistatin complex populates a previously inaccessible conformation of myosin resembling the start of the powerstroke.
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http://dx.doi.org/10.1073/pnas.0907585107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2872372PMC
April 2010

Conservation of the regulated structure of folded myosin 2 in species separated by at least 600 million years of independent evolution.

Proc Natl Acad Sci U S A 2008 Apr 14;105(16):6022-6. Epub 2008 Apr 14.

Institute of Molecular and Cellular Biology and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.

The myosin 2 family of molecular motors includes isoforms regulated in different ways. Vertebrate smooth-muscle myosin is activated by phosphorylation of the regulatory light chain, whereas scallop striated adductor-muscle myosin is activated by direct calcium binding to its essential light chain. The paired heads of inhibited molecules from myosins regulated by phosphorylation have an asymmetric arrangement with motor-motor interactions. It was unknown whether such interactions were a common motif for inactivation used in other forms of myosin-linked regulation. Using electron microscopy and single-particle image processing, we show that indistinguishable structures are indeed found in myosins and heavy meromyosins isolated from scallop striated adductor muscle and turkey gizzard smooth muscle. The similarities extend beyond the shapes of the heads and interactions between them: In both myosins, the tail folds into three segments, apparently at identical sites; all three segments are in close association outside the head region; and two segments are associated in the same way with one head in the asymmetric arrangement. Thus, these organisms, which have different regulatory mechanisms and diverged from a common ancestor >600 Myr ago, have the same quaternary structure. Conservation across such a large evolutionary distance suggests that this conformation is of fundamental functional importance.
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http://dx.doi.org/10.1073/pnas.0707846105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2329715PMC
April 2008