Publications by authors named "Peter D Chantler"

13 Publications

  • Page 1 of 1

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

Myosin VI and Associated Proteins Are Expressed in Human Macrophages but Do Not Play a Role in Foam Cell Formation in THP-1 Cells.

Int J Vasc Med 2013 9;2013:516015. Epub 2013 Jun 9.

Department of Comparative Biomedical Sciences, Royal Veterinary College, Royal College Street, London NW1 0TU, UK.

Myosin VI (Myo6) functions in endocytosis in conjunction with binding partners including adaptor protein (AP)-2, disabled 2 (Dab2), and GAIP interacting protein C terminus 1 (GIPC1). This study aimed to investigate the expression and function of Myo6 in macrophages and its possible role in the endocytosis of lipoproteins during the induction of foam cell formation. Expression of Myo6, AP-2 ( α 2 subunit), and Dab2 in THP-1 macrophages and primary human monocyte-derived macrophages was demonstrated at the mRNA and protein level, but GIPC1 was only detected at the mRNA level. Immunofluorescence showed that Myo6 was distributed similarly to F-actin in both macrophage types. AP-2 α 2 was found to have a similar subcellular distribution to Myo6 and Dab2 in THP-1 cells. Myo6 was located within membrane ruffles and protrusions of the plasma membrane. These results suggest that in macrophages Myo6 is required for several functions including cell adhesion, cell progression, and macropinocytosis. Low-density lipoprotein (LDL) and oxidised LDL (oxLDL) decreased Myo6 and GIPC1 mRNA expression in THP-1 cells, but uptake of the fluorescence-labelled lipoproteins was unaffected by knockdown of the expression of Myo6 or associated proteins with siRNA. Our findings, therefore, do not support the idea that Myo6 plays a major role in foam cell formation.
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http://dx.doi.org/10.1155/2013/516015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3690746PMC
July 2013

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

Conventional myosins - unconventional functions.

Biophys Rev 2010 May 9;2(2):67-82. Epub 2010 Mar 9.

Unit of Molecular and Cellular Biology, Royal Veterinary College, University of London, Royal College Street, London, NW1 0TU, UK.

While the discovery of unconventional myosins raised expectations that their actions were responsible for most aspects of actin-based cell motility, few anticipated the wide range of cellular functions that would remain the purview of conventional two-headed myosins. The three nonsarcomeric, cellular myosins-M2A, M2B and M2C-participate in diverse roles including, but not limited to: neuronal dynamics, axon guidance and synaptic transmission; endothelial cell migration; cell adhesion, polarity, fusion and cytokinesis; vesicle trafficking and viral egress. These three conventional myosins each take on specific, differing functional roles during development and maturity, characteristic of each cell lineage; exact roles depend on the developmental stage of the cell, cellular location, upstream regulatory controls, relative isoform expression, orientation and associated state of the actin cytoscaffolds in which these myosins operate. Here, we discuss the separate yet related roles that characterise the actions of M2A, M2B and M2C in various cell types and show that these conventional myosins are responsible for functions as unconventional as any performed by unconventional myosins.
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http://dx.doi.org/10.1007/s12551-010-0030-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5425674PMC
May 2010

Relocation of myosin and actin, kinesin and tubulin in the acrosome reaction of bovine spermatozoa.

Reprod Fertil Dev 2009 ;21(2):364-77

Royal Veterinary College, Royal College Street, London NW1 0TU, UK.

The mammalian acrosome reaction is a specialised exocytotic event. Although molecular motors are known to be involved in exocytosis in many cell types, their potential involvement in the acrosome reaction has remained unknown. Here, it has been shown that actin is localised within the equatorial segment and in the marginal acrosomal ridge of the heads of unreacted bull spermatozoa. Myosins IIA and IIB are found within the anterior acrosomal margins of virtually all sperm cells and, less prominently, within the equatorial segment. Tubulin was detected in the equatorial segment and around the periphery of the acrosome while kinesin was prominent in the equatorial segment. After induction of the acrosome reaction by means of the calcium ionophore A23187, the number of cells exhibiting actin fluorescence intensity in the anterior acrosomal margin decreased four-fold and those displaying equatorial segment fluorescence decreased 3.5-fold; myosin IIA immunofluorescence decreased in intensity with most spermatozoa losing equatorial staining, whereas there was little change in the distribution or intensity of myosin IIB immunofluorescence, except for approximately 20% decrease in the number of cells exhibiting acrosomal staining. Tubulin became largely undetectable within the head and kinesin staining spread rostrally over the main acrosome region. A possible sequence of events that ties in these observations of molecular motor involvement with the known participation of SNARE proteins is provided.
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http://dx.doi.org/10.1071/rd08166DOI Listing
April 2009

Myosin IIC: a third molecular motor driving neuronal dynamics.

Mol Biol Cell 2008 Sep 9;19(9):3956-68. Epub 2008 Jul 9.

Unit of Molecular and Cellular Biology, Royal Veterinary College, University of London, London NW1 0TU, United Kingdom.

Neuronal dynamics result from the integration of forces developed by molecular motors, especially conventional myosins. Myosin IIC is a recently discovered nonsarcomeric conventional myosin motor, the function of which is poorly understood, particularly in relation to the separate but coupled activities of its close homologues, myosins IIA and IIB, which participate in neuronal adhesion, outgrowth and retraction. To determine myosin IIC function, we have applied a comparative functional knockdown approach by using isoform-specific antisense oligodeoxyribonucleotides to deplete expression within neuronally derived cells. Myosin IIC was found to be critical for driving neuronal process outgrowth, a function that it shares with myosin IIB. Additionally, myosin IIC modulates neuronal cell adhesion, a function that it shares with myosin IIA but not myosin IIB. Consistent with this role, myosin IIC knockdown caused a concomitant decrease in paxillin-phospho-Tyr118 immunofluorescence, similar to knockdown of myosin IIA but not myosin IIB. Myosin IIC depletion also created a distinctive phenotype with increased cell body diameter, increased vacuolization, and impaired responsiveness to triggered neurite collapse by lysophosphatidic acid. This novel combination of properties suggests that myosin IIC must participate in distinctive cellular roles and reinforces our view that closely related motor isoforms drive diverse functions within neuronal cells.
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http://dx.doi.org/10.1091/mbc.e07-08-0744DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2526701PMC
September 2008

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

Accessory gene regulator locus of Staphylococcus intermedius.

Infect Immun 2006 May;74(5):2947-56

Department of Veterinary Clinical Sciences, Royal Veterinary College, University of London, United Kingdom.

The accessory gene regulator (agr) locus, a candidate system for the regulation of the production of virulence factors in Staphylococcus intermedius, has been characterized. Using PCR-based genome walking, we have obtained the first complete sequence (3,436 bp) of the accessory gene regulator (agr) gene in this organism. Sequence analysis of the agr gene has identified five open reading frames (ORFs), agrB, agrD, agrC, agrA, and hld. The translated ORF contained amino acid motifs characteristic of the response regulator and histidine protein kinase signal transducer of the classic two-component regulatory system. Sequencing of the agrD PCR products amplified from DNA from 20 different isolates has facilitated detection of genetic variation in the putative autoinducing peptide (AIP) within the agr gene of S. intermedius, revealing the presence of at least three agr specificity groups within this species. Classification of the agr gene from S. intermedius was supported by phylogenetic analysis. Real-time PCR also revealed that the effector molecule of the agr system, RNAIII, was regulated in an autocrine manner in S. intermedius and demonstrated positive correlation with the temporal gene expression patterns of luk and entC. Transcription of RNAIII was also dependent on self secreted cues. Cyclic self and nonself peptides were synthesized on the basis of the novel AIPs produced by S. intermedius, which lack the cysteine necessary to form the thiolactone ring in analogous peptides from Staphylococcus aureus and Staphylococcus epidermidis. Experiments with these synthetic cyclic peptides indicated that self peptides led to up-regulation of RNAIII--findings in support of the assumption that activation of the agr gene is initiated by growth- and species-specific factors generated during bacterial growth.
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http://dx.doi.org/10.1128/IAI.74.5.2947-2956.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1459752PMC
May 2006

Calcium regulates scallop muscle by changing myosin flexibility.

Eur Biophys J 2006 Apr 11;35(4):302-12. Epub 2006 Jan 11.

Division of Physical Biochemistry, MRC National Institute for Medical Research, Mill Hill, NW7 1AA, London, UK.

Muscle myosins are molecular motors that convert the chemical free energy available from ATP hydrolysis into mechanical displacement of actin filaments, bringing about muscle contraction. Myosin cross-bridges exert force on actin filaments during a cycle of attached and detached states that are coupled to each round of ATP hydrolysis. Contraction and ATPase activity of the striated adductor muscle of scallop is controlled by calcium ion binding to myosin. This mechanism of the so-called "thick filament regulation" is quite different to vertebrate striated muscle which is switched on and off via "thin filament regulation" whereby calcium ions bind to regulatory proteins associated with the actin filaments. We have used an optically based single molecule technique to measure the angular disposition adopted by the two myosin heads whilst bound to actin in the presence and absence of calcium ions. This has allowed us to directly observe the movement of individual myosin heads in aqueous solution at room temperature in real time. We address the issue of how scallop striated muscle myosin might be regulated by calcium and have interpreted our results in terms of the structures of smooth muscle myosin that also exhibit thick filament regulation.
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http://dx.doi.org/10.1007/s00249-005-0036-4DOI Listing
April 2006

Nonmuscle myosins IIA and IIB are present in adult motor nerve terminals.

Neuroreport 2005 Aug;16(11):1143-6

Royal Veterinary College, University of London, Royal College Street, London NW1 0TU, UK.

Throughout life, neuromuscular junctions undergo dynamic changes, remodelling occurring through extension and withdrawal of motor nerve terminals in conjunction with changes in the distribution of acetylcholine receptors at the muscle endplate. However, relatively little is known about the fundamental processes by which nerve terminals are remodelled. These dynamic processes are likely to be driven by molecular motors. Previously, we have implicated myosins IIA and IIB as opposing motors influencing neuronal growth cone dynamics. Using confocal microscopy of neuromuscular junction preparations colabelled for myosin II isoforms and nerve terminal or muscle endplate markers, we demonstrate that both myosin IIA and myosin IIB are localized in nerve terminals. We propose roles for these motor proteins in junctional stabilization and destabilization.
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http://dx.doi.org/10.1097/00001756-200508010-00002DOI Listing
August 2005

Trifluoperazine inhibits the MgATPase activity and in vitro motility of conventional and unconventional myosins.

J Muscle Res Cell Motil 2003 ;24(8):579-85

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

Trifluoperazine, a calmodulin antagonist, has recently been shown to inhibit the MgATPase activity of scallop myosin in the absence of light chain dissociation (Patel et al. (2000) J Biol Chem 275: 4880-4888). To investigate the generality of this observation and the mechanism by which it occurs, we have examined the ability of trifluoperazine to inhibit the enzymatic properties of other conventional and unconventional myosins. We show that trifluoperazine can inhibit the actin-activated MgATPase activity of rabbit skeletal muscle myosin II heavy meromyosin (HMM), phosphorylated turkey gizzard smooth muscle myosin II HMM, phosphorylated human nonmuscle myosin IIA HMM and myosin V subfragment-1 (S1). In all cases half maximal inhibition occurred at 50-75 microM trifluoperazine while light chains (myosin II) or calmodulin (myosin V) remained associated with the heavy chains. In vitro motility of all myosins tested was completely inhibited by trifluoperazine. Chymotryptic digestion of baculovirus-expressed myosin V HMM possessing only two calmodulin binding sites yielded a minimal motor fragment with no bound calmodulin. The MgATPase of this fragment was inhibited by trifluoperazine over the same range of concentrations as the S1 fragment of myosin.
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http://dx.doi.org/10.1023/b:jure.0000009969.04562.58DOI Listing
November 2004

Myosin IIA drives neurite retraction.

Mol Biol Cell 2003 Nov 5;14(11):4654-66. Epub 2003 Sep 5.

Unit of Molecular and Cellular Biology, Royal Veterinary College, University of London, London NW1 0TU, United Kingdom.

Neuritic extension is the resultant of two vectorial processes: outgrowth and retraction. Whereas myosin IIB is required for neurite outgrowth, retraction is driven by a motor whose identity has remained unknown until now. Preformed neurites in mouse Neuro-2A neuroblastoma cells undergo immediate retraction when exposed to isoform-specific antisense oligonucleotides that suppress myosin IIB expression, ruling out myosin IIB as the retraction motor. When cells were preincubated with antisense oligonucleotides targeting myosin IIA, simultaneous or subsequent addition of myosin IIB antisense oligonucleotides did not elicit neurite retraction, both outgrowth and retraction being curtailed. Even during simultaneous application of antisense oligonucleotides against both myosin isoforms, lamellipodial spreading continued despite the complete inhibition of neurite extension, indicating an uncoupling of lamellipodial dynamics from movement of the neurite. Significantly, lysophosphatidate- or thrombin-induced neurite retraction was blocked not only by the Rho-kinase inhibitor Y27632 but also by antisense oligonucleotides targeting myosin IIA. Control oligonucleotides or antisense oligonucleotides targeting myosin IIB had no effect. In contrast, Y27632 did not inhibit outgrowth, a myosin IIB-dependent process. We conclude that the conventional myosin motor, myosin IIA, drives neurite retraction.
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http://dx.doi.org/10.1091/mbc.e03-03-0187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC266780PMC
November 2003

Evaluation of the symmetric model for myosin-linked regulation: effect of site-directed mutations in the regulatory light chain on scallop myosin.

Biochem J 2003 Aug;374(Pt 1):89-96

Unit of Molecular and Cellular Biology, Royal Veterinary College, University of London, Royal College Street, London NW1 0TU, UK.

Regulatory myosins are controlled through mechanisms intrinsic to their structures and can alternate between activated and inhibited states. However, the structural difference between these two states is unclear. Scallop (Pecten maximus) striated adductor myosin is activated directly by calcium. It has been proposed that the two heads of scallop myosin are symmetrically arranged and interact through their regulatory light chains [Offer and Knight (1996) J. Mol. Biol. 256, 407-416], the interface being strengthened in the inhibited state. By contrast, vertebrate smooth-muscle myosin is activated by phosphorylation. Its structure in the inhibited state has been determined from two-dimensional crystalline arrays [Wendt, Taylor, Trybus and Taylor (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 4361-4366] and is asymmetric, requiring no interaction between regulatory light chains. Using site-directed mutagenesis of the scallop regulatory light chain, we have tested the symmetric model for scallop adductor muscle myosin. Specifically, we have made myosin hybrid molecules from scallop (P. maximus) myosin, in which the normal regulatory light chains have been replaced by expressed light chains containing mutations in three residues proposed to participate in the interaction between regulatory light chains. The mutations were R126A (Arg126-->Ala), K130A and E131A; made singly, in pairs or all three together, these mutations were designed to eliminate hydrogen bonding or salt linkages between heads, which are key features of this model. Functional assays to address the competence of these hybrid myosins to bind calcium specifically, to exhibit a calcium-regulated myofibrillar Mg-ATPase and to display calcium-dependent actin sliding were performed. We conclude that the symmetrical model does not describe the inhibited state of scallop regulatory myosin and that an asymmetric structure is a plausible alternative.
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http://dx.doi.org/10.1042/BJ20030404DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1223580PMC
August 2003