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P D Chantler

Publications and source records attributed to P D Chantler.

At least 19 recordsLinked to original sources
PubMed2000

Locking regulatory myosin in the off-state with trifluoperazine.

Scallop striated adductor muscle myosin is a regulatory myosin, its activity being controlled directly through calcium binding. Here, we show that millimolar concentrations of trifluoperazine were effective at removal of all regulatory light chains from scallop myosin or myofibrils. More important, 200 microM trifluoperazine, a concentration 10-fold less than that required for light-chain removal, resulted in the reversible elimination of actin-activated and intrinsic ATPase activities. Unlike desensitization induced by metal ion chelation, which leads to an elevation of activity in the absence of calcium concurrent with regulatory light-chain removal, trifluoperazine caused a decline in actin-activated MgATPase activity both in the presence and absence of calcium. Procedures were equally effective with respect to scallop myosin, myofibrils, subfragment-1, or desensitized myofibrils. Increased alpha-helicity could be induced in the isolated essential light chain through addition of 100-200 microM trifluoperazine. We propose that micromolar concentrations of trifluoperazine disrupt regulation by binding to a single high-affinity site located in the C-terminal domain of the essential light chain, which locks scallop myosin in a conformation resembling the off-state. At millimolar trifluoperazine concentrations, additional binding sites on both light chains would be filled, leading to regulatory light-chain displacement.

Adenosine Triphosphatases↗
PubMed1999

Myofibrillar protein structure and assembly during idiopathic dilated cardiomyopathy.

A neutral protease, mekratin, active in human hearts at end stage idiopathic dilated cardiomyopathy (IDC), mediates the breakdown of cardiac myosin LC2. Myosin purified from IDC heart tissue forms unusually short synthetic thick filaments. Therefore, determination of filament length and mekratin distribution in IDC heart muscle were initiated. Native thick filaments were prepared directly from control and IDC tissues and analyzed. Also, paraffin-embedded tissue sections were stained with a fluorescently-labeled anti-protease antibody to establish its distribution in myocardial tissues. Control sections had only very weak, background levels of fluorescence whereas IDC sections stained intensely throughout, indicating a wide ranging distribution of the protease within the myocyte cytoplasm. SDS-PAGE revealed LC2 to be present in stoichiometric amounts in control but greatly reduced in IDC heart muscle. Native thick filaments from control myocardium were structurally stable. They had a median length of 1.65 microm with well-defined bare zones and displayed the 43 nm helical periodicity typical of the relaxed arrangement of myosin heads close to the filaments' shafts. In contrast, native IDC filaments were less stable, and had a median length of 0.9 microm. These filaments were highly disordered: they had no surface periodicity and myosin heads were positioned away from the filaments' shafts. The shorter, less stable, aperiodic thick filaments from IDC hearts appear to result from depletion of LC2 caused by increased activity of mekratin in the IDC myocardium.

Actin Cytoskeleton↗
PubMed1999

Calcium regulation in the human myocardium affected by dilated cardiomyopathy: a structural basis for impaired Ca2+-sensitivity.

Calcium regulation in the human heart is impaired during idiopathic dilated cardiomyopathy (IDC). Here, we analyze the structural basis for impairment in the regulatory mechanism. Regulation of contractility was monitored by MgATPase and Ca2+-binding assays as a function of calcium. Myofibrillar proteolysis and expression of troponin T isoforms were established by gel electrophoresis and by Western blots. Myofibrillar ATPase assays in low salt however, revealed a drastic lowering of calcium sensitivity in IDC myofibrils as indicated by reductions in both activation by high calcium and in EGTA-mediated inhibition of MgATPase. Structural changes in myofilament proteins were found in most IDC hearts, specifically proteolysis of myosin light chain 2 (LC2), troponin T and I (TnT and TnI), and sometimes a large isoform shift in TnT. IDC did not induce mutations in LC2 and troponin C (TnC), as established by cDNA sequence data from IDC cases, thus, calcium binding to IDC myofibrils was unaffected. Reassociation of IDC myofibrils with native LC2 raised MgATPase activation at high Ca2+ to control levels, while repletion with intact, canine TnI/TnT restored inhibition at low Ca2+. A model, identifying possible steps in the steric blocking mechanism of regulation, is proposed to explain IDC-induced changes in Ca2+-regulation. Moreover, shifts in TnT isoforms may imply either a genetic or a compensatory factor in the development and pathogenesis of some forms of IDC.

Adenosine Triphosphatases↗
PubMed1998

A conventional myosin motor drives neurite outgrowth.

Neuritic outgrowth is a striking example of directed motility, powered through the actions of molecular motors. Members of the myosin superfamily of actin-associated motors have been implicated in this complex process. Although conventional myosin II is known to be present in neurons, where it is localized at the leading edge of growth cones and in the cell cortex close to the plasma membrane, its functional involvement in growth cone motility has remained unproven. Here, we show that antisense oligodeoxyribonucleotides, complementary to a specific isoform of conventional myosin (myosin IIB), attenuate filopodial extension whereas sense and scrambled control oligodeoxyribonucleotides have no effect. Attenuation is shown to be reversible, neurite outgrowth being restored after cessation of the antisense regimen. Myosin IIB mRNA was present during active neurite extension, but levels were minimal in phenotypically rounded cells before neurite outgrowth and message levels decreased during antisense treatment. By contrast, the myosin IIA isoform is shown to be expressed constitutively both before and during neurite outgrowth and throughout exposure to myosin IIB antisense oligodeoxyribonucleotides. These results provide direct evidence that a conventional two-headed myosin is required for growth cone motility and is responsible, at least in part, for driving neuritic process outgrowth.

Animals↗
PubMed1995

Human cardiac myosin light chains: sequence comparisons between myosin LC1 and LC2 from normal and idiopathic dilated cardiomyopathic hearts.

The primary structures of light chains isolated from the human myocardium with idiopathic dilated cardiomyopathy (IDC) were determined and compared with the sequence structures of myosin light chains obtained from control human heart myosin. Sequences were determined by chemical analysis and the identity of N-terminal residues established by mass spectrometry. The N-terminal residues in essential (ELC) and regulatory (RLC) light chains were blocked and were identified to be trimethyl alanine. The amino acid sequences of ELC and RLC from control human myosin revealed a high degree of homology with those purified from rat and chicken cardiac myosin. Comparison with a published partial chemical sequence of the human heart myosin light chains revealed significant variations. However, there was very good agreement with published sequences obtained by molecular biological techniques. Sequences of the light chains from cardiomyopathic myosin revealed no difference in the primary structures when compared with control human heart myosin light chains indicating IDC had no influence on, nor was caused by, altered myosin light chain gene expression.

Amino Acid Sequence↗
PubMed1995

Cloning and in vivo expression of the pig MyoD gene.

The complete sequence of the pig MyoD gene has been determined through the isolation and characterization of a 17 kb genomic clone. The deduced amino acid sequence shows relative conservation of about 90% with the MyoD sequences determined in other mammalian species, though there are several non-conservative changes scattered throughout the C-terminus. In situ hybridization on pig embryos (20-30 days post coitum) showed that MyoD expression was confined to myotomes and skeletal muscle masses.

Amino Acid Sequence↗
PubMed1994

Functional analysis of individual brain myosin II isoforms through hybrid formation.

We have used a scallop hybrid myosin test system in an attempt to determine the regulatory properties of an individual myosin II isoform from rat brain. The complete coding region of cDNA corresponding to a regulatory light chain isoform previously shown to be expressed in brain [Feinstein, Durand and Milner (1991) Mol. Brain Res. 10, 97-105] was ligated within the prokaryotic expression vector, pAED4, overexpressed in bacteria, and the purified light chain incorporated within a scallop hybrid myosin. Actin activation was calcium insensitive for all hybrids tested, irrespective of whether light chain phosphorylation had taken place before, or subsequent to, hybrid formation. We discuss the implications of these results, including the possibility that these results constitute evidence for a myosin II isoform within brain that is regulated at the level of the thin filament. In addition, evidence is presented for the presence of an additional, novel isoform of regulatory light chain expressed in rat brain.

Amino Acid Sequence↗
PubMed1994

Association of a cellular myosin II with anionic phospholipids and the neuronal plasma membrane.

Myosin II has been observed in close proximity to the neuronal plasma membrane, suggesting the possibility that at least one isoform of neuronal myosin II may be capable of direct association. Here, we demonstrate that a significant fraction (> 30%, saturable around 90%) of brain myosin II, but not myosins from skeletal or cardiac muscle, can bind to lipid vesicles composed of the anionic phospholipid L-alpha-phosphatidyl-L-serine but not with vesicles made from the neutral phospholipid L-alpha-phosphatidylcholine. Binding to lipid vesicles made from L-alpha-phosphatidyl-L-serine is enhanced in the presence of millimolar amounts of free calcium. ATPase activity remains unimpaired after vesicle association. Myosin II was also shown to remain in tight association with purified plasma membranes, even after depletion of actin. The above observations suggest that mechanisms involving membrane-bound myosin II are required to facilitate metazoan cell motility.

Animals↗
PubMed1992

Regulatory light-chain Cys-55 sites on the two heads of myosin can come within 2A of each other.

The di-thiol reagent, 5,5'-dithiobis (2-nitrobenzoic acid) is shown to induce disulfide bond formation between Mercenaria regulatory light-chain Cys-55 sites on either head of scallop hybrid myosin. This indicates that these two sites on opposite heads of myosin can come within 2A of each other and this confirms a prediction based on earlier data [Chantler, Tao and Stafford (1991) Biophys. J. 59, 1242-1250]. Results demonstrate that myosin heads in solution show a considerable mutual freedom of movement which can be monitored by probes in the vicinity of regulatory light-chain residue 55. Implications for light-chain movement on the myosin head are discussed.

Animals↗
PubMed1992

Cloning of the cDNA encoding a neuronal myosin heavy chain from mammalian brain and its differential expression within the central nervous system.

The complete amino acid sequence of a neuronal myosin heavy chain (MHC) from mammalian brain (1999 amino acids, 230 kDa) has been deduced by sequencing cDNA clones isolated from a rat brain cDNA library. The library was screened using an affinity-purified polyclonal antibody that had been raised against myosin purified from a neuronally-derived cell line (Neuro-2A). Restriction digests of genomic DNA from Neuro-2A cells and rat brain are consistent with an identity of the sequenced isoform from these two sources. RNA blot analysis demonstrates this myosin to exhibit differential expression within the cerebral cortex and spinal cord. No expression was observed in liver, kidney, heart, spleen or skeletal muscle, or even within other regions of the brain. The sequence of this neuronal MHC is compared with those of other non-muscle MHCs, to which it shows an overall similarity of structure, especially with respect to conserved regions within the head (ATP binding site, actin binding site, reactive thiols) and the presence of an alpha-helical coiled-coil tail that can be arranged as 28-residue repeating units plus four skip residues. A unique non-helical tailpiece composed of 72 amino acid residues marks the C-terminus of this neuronal myosin isoform.

Amino Acid Sequence↗
PubMed1992

Myosin II distribution in neurons is consistent with a role in growth cone motility but not synaptic vesicle mobilization.

We have generated a polyclonal antibody against myosin II from a neuronally derived cell line in order to assess potential roles for myosin II in growth cone movement and synaptic transmission. The distribution of neuronal myosin II, in isolated cells as well as in tissues of the adult rat brain and spinal cord, was examined at the light microscopic and ultrastructural levels. In isolated neuroblastoma cells and dorsal root ganglion neurons, myosin II was found at the leading edge of growth cones, within neuritic processes and cell soma, and adjacent to the plasma membrane. The subcellular distribution of myosin II overlapped significantly with that of both actin and single-headed myosin I. These results implicate both myosin I and myosin II as molecular motors required for neurite elongation and growth cone motility. An exclusive postsynaptic distribution of myosin II in neurons of the mature central nervous system suggests that myosin II cannot play a role in the mobilization of synaptic vesicles, but could participate in synaptic plasticity.

Animals↗
PubMed1992

Evidence for a new member of the myosin I family from mammalian brain.

Myosin I is an actin-based motor responsible for powering a wide variety of motile activities in amebae and slime molds and has been found previously in vertebrates as the lateral bridges within intestinal epithelial cell microvilli. Although neurons exhibit extensive cellular and intracellular motility, including the production of ameboid-like growth cones during development, the proteins responsible for the motor in these processes are unknown. Here, we report the isolation of a partially purified protein fraction from bovine brain that is enriched for a 150-kDa protein; immunochemical and biochemical analyses suggest that this protein possesses a number of functional properties that have been ascribed to myosin I from various sources. These properties include an elevated K(+)-EDTA ATPase, a modest actin-activated Mg(2+)-ATPase, the ability to bind calmodulin, and a ready association with phospholipid vesicles made from phosphatidylserine, but not from phosphatidylcholine. The combination of these properties, together with a molecular mass of 150 kDa (most myosin I molecules found to date have molecular masses in the range 110-130 kDa) yet recognition by an anti-myosin I antibody, suggests the presence of a new member of the myosin I family within mammalian brain.

Actins↗
PubMed1991

Proximity relationships between sites on myosin and actin. Resonance energy transfer determination of the following distances, using a hybrid myosin: those between Cys-55 on the Mercenaria regulatory light chain, SH-1 on the Aequipecten myosin heavy chain, and Cys-374 of actin.

Resonance energy transfer measurements have been made on hybrid myosins in order to map distances between sites on the regulatory light chain, heavy chain, and actin as well as to assess potential conformational changes of functional importance. Using scallop (Aequipecten) myosin hybrid molecules possessing clam (Mercenaria) regulatory light chains, we have been able to map the distance between Cys-55 on the regulatory light chain and the fast-reacting thiol on the myosin heavy chain (SH-1). This distance is shown to be approximately 6.4 nm, and it is not altered by the presence or absence of Ca2+, MgATP, or actin. Experiments performed at low ionc strength on heavy meromyosin (HMM) derived from these hybrid myosins gave results similar to those performed on the soluble parent myosin preparations. The distances between Cys-374 on actin and each of the above sites were also measured. Mercenaria regulatory light-chain Cys-55, within the hybrid myosin molecule, was found to be greater than 8.0 nm away from actin Cys-374. Scallop heavy-chain SH-1 is shown to be approximately 4.5 nm away from actin Cys-374, in broad agreement with earlier measurements made by others in nonregulatory myosins. The significance of our results is discussed with respect to putative conformational changes within the region of the heavy chain connecting SH-1 to the N-terminal region of the light chain.

Actins↗
PubMed1991

A unique cellular myosin II exhibiting differential expression in the cerebral cortex.

Clones possessing inserts of brain myosin II have been obtained by screening a rat brain cDNA expression library with a polyclonal antibody, raised against myosin II from the mouse neuroblastoma cell line, Neuro-2A. A partial sequence comprising the 3' coding and non-coding regions of the myosin message has been determined which is markedly different from other myosin sequences. The derived amino-acid sequence comprises the C-terminal 90 amino acids: VSS(PO4)LKNKLRRGDLPFVVTRRLVRKGTLELS(PO4)DDDDESKASLINETQPPQCLDQQ LDQQ LDQLFNWPVNAGCVCGWGVEQTQGEEAVHKCRT(CO2H). This sequence encompasses regions homologous to both the casein kinase II and protein kinase C heavy-chain phosphorylation sites. The non-helical "tail-piece" is considerably longer (an additional 39 amino acid residues) than found in other myosins. Northern blot analysis demonstrates this myosin II message to be unique to cerebral cortex, with no expression in all other non-cortical brain regions and peripheral tissues tested. Our results suggest functional diversity for myosin II isozymes within the brain.

Amino Acid Sequence↗
PubMed1991

On the relationship between distance information derived from cross-linking and from resonance energy transfer, with specific reference to sites located on myosin heads.

The techniques of fluorescence resonance energy transfer (FRET) and cross-linking can provide complementary information concerning the relative separation of a pair of sites. Cross-linking experiments provide an assessment of the distance of closest approach between a pair of sites. FRET measurements, by contrast, yield information about the average distance between the pair of sites. We have taken advantage of hybrid myosins to understand the relationship between distances obtained for a pair of equivalent sites, one on each myosin head, using both FRET (steady-state and time-decay) and cross-linking techniques. The rigid cross-linker, 4-4'-dimaleimidyl-stilbene-2-2'-disulfonic acid (DMSDS), can efficiently cross-link the two myosin regulatory light-chains, each at residue Cys50 of the Mercenaria regulatory light chain (Chantler, P.D., and S. M. Bower. 1988. J. Biol. Chem. 263:938-944), indicating that these sites can come within 18 +/- 2 A of each other. In a complementary set of experiments, steady-state and time-decay measurements using fluorescence donor/acceptor pairs located at these same sites indicate transfer efficiencies of somewhat less than 20%, suggesting an average separation of greater than 50 A between sites (Chantler, P. D., and T. Tao. 1986. J. Mol. Biol. 192:87-99). Here, we present theoretical calculations which show that efficient cross-linking can be achieved readily in dynamic systems such as the heads of myosin, even though the necessary subpopulation of proximate molecules at any instant may be below the detection limits of time-decay-FRET. Therefore, cross-linking experiments can provide important ancillary information about the extent of motions within a marcomolecular system when used in conjunction with FRET.As a corollary, demonstration of extensive cross-linking does not necessarily indicate a static proximity; the mean separation distance should be ascertained by other methods such as FRET.

Binding Sites↗