Publications by authors named "Thomas P Burghardt"

47 Publications

The frequency-dependent effect of electrical fields on the mobility of intracellular vesicles in astrocytes.

Biochem Biophys Res Commun 2021 01 3;534:429-435. Epub 2020 Dec 3.

Neurology Department, Mayo Clinic, Rochester, MN, USA; Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA. Electronic address:

Slow-wave sleep, defined by low frequency (<4 Hz) electrical brain activity, is a basic brain function affecting metabolite clearance and memory consolidation. The origin of low-frequency activity is related to cortical up and down states, but the underlying cellular mechanism of how low-frequency activities affect metabolite clearance and memory consolidation has remained elusive. We applied electrical stimulation with voltages comparable to in vivo sleep recordings over a range of frequencies to cultured glial astrocytes while monitored the trafficking of GFP-tagged intracellular vesicles using total internal reflection fluorescence microscopy (TIRFM). We found that during low frequency (2 Hz) electrical stimulation the mobility of intracellular vesicle increased more than 20%, but remained unchanged under intermediate (20 Hz) or higher (200 Hz) frequency stimulation. We demonstrated a frequency-dependent effect of electrical stimulation on the mobility of astrocytic intracellular vesicles. We suggest a novel mechanism of brain modulation that electrical signals in the lower range frequencies embedded in brainwaves modulate the functionality of astrocytes for brain homeostasis and memory consolidation. The finding suggests a physiological mechanism whereby endogenous low-frequency brain oscillations enhance astrocytic function that may underlie some of the benefits of slow-wave sleep and highlights possible medical device approach for treating neurological diseases.
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http://dx.doi.org/10.1016/j.bbrc.2020.11.064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8215681PMC
January 2021

Demographic model for inheritable cardiac disease.

Arch Biochem Biophys 2019 09 26;672:108056. Epub 2019 Jul 26.

Department of Biochemistry and Molecular Biology and Physiology and Biomedical Engineering, 200 First St. SW, Mayo Clinic Rochester, Rochester, MN, 55905, USA. Electronic address:

The cardiac muscle proteins, generating and regulating energy transduction during a heartbeat, assemble in the sarcomere into a cyclical machine repetitively translating actin relative to myosin filaments. Myosin is the motor transducing ATP free energy into actin movement against resisting force. Cardiac myosin binding protein C (mybpc3) regulates shortening velocity probably by transient N-terminus binding to actin while its C-terminus strongly binds the myosin filament. Inheritable heart disease associated mutants frequently modify these proteins involving them in disease mechanisms. Nonsynonymous single nucleotide polymorphisms (SNPs) cause single residue substitutions with independent characteristics (sequence location, residue substitution, human demographic, and allele frequency) hypothesized to decide dependent phenotype and pathogenicity characteristics in a feed-forward neural network model. Trial models train and validate on a dynamic worldwide SNP database for cardiac muscle proteins then predict phenotype and pathogenicity for any single residue substitution in myosin, mybpc3, or actin. A separate Bayesian model formulates conditional probabilities for phenotype or pathogenicity given independent SNP characteristics. Neural/Bayes forecasting tests SNP pathogenicity vs (in)dependent SNP characteristics to assess individualized disease risk and in particular to elucidate gender and human subpopulation bias in disease. Evident subpopulation bias in myosin SNP pathogenicities imply myosin normally engages multiple sarcomere proteins functionally. Consistent with this observation, mybpc3 forms a third actomyosin interaction competing with myosin essential light chain N-terminus suggesting a novel strain-dependent mechanism adapting myosin force-velocity to load dynamics. The working models, and the integral myosin/mybpc3 motor concept, portends the wider considerations involved in understanding heart disease as a systemic maladaptation.
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http://dx.doi.org/10.1016/j.abb.2019.07.021DOI Listing
September 2019

Cardiac and skeletal actin substrates uniquely tune cardiac myosin strain-dependent mechanics.

Open Biol 2018 11 21;8(11). Epub 2018 Nov 21.

Department of Biochemistry and Molecular Biology, Mayo Clinic Rochester, Rochester, MN 55905, USA

Cardiac ventricular myosin (βmys) translates actin by transducing ATP free energy into mechanical work during muscle contraction. Unitary βmys translation of actin is the step-size. and βmys regulates contractile force and velocity autonomously by remixing three different step-sizes with adaptive stepping frequencies. Cardiac and skeletal actin isoforms have a specific 1 : 4 stoichiometry in normal adult human ventriculum. Human adults with inheritable hypertrophic cardiomyopathy (HCM) upregulate skeletal actin in ventriculum probably compensating the diseased muscle's inability to meet demand by adjusting βmys force-velocity characteristics. βmys force-velocity characteristics were compared for skeletal versus cardiac actin substrates using ensemble motility and single myosin assays. Two competing myosin strain-sensitive mechanisms regulate step-size choices dividing single βmys mechanics into low- and high-force regimes. The actin isoforms alter myosin strain-sensitive regulation such that onset of the high-force regime, where a short step-size is a large or major contributor, is offset to higher loads probably by the unique cardiac essential light chain (ELC) N-terminus/cardiac actin contact at Glu6/Ser358. It modifies βmys force-velocity by stabilizing the ELC N-terminus/cardiac actin association. Uneven onset of the high-force regime for skeletal versus cardiac actin modulates force-velocity characteristics as skeletal/cardiac actin fractional content increases in diseased muscle.
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http://dx.doi.org/10.1098/rsob.180143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6282072PMC
November 2018

Uncured PDMS inhibits myosin in vitro motility in a microfluidic flow cell.

Anal Biochem 2018 12 6;563:56-60. Epub 2018 Oct 6.

Department of Biochemistry and Molecular Biology, Mayo Clinic Rochester, Rochester, MN, 55905, USA; Department of Physiology and Biomedical Engineering, Mayo Clinic Rochester, Rochester, MN, 55905, USA. Electronic address:

The myosin motor powers cardiac contraction and is frequently implicated in hereditary heart disease by its mutation. Principal motor function characteristics include myosin unitary step size, duty cycle, and force-velocity relationship for translating actin under load. These characteristics are sometimes measured in vitro with a motility assay detecting fluorescent labeled actin filament gliding velocity over a planar array of surface immobilized myosin. Assay miniaturization in a polydimethylsiloxane/glass (PDMS/glass) hybrid microfluidic flow channel is an essential component to a small sample volume assay applicable to costly protein samples however the PDMS substrate dramatically inhibits myosin motility. Myosin in vitro motility in a PDMS/glass hybrid microfluidic flow cell was tested under a variety of conditions to identify and mitigate the effect of PDMS on myosin. Substantial contamination by unpolymerized species in the PDMS flow cells is shown to be the cause of myosin motility inhibition. Normal myosin motility recovers by either extended cell aging (~20 days) to allow more complete polymerization or by direct chemical extraction of the unpolymerized species from the polymer substrate. PDMS flow cell aging is the low cost alternative compatible with the other PDMS and glass modifications needed for in vitro myosin motility assaying.
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http://dx.doi.org/10.1016/j.ab.2018.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6689414PMC
December 2018

Single cardiac ventricular myosins are autonomous motors.

Open Biol 2018 04;8(4)

Department of Biochemistry and Molecular Biology, Mayo Clinic Rochester, 200 First Street SW, Rochester, MN 55905, USA

Myosin transduces ATP free energy into mechanical work in muscle. Cardiac muscle has dynamically wide-ranging power demands on the motor as the muscle changes modes in a heartbeat from relaxation, via auxotonic shortening, to isometric contraction. The cardiac power output modulation mechanism is explored by assessing single cardiac myosin step-size selection versus load. Transgenic mice express human ventricular essential light chain (ELC) in wild- type (WT), or hypertrophic cardiomyopathy-linked mutant forms, A57G or E143K, in a background of mouse α-cardiac myosin heavy chain. Ensemble motility and single myosin mechanical characteristics are consistent with an A57G that impairs ELC N-terminus actin binding and an E143K that impairs lever-arm stability, while both species down-shift average step-size with increasing load. Cardiac myosin down-shifts velocity/force ratio with increasing load by changed unitary step-size selections. Here, the loaded single myosin assay indicates quantitative complementarity with the mechanism. Both have two embedded regulatory transitions, one inhibiting ADP release and a second novel mechanism inhibiting actin detachment via strain on the actin-bound ELC N-terminus. Competing regulators filter unitary step-size selection to control force-velocity modulation without myosin integration into muscle. Cardiac myosin is muscle in a molecule.
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http://dx.doi.org/10.1098/rsob.170240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5936712PMC
April 2018

Neural/Bayes network predictor for inheritable cardiac disease pathogenicity and phenotype.

J Mol Cell Cardiol 2018 06 11;119:19-27. Epub 2018 Apr 11.

Department of Biochemistry and Molecular Biology, 200 First St. SW, Mayo Clinic Rochester, Rochester, MN 55905, United States.

The cardiac muscle sarcomere contains multiple proteins contributing to contraction energy transduction and its regulation during a heartbeat. Inheritable heart disease mutants affect most of them but none more frequently than the ventricular myosin motor and cardiac myosin binding protein c (mybpc3). These co-localizing proteins have mybpc3 playing a regulatory role to the energy transducing motor. Residue substitution and functional domain assignment of each mutation in the protein sequence decides, under the direction of a sensible disease model, phenotype and pathogenicity. The unknown model mechanism is decided here using a method combing neural and Bayes networks. Missense single nucleotide polymorphisms (SNPs) are clues for the disease mechanism summarized in an extensive database collecting mutant sequence location and residue substitution as independent variables that imply the dependent disease phenotype and pathogenicity characteristics in 4 dimensional data points (4ddps). The SNP database contains entries with the majority having one or both dependent data entries unfulfilled. A neural network relating causes (mutant residue location and substitution) and effects (phenotype and pathogenicity) is trained, validated, and optimized using fulfilled 4ddps. It then predicts unfulfilled 4ddps providing the implicit disease model. A discrete Bayes network interprets fulfilled and predicted 4ddps with conditional probabilities for phenotype and pathogenicity given mutation location and residue substitution thus relating the neural network implicit model to explicit features of the motor and mybpc3 sequence and structural domains. Neural/Bayes network forecasting automates disease mechanism modeling by leveraging the world wide human missense SNP database that is in place and expanding.
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http://dx.doi.org/10.1016/j.yjmcc.2018.04.006DOI Listing
June 2018

Auxotonic to isometric contraction transitioning in a beating heart causes myosin step-size to down shift.

PLoS One 2017 19;12(4):e0174690. Epub 2017 Apr 19.

Department of Biochemistry and Molecular Biology, Mayo Clinic Rochester, Rochester, Minnesota, United States of America.

Myosin motors in cardiac ventriculum convert ATP free energy to the work of moving blood volume under pressure. The actin bound motor cyclically rotates its lever-arm/light-chain complex linking motor generated torque to the myosin filament backbone and translating actin against resisting force. Previous research showed that the unloaded in vitro motor is described with high precision by single molecule mechanical characteristics including unitary step-sizes of approximately 3, 5, and 8 nm and their relative step-frequencies of approximately 13, 50, and 37%. The 3 and 8 nm unitary step-sizes are dependent on myosin essential light chain (ELC) N-terminus actin binding. Step-size and step-frequency quantitation specifies in vitro motor function including duty-ratio, power, and strain sensitivity metrics. In vivo, motors integrated into the muscle sarcomere form the more complex and hierarchically functioning muscle machine. The goal of the research reported here is to measure single myosin step-size and step-frequency in vivo to assess how tissue integration impacts motor function. A photoactivatable GFP tags the ventriculum myosin lever-arm/light-chain complex in the beating heart of a live zebrafish embryo. Detected single GFP emission reports time-resolved myosin lever-arm orientation interpreted as step-size and step-frequency providing single myosin mechanical characteristics over the active cycle. Following step-frequency of cardiac ventriculum myosin transitioning from low to high force in relaxed to auxotonic to isometric contraction phases indicates that the imposition of resisting force during contraction causes the motor to down-shift to the 3 nm step-size accounting for >80% of all the steps in the near-isometric phase. At peak force, the ATP initiated actomyosin dissociation is the predominant strain inhibited transition in the native myosin contraction cycle. The proposed model for motor down-shifting and strain sensing involves ELC N-terminus actin binding. Overall, the approach is a unique bottom-up single molecule mechanical characterization of a hierarchically functional native muscle myosin.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0174690PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5396871PMC
April 2017

Hypercontractile mutant of ventricular myosin essential light chain leads to disruption of sarcomeric structure and function and results in restrictive cardiomyopathy in mice.

Cardiovasc Res 2017 Aug;113(10):1124-1136

Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, FL 33136, USA.

Aims: The E143K (Glu → Lys) mutation in the myosin essential light chain has been associated with restrictive cardiomyopathy (RCM) in humans, but the mechanisms that underlie the development of defective cardiac function are unknown. Using transgenic E143K-RCM mice, we sought to determine the molecular and cellular triggers of E143K-induced heart remodelling.

Methods And Results: The E143K-induced abnormalities in cardiac function and morphology observed by echocardiography and invasive haemodynamics were paralleled by augmented active and passive tension measured in skinned papillary muscle fibres compared with wild-type (WT)-generated force. In vitro, E143K-myosin had increased duty ratio and binding affinity to actin compared with WT-myosin, increased actin-activated ATPase activity and slower rates of ATP-dependent dissociation of the acto-myosin complex, indicating an E143K-induced myosin hypercontractility. E143K was also observed to reduce the level of myosin regulatory light chain phosphorylation while that of troponin-I remained unchanged. Small-angle X-ray diffraction data showed a decrease in the filament lattice spacing (d1,0) with no changes in the equatorial reflections intensity ratios (I1,1/I1,0) in E143K vs. WT skinned papillary muscles. The hearts of mutant-mice demonstrated ultrastructural defects and fibrosis that progressively worsened in senescent animals and these changes were hypothesized to contribute to diastolic disturbance and to mild systolic dysfunction. Gene expression profiles of E143K-hearts supported the histopathology results and showed an upregulation of stress-response and collagen genes. Finally, proteomic analysis evidenced RCM-dependent metabolic adaptations and higher energy demands in E143K vs. WT hearts.

Conclusions: As a result of the E143K-induced myosin hypercontractility, the hearts of RCM mice model exhibited cardiac dysfunction, stiff ventricles and physiological, morphologic, and metabolic remodelling consistent with the development of RCM. Future efforts should be directed toward normalization of myosin motor function and the use of myosin-specific therapeutics to avert the hypercontractile state of E143K-myosin and prevent pathological cardiac remodelling.
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http://dx.doi.org/10.1093/cvr/cvx060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5852631PMC
August 2017

In vivo myosin step-size from zebrafish skeletal muscle.

Open Biol 2016 05 25;6(5). Epub 2016 May 25.

Department of Biochemistry and Molecular Biology, Mayo Clinic Rochester, Rochester, MN 55905, USA.

Muscle myosins transduce ATP free energy into actin displacement to power contraction. In vivo, myosin side chains are modified post-translationally under native conditions, potentially impacting function. Single myosin detection provides the 'bottom-up' myosin characterization probing basic mechanisms without ambiguities inherent to ensemble observation. Macroscopic muscle physiological experimentation provides the definitive 'top-down' phenotype characterizations that are the concerns in translational medicine. In vivo single myosin detection in muscle from zebrafish embryo models for human muscle fulfils ambitions for both bottom-up and top-down experimentation. A photoactivatable green fluorescent protein (GFP)-tagged myosin light chain expressed in transgenic zebrafish skeletal muscle specifically modifies the myosin lever-arm. Strychnine induces the simultaneous contraction of the bilateral tail muscles in a live embryo, causing them to be isometric while active. Highly inclined thin illumination excites the GFP tag of single lever-arms and its super-resolution orientation is measured from an active isometric muscle over a time sequence covering many transduction cycles. Consecutive frame lever-arm angular displacement converts to step-size by its product with the estimated lever-arm length. About 17% of the active myosin steps that fall between 2 and 7 nm are implicated as powerstrokes because they are beyond displacements detected from either relaxed or ATP-depleted (rigor) muscle.
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http://dx.doi.org/10.1098/rsob.160075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4892436PMC
May 2016

In vitro and in vivo single myosin step-sizes in striated muscle.

J Muscle Res Cell Motil 2015 Dec 4;36(6):463-77. Epub 2016 Jan 4.

Department of Biochemistry and Molecular Biology, Mayo Clinic Rochester, Rochester, MN, 55905, USA.

Myosin in muscle transduces ATP free energy into the mechanical work of moving actin. It has a motor domain transducer containing ATP and actin binding sites, and, mechanical elements coupling motor impulse to the myosin filament backbone providing transduction/mechanical-coupling. The mechanical coupler is a lever-arm stabilized by bound essential and regulatory light chains. The lever-arm rotates cyclically to impel bound filamentous actin. Linear actin displacement due to lever-arm rotation is the myosin step-size. A high-throughput quantum dot labeled actin in vitro motility assay (Qdot assay) measures motor step-size in the context of an ensemble of actomyosin interactions. The ensemble context imposes a constant velocity constraint for myosins interacting with one actin filament. In a cardiac myosin producing multiple step-sizes, a "second characterization" is step-frequency that adjusts longer step-size to lower frequency maintaining a linear actin velocity identical to that from a shorter step-size and higher frequency actomyosin cycle. The step-frequency characteristic involves and integrates myosin enzyme kinetics, mechanical strain, and other ensemble affected characteristics. The high-throughput Qdot assay suits a new paradigm calling for wide surveillance of the vast number of disease or aging relevant myosin isoforms that contrasts with the alternative model calling for exhaustive research on a tiny subset myosin forms. The zebrafish embryo assay (Z assay) performs single myosin step-size and step-frequency assaying in vivo combining single myosin mechanical and whole muscle physiological characterizations in one model organism. The Qdot and Z assays cover "bottom-up" and "top-down" assaying of myosin characteristics.
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http://dx.doi.org/10.1007/s10974-015-9440-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4764389PMC
December 2015

N-Terminus of Cardiac Myosin Essential Light Chain Modulates Myosin Step-Size.

Biochemistry 2016 Jan 29;55(1):186-98. Epub 2015 Dec 29.

Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine , Miami, Florida 33136, United States.

Muscle myosin cyclically hydrolyzes ATP to translate actin. Ventricular cardiac myosin (βmys) moves actin with three distinct unitary step-sizes resulting from its lever-arm rotation and with step-frequencies that are modulated in a myosin regulation mechanism. The lever-arm associated essential light chain (vELC) binds actin by its 43 residue N-terminal extension. Unitary steps were proposed to involve the vELC N-terminal extension with the 8 nm step engaging the vELC/actin bond facilitating an extra ∼19 degrees of lever-arm rotation while the predominant 5 nm step forgoes vELC/actin binding. A minor 3 nm step is the unlikely conversion of the completed 5 to the 8 nm step. This hypothesis was tested using a 17 residue N-terminal truncated vELC in porcine βmys (Δ17βmys) and a 43 residue N-terminal truncated human vELC expressed in transgenic mouse heart (Δ43αmys). Step-size and step-frequency were measured using the Qdot motility assay. Both Δ17βmys and Δ43αmys had significantly increased 5 nm step-frequency and coincident loss in the 8 nm step-frequency compared to native proteins suggesting the vELC/actin interaction drives step-size preference. Step-size and step-frequency probability densities depend on the relative fraction of truncated vELC and relate linearly to pure myosin species concentrations in a mixture containing native vELC homodimer, two truncated vELCs in the modified homodimer, and one native and one truncated vELC in the heterodimer. Step-size and step-frequency, measured for native homodimer and at two or more known relative fractions of truncated vELC, are surmised for each pure species by using a new analytical method.
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http://dx.doi.org/10.1021/acs.biochem.5b00817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4727542PMC
January 2016

In vivo orientation of single myosin lever arms in zebrafish skeletal muscle.

Biophys J 2014 Sep;107(6):1403-14

Department of Biochemistry and Molecular Biology, Mayo Clinic Rochester, Rochester, Minnesota; Department of Physiology and Biomedical Engineering, Mayo Clinic Rochester, Rochester, Minnesota. Electronic address:

Cardiac and skeletal myosin assembled in the muscle lattice power contraction by transducing ATP free energy into the mechanical work of moving actin. Myosin catalytic/lever-arm domains comprise the transduction/mechanical coupling machinery that move actin by lever-arm rotation. In vivo, myosin is crowded and constrained by the fiber lattice as side chains are mutated and otherwise modified under normal, diseased, or aging conditions that collectively define the native myosin environment. Single-myosin detection uniquely defines bottom-up characterization of myosin functionality. The marriage of in vivo and single-myosin detection to study zebrafish embryo models of human muscle disease is a multiscaled technology that allows one-to-one registration of a selected myosin molecular alteration with muscle filament-sarcomere-cell-fiber-tissue-organ- and organism level phenotypes. In vivo single-myosin lever-arm orientation was observed at superresolution using a photoactivatable-green-fluorescent-protein (PAGFP)-tagged myosin light chain expressed in zebrafish skeletal muscle. By simultaneous observation of multiphoton excitation fluorescence emission and second harmonic generation from myosin, we demonstrated tag specificity for the lever arm. Single-molecule detection used highly inclined parallel beam illumination and was verified by quantized photoactivation and photobleaching. Single-molecule emission patterns from relaxed muscle in vivo provided extensive superresolved dipole orientation constraints that were modeled using docking scenarios generated for the myosin (S1) and GFP crystal structures. The dipole orientation data provided sufficient constraints to estimate S1/GFP coordination. The S1/GFP coordination in vivo is rigid and the lever-arm orientation distribution is well-ordered in relaxed muscle. For comparison, single myosins in relaxed permeabilized porcine papillary muscle fibers indicated slightly differently oriented lever arms and rigid S1/GFP coordination. Lever arms in both muscles indicated one preferred spherical polar orientation and widely distributed azimuthal orientations relative to the fiber symmetry axis. Cardiac myosin is more radially displaced from the fiber axis. Probe rigidity implies the PAGFP tag reliably indicates cross-bridge orientation in situ and in vivo.
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http://dx.doi.org/10.1016/j.bpj.2014.07.055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4167300PMC
September 2014

Analytical comparison of natural and pharmaceutical ventricular myosin activators.

Biochemistry 2014 Aug 7;53(32):5298-306. Epub 2014 Aug 7.

Department of Biochemistry and Molecular Biology and ‡Department of Physiology and Biomedical Engineering, Mayo Clinic Rochester , Rochester, Minnesota 55905, United States.

Ventricular myosin (βMys) is the motor protein in cardiac muscle generating force using ATP hydrolysis free energy to translate actin. In the cardiac muscle sarcomere, myosin and actin filaments interact cyclically and undergo rapid relative translation facilitated by the low duty cycle motor. It contrasts with high duty cycle processive myosins for which persistent actin association is the priority. The only pharmaceutical βMys activator, omecamtive mecarbil (OM), upregulates cardiac contractility in vivo and is undergoing testing for heart failure therapy. In vitro βMys step-size, motility velocity, and actin-activated myosin ATPase were measured to determine duty cycle in the absence and presence of OM. A new parameter, the relative step-frequency, was introduced and measured to characterize βMys motility due to the involvement of its three unitary step-sizes. Step-size and relative step-frequency were measured using the Qdot assay. OM decreases motility velocity 10-fold without affecting step-size, indicating a large increase in duty cycle converting βMys to a near processive myosin. The OM conversion dramatically increases force and modestly increases power over the native βMys. Contrasting motility modification due to OM with that from the natural myosin activator, specific βMys phosphorylation, provides insight into their respective activation mechanisms and indicates the boilerplate screening characteristics desired for pharmaceutical βMys activators. New analytics introduced here for the fast and efficient Qdot motility assay create a promising method for high-throughput screening of motor proteins and their modulators.
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http://dx.doi.org/10.1021/bi500730tDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4139156PMC
August 2014

Ventricular myosin modifies in vitro step-size when phosphorylated.

J Mol Cell Cardiol 2014 Jul 12;72:231-7. Epub 2014 Apr 12.

Department of Biochemistry and Molecular Biology, United States; Department of Physiology and Biomedical Engineering, United States. Electronic address:

Cardiac and skeletal muscle myosins have the central role in contraction transducing ATP free energy into the mechanical work of moving actin. Myosin has a motor domain containing ATP and actin binding sites and a lever-arm that undergoes rotation impelling bound actin. The lever-arm converts torque generated in the motor into the linear displacement known as step-size. The myosin lever-arm is stabilized by bound essential and regulatory light chains (ELC and RLC). RLC phosphorylation at S15 is linked to modified lever-arm mechanical characteristics contributing to myosin filament based contraction regulation and to the response of the muscle to disease. Myosin step-size was measured using a novel quantum dot (Qdot) assay that previously confirmed a 5nm step-size for fast skeletal myosin and multiple unitary steps, most frequently 5 and 8nm, and a rare 3nm displacement for β cardiac myosin (βMys). S15 phosphorylation in βMys is now shown to change step-size distribution by advancing the 8nm step frequency. After phosphorylation, the 8nm step is the dominant myosin step-size resulting in significant gain in the average step-size. An increase in myosin step-size will increase the amount of work produced per ATPase cycle. The results indicate that RLC phosphorylation modulates work production per ATPase cycle suggesting the mechanism for contraction regulation by the myosin filament.
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http://dx.doi.org/10.1016/j.yjmcc.2014.03.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4037356PMC
July 2014

The Qdot-labeled actin super-resolution motility assay measures low-duty cycle muscle myosin step size.

Biochemistry 2013 Mar 21;52(9):1611-21. Epub 2013 Feb 21.

Department of Biochemistry and Molecular Biology, Mayo Clinic Rochester, Rochester, Minnesota 55905, USA.

Myosin powers contraction in heart and skeletal muscle and is a leading target for mutations implicated in inheritable muscle diseases. During contraction, myosin transduces ATP free energy into the work of muscle shortening against resisting force. Muscle shortening involves relative sliding of myosin and actin filaments. Skeletal actin filaments were fluorescently labeled with a streptavidin conjugate quantum dot (Qdot) binding biotin-phalloidin on actin. Single Qdots were imaged in time with total internal reflection fluorescence microscopy and then spatially localized to 1-3 nm using a super-resolution algorithm as they translated with actin over a surface coated with skeletal heavy meromyosin (sHMM) or full-length β-cardiac myosin (MYH7). The average Qdot-actin velocity matches measurements with rhodamine-phalloidin-labeled actin. The sHMM Qdot-actin velocity histogram contains low-velocity events corresponding to actin translation in quantized steps of ~5 nm. The MYH7 velocity histogram has quantized steps at 3 and 8 nm in addition to 5 nm and larger compliance compared to that of sHMM depending on the MYH7 surface concentration. Low-duty cycle skeletal and cardiac myosin present challenges for a single-molecule assay because actomyosin dissociates quickly and the freely moving element diffuses away. The in vitro motility assay has modestly more actomyosin interactions, and methylcellulose inhibited diffusion to sustain the complex while preserving a subset of encounters that do not overlap in time on a single actin filament. A single myosin step is isolated in time and space and then characterized using super-resolution. The approach provides a quick, quantitative, and inexpensive step size measurement for low-duty cycle muscle myosin.
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http://dx.doi.org/10.1021/bi301702pDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3616449PMC
March 2013

Regulatory light chain mutants linked to heart disease modify the cardiac myosin lever arm.

Biochemistry 2013 Feb 6;52(7):1249-59. Epub 2013 Feb 6.

Department of Biochemistry and Molecular Biology, Mayo Clinic Rochester, Rochester, MN 55905, USA.

Myosin is the chemomechanical energy transducer in striated heart muscle. The myosin cross-bridge applies impulsive force to actin while consuming ATP chemical energy to propel myosin thick filaments relative to actin thin filaments in the fiber. Transduction begins with ATP hydrolysis in the cross-bridge driving rotary movement of a lever arm converting torque into linear displacement. Myosin regulatory light chain (RLC) binds to the lever arm and modifies its ability to translate actin. Gene sequencing implicated several RLC mutations in heart disease, and three of them are investigated here using photoactivatable GFP-tagged RLC (RLC-PAGFP) exchanged into permeabilized papillary muscle fibers. A single-lever arm probe orientation is detected in the crowded environment of the muscle fiber by using RLC-PAGFP with dipole orientation deduced from the three-spatial dimension fluorescence emission pattern of the single molecule. Symmetry and selection rules locate dipoles in their half-sarcomere, identify those at the minimal free energy, and specify active dipole contraction intermediates. Experiments were performed in a microfluidic chamber designed for isometric contraction, total internal reflection fluorescence detection, and two-photon excitation second harmonic generation to evaluate sarcomere length. The RLC-PAGFP reports apparently discretized lever arm orientation intermediates in active isometric fibers that on average produce the stall force. Disease-linked mutants introduced into RLC move intermediate occupancy further down the free energy gradient, implying lever arms rotate more to reach stall force because mutant RLC increases lever arm shear strain. A lower free energy intermediate occupancy involves a lower energy conversion efficiency in the fiber relating a specific myosin function modification to the disease-implicated mutant.
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http://dx.doi.org/10.1021/bi301500dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3587134PMC
February 2013

Human Tonic and Phasic Smooth Muscle Myosin Isoforms Are Unresponsive to the Loop 1 Insert.

ISRN Struct Biol 2013 Jan;2013:634341

Department of Biochemistry and Molecular Biology, Mayo Clinic Rochester, 200 First Street SW, Rochester, MN 55905, USA ; Department of Physiology and Biomedical Engineering, Mayo Clinic Rochester, 200 First Street SW, Rochester, MN 55905, USA.

Smooth muscle myosin gene products include two isoforms, SMA and SMB, differing by a 7-residue peptide in loop 1 (i7) at the myosin active site where ATP is hydrolyzed. Using chicken isoforms, previous work indicated that the i7 deletion in SMA prolongs strong actin binding by inhibiting active site ingress and egress of nucleotide when compared to i7 inserted SMB. Additionally, i7 deletion inhibits Pi release associated with the switch 2 closed → open transition in actin-activated ATPase. Switch 2 is far from loop 1 indicating i7 deletion has an allosteric effect on Pi release. Chicken SMA and SMB have unknown and robust nucleotide-sensitive tryptophan (NST) fluorescence increments, respectively. Human SMA and SMB both lack NST increments while Pi release in Ca ATPase is not impacted by i7 deletion. The NST reports relay helix movement following conformation change in switch 2 but in the open → closed transition. The NST is common to all known myosin isoforms except human smooth muscle. Other independent works on human SMA and SMB motility indicate no functional effect of i7 deletion. Smooth muscle myosin is a stunning example of species-specific myosin structure/function divergence underscoring the danger in extrapolating disease-linked mutant effects on myosin across species.
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http://dx.doi.org/10.1155/2013/634341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3938199PMC
January 2013

Measuring incidence angle for through-the-objective total internal reflection fluorescence microscopy.

J Biomed Opt 2012 Dec;17(12):126007

Mayo Clinic Rochester, Department of Biochemistry and Molecular Biology, Rochester, Minnesota 55905, USA.

Total internal reflection fluorescence (TIRF) microscopy has the exciting laser beam incident beyond critical angle from the glass side of a glass/aqueous interface formed by the coverslip and aqueous sample. The aqueous side evanescent field decays exponentially with distance from the interface with penetration depth depending on incidence angle. Through-the-objective TIRF has the exciting laser focused at the back focal plane (BFP) creating a refracted parallel beam approaching the interface in the small gap between objective and coverslip, making incidence angle challenging to measure. Objective axial scanning does not affect incidence angle but translates beam and interface intersection detected by the fluorescence center of mass from fluorescent spheres attached to the aqueous side of the interface. Center of mass translation divided by the axial translation is the tangent of the incidence angle that is sampled repeatedly over objective trajectory to obtain a best estimate. Incidence angle is measured for progressively larger radial positions of the focused beam on the BFP. A through-the-objective TIRF microscope, utilizing a micrometer and relay lenses to position the focused beam at the BFP, is calibrated for incidence angle. Calibration depends on microscope characteristics and TIRF objective and is applicable to any interface or sample.
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http://dx.doi.org/10.1117/1.JBO.17.12.126007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3512109PMC
December 2012

Cell biology. Heart brakes.

Science 2012 Sep;337(6099):1182-3

Biochemistry and Molecular Biology, Mayo Clinic Rochester, Rochester, MN 55905, USA.

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http://dx.doi.org/10.1126/science.1227943DOI Listing
September 2012

Evanescent field shapes excitation profile under axial epi-illumination.

J Biomed Opt 2012 Jun;17(6):066021

Mayo Clinic Rochester, Department of Biochemistry and Molecular Biology, Rochester, Minnesota 55905, USA.

Axial epi-illuminating light transmitting a >1.3-numerical-aperture microscope objective creates an excitation volume at focus with size and shape dictated by diffraction and due to refraction by the objective and by the coverslip interface separating a specimen in aqueous buffer from the oil immersion objective. The evanescent field on the coverslip aqueous side affects primarily the excitation volume axial dimension as the specimen in focus approaches the interface to within a few hundred nanometers. Following excitation, an excited stationary dipole moment emits fluorescence in a spatially varying pattern collected over the large objective aperture. Collected light propagates in parallel rays toward the tube lens that forms a real three-dimensional image that is decoded to identify dipole orientation. An integral representation of the excitation and emitted fields for infinity-corrected optics--including effects of finite conjugate illumination, fluorescence emission near an interface, emitter dipole orientation, spherical aberration, light transmission through a dichroic filter, and for real microscopic specifications--accurately models observed field intensities including the substantial excitation from the evanescent field. The goal is to develop and verify the practical depiction of excitation and emission in a real microscope for quantitative interpretation of the 3-D emission pattern.
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http://dx.doi.org/10.1117/1.JBO.17.6.066021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3381040PMC
June 2012

Smooth muscle myosin light chain kinase efficiently phosphorylates serine 15 of cardiac myosin regulatory light chain.

Biochem Biophys Res Commun 2011 Dec 19;416(3-4):367-71. Epub 2011 Nov 19.

Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905, USA.

Specific phosphorylation of the human ventricular cardiac myosin regulatory light chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca(2+) sensitivity of contraction in cardiac tissue. Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory light chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory light chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis-Menten V(max) and K(M) for regulatory light chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant kinase in cardiac tissue on the basis of its specificity, kinetics, and tissue expression.
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http://dx.doi.org/10.1016/j.bbrc.2011.11.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3242870PMC
December 2011

Single myosin cross-bridge orientation in cardiac papillary muscle detects lever-arm shear strain in transduction.

Biochemistry 2011 Sep 18;50(36):7809-21. Epub 2011 Aug 18.

Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, United States.

Myosin motors transduce ATP free energy into mechanical work. Transduction models allocate specific functions to motor structural domains beginning with ATP hydrolysis in the active site and ending in a lever-arm rotating power-stroke. Myosin light chains, regulatory (RLC) and essential (ELC), bind IQ-domains on the lever-arm and track its movement. Strong evidence exists that light chains stabilize the lever-arm and that light chain mutation undermines stability. Human ventricular RLC tagged with photoactivatable GFP (HCRLC-PAGFP) replaces native RLC in porcine papillary muscle fibers, restores native contractility, and situates PAGFP for single molecule orientation tracking within the crowded fiber lattice. The spatial emission pattern from single photoactivated PAGFP tagged myosins was observed in z-stacks fitted simultaneously to maximize accuracy in estimated dipole orientation. Emitter dipole polar and azimuthal angle pair scatter plots identified an area where steric and molecular crowding constraints depopulated orientations unfavorable for actin interaction. Transitions between pre- and post-power-stroke states represent the lever-arm trajectory sampled by the data and quantify lever-arm shear strain in transduction at three tension levels. These data identify forces acting on myosin in the in situ fiber system due to crowding, steric hindrance, and actomyosin interaction. They induce lever-arm shear strain observed with single molecule orientation detection. A single myosin work histogram reveals discretized power-stroke substates reminiscent of the Huxley-Simmons model for myosin based contraction [Huxley and Simmons ( 1971 ) Nature 233 , 533]. RLC or ELC mutation, should it impact lever-arm shear strain, will be detected as changes in single myosin shear strain or power-stroke substate distribution.
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http://dx.doi.org/10.1021/bi2008992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3177300PMC
September 2011

Single molecule fluorescence image patterns linked to dipole orientation and axial position: application to myosin cross-bridges in muscle fibers.

PLoS One 2011 Feb 8;6(2):e16772. Epub 2011 Feb 8.

Department of Biochemistry and Molecular Biology, Mayo Clinic Rochester, Rochester, Minnesota, United States of America.

Background: Photoactivatable fluorescent probes developed specifically for single molecule detection extend advantages of single molecule imaging to high probe density regions of cells and tissues. They perform in the native biomolecule environment and have been used to detect both probe position and orientation.

Methods And Findings: Fluorescence emission from a single photoactivated probe captured in an oil immersion, high numerical aperture objective, produces a spatial pattern on the detector that is a linear combination of 6 independent and distinct spatial basis patterns with weighting coefficients specifying emission dipole orientation. Basis patterns are tabulated for single photoactivated probes labeling myosin cross-bridges in a permeabilized muscle fiber undergoing total internal reflection illumination. Emitter proximity to the glass/aqueous interface at the coverslip implies the dipole near-field and dipole power normalization are significant affecters of the basis patterns. Other characteristics of the basis patterns are contributed by field polarization rotation with transmission through the microscope optics and refraction by the filter set. Pattern recognition utilized the generalized linear model, maximum likelihood fitting, for Poisson distributed uncertainties. This fitting method is more appropriate for treating low signal level photon counting data than χ(2) minimization.

Conclusions: Results indicate that emission dipole orientation is measurable from the intensity image except for the ambiguity under dipole inversion. The advantage over an alternative method comparing two measured polarized emission intensities using an analyzing polarizer is that information in the intensity spatial distribution provides more constraints on fitted parameters and a single image provides all the information needed. Axial distance dependence in the emission pattern is also exploited to measure relative probe position near focus. Single molecule images from axial scanning fitted simultaneously boost orientation and axial resolution in simulation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0016772PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3035662PMC
February 2011

Single-molecule fluorescence characterization in native environment.

Biophys Rev 2010 Dec;2(4):159-167

Department of Biochemistry and Molecular Biology, Mayo Clinic Rochester, Rochester, MN 55905, USA.

Single-molecule detection (SMD) with fluorescence is a widely used microscopic technique for biomolecule structure and function characterization. The modern light microscope with high numerical aperture objective and sensitive CCD camera can image the brightly emitting organic and fluorescent protein tags with reasonable time resolution. Single-molecule imaging gives an unambiguous bottom-up biomolecule characterization that avoids the "missing information" problem characteristic of ensemble measurements. It has circumvented the diffraction limit by facilitating single-particle localization to ~1 nm. Probes developed specifically for SMD applications extend the advantages of single-molecule imaging to high probe density regions of cells and tissues. These applications perform under conditions resembling the native biomolecule environment and have been used to detect both probe position and orientation. Native, high density SMD may have added significance if molecular crowding impacts native biomolecule behavior as expected inside the cell.
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http://dx.doi.org/10.1007/s12551-010-0038-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3004222PMC
December 2010

Myosin individualized: single nucleotide polymorphisms in energy transduction.

BMC Genomics 2010 Mar 15;11:172. Epub 2010 Mar 15.

Department of Biochemistry and Molecular Biology, Mayo Clinic Rochester, 200 First Street SW, Rochester, MN 55905, USA.

Background: Myosin performs ATP free energy transduction into mechanical work in the motor domain of the myosin heavy chain (MHC). Energy transduction is the definitive systemic feature of the myosin motor performed by coordinating in a time ordered sequence: ATP hydrolysis at the active site, actin affinity modulation at the actin binding site, and the lever-arm rotation of the power stroke. These functions are carried out by several conserved sub-domains within the motor domain. Single nucleotide polymorphisms (SNPs) affect the MHC sequence of many isoforms expressed in striated muscle, smooth muscle, and non-muscle tissue. The purpose of this work is to provide a rationale for using SNPs as a functional genomics tool to investigate structurefunction relationships in myosin. In particular, to discover SNP distribution over the conserved sub-domains and surmise what it implies about sub-domain stability and criticality in the energy transduction mechanism.

Results: An automated routine identifying human nonsynonymous SNP amino acid missense substitutions for any MHC gene mined the NCBI SNP data base. The routine tested 22 MHC genes coding muscle and non-muscle isoforms and identified 89 missense mutation positions in the motor domain with 10 already implicated in heart disease and another 8 lacking sequence homology with a skeletal MHC isoform for which a crystallographic model is available. The remaining 71 SNP substitutions were found to be distributed over MHC with 22 falling outside identified functional sub-domains and 49 in or very near to myosin sub-domains assigned specific crucial functions in energy transduction. The latter includes the active site, the actin binding site, the rigid lever-arm, and regions facilitating their communication. Most MHC isoforms contained SNPs somewhere in the motor domain.

Conclusions: Several functional-crucial sub-domains are infiltrated by a large number of SNP substitution sites suggesting these domains are engineered by evolution to be too-robust to be disturbed by otherwise intrusive sequence changes. Two functional sub-domains are SNP-free or relatively SNP-deficient but contain many disease implicated mutants. These sub-domains are apparently highly sensitive to any missense substitution suggesting they have failed to evolve a robust sequence paradigm for performing their function.
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http://dx.doi.org/10.1186/1471-2164-11-172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2848645PMC
March 2010

Around-the-objective total internal reflection fluorescence microscopy.

Appl Opt 2009 Nov;48(32):6120-31

Department of Biochemistry and Molecular Biology, Mayo Clinic Rochester, Rochester, Minnesota 55905, USA.

Total internal reflection fluorescence (TIRF) microscopy uses the evanescent field on the aqueous side of a glass/aqueous interface to selectively illuminate fluorophores within approximately 100 nm of the interface. Applications of the method include epi-illumination TIRF, where the exciting light is refracted by the microscope objective to impinge on the interface at incidence angles beyond critical angle, and prism-based TIRF, where exciting light propagates to the interface externally to the microscope optics. The former has higher background autofluorescence from the glass elements of the objective where the exciting beam is focused, and the latter does not collect near-field emission from the fluorescent sample. Around-the-objective TIRF, developed here, creates the evanescent field by conditioning the exciting laser beam to propagate through the submillimeter gap created by the oil immersion high numerical aperture objective and the glass coverslip. The approach eliminates background light due to the admission of the laser excitation to the microscopic optics while collecting near-field emission from the dipoles excited by the approximately 50 nm deep evanescent field.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2802224PMC
http://dx.doi.org/10.1364/AO.48.006120DOI Listing
November 2009

Mapping microscope object polarized emission to the back focal plane pattern.

J Biomed Opt 2009 May-Jun;14(3):034036

Mayo Clinic Rochester, Department of Biochemistry, 200 First Street South West, Rochester, Minnesota 55905, USA.

The back focal plane (BFP) intensity pattern from a high-aperture objective separately maps far- and near-field emission from dipoles near a bare glass or metal-film-coated glass/aqueous interface. Total internal reflection (TIR) excitation of a fluorescent sample gave a BFP pattern interpreted in terms of fluorescent dipole orientation and distance from the interface. Theoretical consideration of this system led to identification of emission characteristics that remove a dipole orientation degeneracy in conventional microscope fluorescence polarization measurements. BFP pattern inspection removes the degeneracy. Alternatively, a BFP mask blocking a small fraction of emitted light in a standard imaging microscope prevents uniform collection of the BFP intensity and also eliminates the degeneracy. The BFP pattern from a single photoactivated photoactivatable green fluorescent protein (PAGFP) tagged myosin in a muscle fiber was observed despite the large background light from the highly concentrated myosin tagged with unphotoactivated PAGFP. This was accomplished by imaging the pattern from a nontelecentric plane, where most of the background intensity's pattern was translated laterally from the single-molecule object's pattern. TIR/BFP pattern imaging requires a simple alteration of the fluorescence microscope and is consistent with single-molecule imaging in a fluorophore dense three-dimensional object like a muscle fiber.
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http://dx.doi.org/10.1117/1.3155520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2745092PMC
September 2009

The myosin C-loop is an allosteric actin contact sensor in actomyosin.

Biochemistry 2009 Jun;48(23):5263-75

Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA.

Actin and myosin form the molecular motor in muscle. Myosin is the enzyme performing ATP hydrolysis under the allosteric control of actin such that actin binding initiates product release and force generation in the myosin power stroke. Non-equilibrium Monte Carlo molecular dynamics simulation of the power stroke suggested that a structured surface loop on myosin, the C-loop, is the actin contact sensor initiating actin activation of the myosin ATPase. Previous experimental work demonstrated C-loop binds actin and established the forward and reverse allosteric link between the C-loop and the myosin active site. Here, smooth muscle heavy meromyosin C-loop chimeras were constructed with skeletal (sCl) and cardiac (cCl) myosin C-loops substituted for the native sequence. In both cases, actin-activated ATPase inhibition is indicated mainly by the lower V(max). In vitro motility was also inhibited in the chimeras. Motility data were collected as a function of myosin surface density, with unregulated actin, and with skeletal and cardiac isoforms of tropomyosin-bound actin for the wild type, cCl, and sCl. Slow and fast subpopulations of myosin velocities in the wild-type species were discovered and represent geometrically unfavorable and favorable actomyosin interactions, respectively. Unfavorable interactions are detected at all surface densities tested. Favorable interactions are more probable at higher myosin surface densities. Cardiac tropomyosin-bound actin promotes the favorable actomyosin interactions by lowering the inhibiting geometrical constraint barriers with a structural effect on actin. Neither higher surface density nor cardiac tropomyosin-bound actin can accelerate motility velocity in cCl or sCl, suggesting the element initiating maximal myosin activation by actin resides in the C-loop.
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http://dx.doi.org/10.1021/bi900584qDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2759872PMC
June 2009

PDLIM4, an actin binding protein, suppresses prostate cancer cell growth.

Cancer Invest 2009 Mar;27(3):264-72

Department of Urology, Mayo Clinic College of Medicine, Mayo Clinic, Rochester, MN 55905, USA.

We investigated the molecular function of PDLIM4 in prostate cancer cells. PDLIM4 mRNA and protein-expression levels were reduced in LNCaP, LAPC4, DU145, CWR22, and PC3 prostate cancer cells. The re-expression of PDLIM4 in prostate cancer cells has significantly reduced the cell growth and clonogenicity with G1 phase of cell-cycle arrest. We have shown the direct interaction of PDLIM4 with F-actin. Restoration of PDLIM4 expression resulted in reduction of tumor growth in xenografts. These results suggest that PDLIM4 may function as a tumor suppressor, involved in the control of cell proliferation by associating with actin in prostate cancer cells.
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http://dx.doi.org/10.1080/07357900802406319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3086358PMC
March 2009

Single myosin lever arm orientation in a muscle fiber detected with photoactivatable GFP.

Biochemistry 2009 Feb;48(4):754-65

Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA.

Myosin 2 is the molecular motor in muscle. It binds actin and executes a power stroke by rotating its lever arm through an angle of approximately 70 degrees to translate actin against resistive force. Myosin 2 has evolved to function optimally under crowded conditions where rates and equilibria of macromolecular reactions undergo major shifts relative to those measured in dilute solution. Hence, an important research objective is to detect in situ the lever arm orientation. Single-molecule measurements are preferred because they clarify ambiguities that are unavoidable with ensemble measurements; however, detecting single molecules in the condensed tissue medium where the myosin concentration exceeds 100 muM is challenging. A myosin light chain (MLC) tagged with photoactivatable green fluorescent protein (PAGFP) was constructed. The recombinant MLC physically and functionally replaced native MLC on the myosin lever arm in a permeabilized skeletal muscle fiber. Probe illumination volume was minimized using total internal reflection fluorescence microscopy, and PAGFP was sparsely photoactivated such that polarized fluorescence identified a single probe orientation. Several physiological states of the muscle fiber were characterized, revealing two distinct orientation populations in all states called straight and bent conformations. Conformation occupancy probability varies among fiber states with rigor and isometric contraction at extremes where straight and bent conformations predominate, respectively. Comparison to previous work on single rigor cross-bridges at the A-band periphery where the myosin concentration is low suggests molecular crowding in the A-band promotes occupancy of the straight myosin conformation [Burghardt, T. P., et al. (2007) Biophys. J. 93, 2226]. The latter may have a role in contraction because it provides additional free energy favoring completion of the cross-bridge power stroke.
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http://dx.doi.org/10.1021/bi8017703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2709297PMC
February 2009