The Qdot-Labeled Actin Super-Resolution Motility Assay Measures Low-Duty Cycle Muscle Myosin Step Size

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

Analytical Comparison of Natural and Pharmaceutical Ventricular Myosin Activators

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 <i>in vivo</i> and is undergoing testing for heart failure therapy. <i>In vitro</i> 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

Analytical Comparison of Natural and Pharmaceutical Ventricular Myosin Activators

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 <i>in vivo</i> and is undergoing testing for heart failure therapy. <i>In vitro</i> 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

The Qdot-Labeled Actin Super-Resolution Motility Assay Measures Low-Duty Cycle Muscle Myosin Step Size

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

Diagram summarizes the inter-regulation between ASPP2 and RAS.

<p>ASPP2 binds active RAS at the plasma membrane, thereby increasing RAS signaling to its downstream pathway effectors Raf/MAPK. Activated MAPK phosphorylates ASPP2 which can then relocate to the nucleus and activate p53 pro-apoptotic signaling. </p

Suppression and Revival of Superconducting Phase Coherence in Monolayer FeSe/SrTiO₃

Monolayer FeSe grown on SrTiO3 (FeSe/STO) is an interfacial high-temperature superconductor distinctively different from bulk FeSe. However, the superconducting phase coherence of the interface is challenging to probe due to its fragility in the atmosphere. Here, we perform in situ mutual inductance under ultrahigh vacuum on FeSe/STO in combination with band mapping by angle-resolved photoemission spectroscopy. We find that even though the monolayer shows a gap-closing temperature above 50 K, no diamagnetism is visible down to 5 K. This is the case for few-layer FeSe/STO until it exceeds a critical number of five layers, where diamagnetism suddenly appears. The suppression of diamagnetism in the monolayer is also lifted by depositing a top FeTe layer. However, Tc and superfluid density both decrease with thicker FeTe, suggesting unconventional electron pairing and phase coherence competition. Our observation may be understood by a scenario in which the interfacial superconducting phase coherence is highly anisotropic

The Sypro Ruby stained and immunoblotted SDS-PAGE gels.

<p>The Sypro Ruby gel band intensities establish ZRLC content in a zebrafish adult heart extract relative to a known amount of in vitro expressed HCRLC (panel a). These samples produce calibrated blot intensities for HCRLC and ZRLC under a standardized protein immunoblotting protocol using the RLC antibody as described in Methods and previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174690#pone.0174690.ref011" target="_blank">11</a>]. The standards are compared to immunoblots from HCRLC-GFP and ZRLC in WT and TgGFP(+) embryos (<b>panel b</b>) to measure relative fractions of HCRLC-GFP and ZRLC content (ZRLC_rep, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174690#pone.0174690.e001" target="_blank">Eq 1</a>). The relative amount of ZRLC in WT and TgGFP(+) embryo hearts confirmed ZRLC content removed (ZRLC_rem, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174690#pone.0174690.e002" target="_blank">Eq 2</a>) by their comparison to control β-actin expression (<b>panel c</b>).</p

Wild-type ASPP2, but not mutant ASPP2 (S827A), translocates to the cytosol and nucleus upon oncogenic RAS activation and this results in an increased interaction with p53.

<p>(<b>A</b>) RAS activation induces cytoplasmic and nuclear translocation of wild-type ASPP2 but not ASPP2 (S827A) in HKe3 ER:HRAS12 cells as detected by immunofluorescence. Arrows indicate cell membrane and stars indicate cytosol. (<b>B</b>) RAS activation enhances the binding of wild-type ASPP2 but not ASPP2 (S827A) to p53. Total cell lysates from HKe3 ER:HRASV12 cells treated with or without 4-OHT were immunoprecipitated with an anti-p53 antibody or control IgG as indicated. </p

Координация деятельности субъектов профессиональной ориентации в общеобразовательной организации

<div><p>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.</p><p>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.</p><p>Overall, the approach is a unique bottom-up single molecule mechanical characterization of a hierarchically functional native muscle myosin.</p></div

Myosin flux through the 4-pathway network contrasting 3 phases of muscle contraction in the beating heart.

<p>Myosin begins and ends detached from actin and with ATP bound in the contraction cycle. The green box with dashed line boundaries group the strong actomyosin bound states. Blue vector <b>v</b> at the end of the myosin lever-arm is positive net force on, and positive velocity of, the thick filament in units where amplitudes are equal. Fluxes through the network, f_i, differ depending on contraction phases (values in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174690#pone.0174690.t001" target="_blank">Table 1</a>). Measured values for f_i are in green while computed values are indicated in black. Four pathways cross from beginning to end of the contraction cycle. The top pathway populated by flux f₁ executes an 8 nm step-size. The middle pathway populated by flux f₃ executes a 3 nm step-size. It releases Pi while weakly actin bound without doing work. The bottom pathway populated by flux f₄ is branched and executes 5 or 5+3 nm step-sizes. The branch from the bottom pathway is populated by flux f₇ and executes the 5+3 nm step-size. Strain sensitivity is modeled with mechanisms in two subpathways within the shaded regions. The upper mechanism is populated by fluxes f₂ and f₈ from the 8 and 3 nm steps when the ELC N-terminus binds actin for actomyosin in rigor. The taut (blue line), intermediate (green curve), and slack (red wave) ELC N-terminus for muscle in near-isometric, auxotonic, or unloaded phases have high, modest, or zero strain when net force <b>v</b> is zero in isometric, intermediate in auxotonic, or high in unloaded phase. The linear (blue) actin bound ELC N-terminus is proposed to inhibit ATP binding by lowering active site accessibility for ATP at the small arrow near the myosin head. Inhibited ATP binding extends actomyosin attachment time indicated by the clock icon and quantitated in our single myosin measurements as a 0 length step. The lower mechanism is populated by flux f₆ from the 5 nm step with ADP bound. Near-isometric, auxotonic, or unloaded phases have high, intermediate, or zero strain (of an unspecified myosin element) when net force is zero, intermediate, or high. Strain lowers ADP release rate. Short duration ADP rate inhibition flux, f₇, leaves to continue with the 3 nm step. Long duration ADP rate inhibition flux, f₆ − f₇, continues with the 0 length step. For either the ATP accessibility or ADP release rate mediated mechanisms (top or bottom strain sensing mechanisms), low net force inhibits myosin cycling by extending the time myosin is strongly actin bound by >2Δt.</p