35 research outputs found
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/SrTiO<sub>3</sub>
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<sub>rep</sub>, <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<sub>rem</sub>, <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<sub>i</sub>, 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<sub>i</sub> 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 f<sub>1</sub> executes an 8 nm step-size. The middle pathway populated by flux f<sub>3</sub> executes a 3 nm step-size. It releases Pi while weakly actin bound without doing work. The bottom pathway populated by flux f<sub>4</sub> is branched and executes 5 or 5+3 nm step-sizes. The branch from the bottom pathway is populated by flux f<sub>7</sub> 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 f<sub>2</sub> and f<sub>8</sub> 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 f<sub>6</sub> 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, f<sub>7</sub>, leaves to continue with the 3 nm step. Long duration ADP rate inhibition flux, f<sub>6</sub> − f<sub>7</sub>, 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