Browse
Submit Data
Databases
API
Help

Dataset Information

15 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Adaptability of myosin V studied by simultaneous detection of position and orientation.

ABSTRACT: We studied the structural dynamics of chicken myosin V by combining the localization power of fluorescent imaging with one nanometer accuracy (FIONA) with the ability to detect angular changes of a fluorescent probe. The myosin V was labeled with bifunctional rhodamine on one of its calmodulin light chains. For every 74 nm translocation, the probe exhibited two reorientational motions, associated with alternating smaller and larger translational steps. Molecules previously identified as stepping alternatively 74-0 nm were found to actually step 64-10 nm. Additional tilting often occurred without full steps, possibly indicating flexibility of the attached myosin heads or probing of their vicinity. Processive myosin V molecules sometimes shifted from the top to the side of actin, possibly to avoid an obstacle. The data indicate marked adaptability of this molecular motor to a nonuniform local environment and provide strong support for a straight-neck model of myosin V in which the lever arm of the leading head is tilted backwards at the prepowerstoke angle.

SUBMITTER: Syed S

PROVIDER: S-EPMC1456946 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Defocused orientation and position imaging (DOPI) of myosin V.

Project description:The centroid of a fluorophore can be determined within approximately 1.5-nm accuracy from its focused image through fluorescence imaging with one-nanometer accuracy (FIONA). If, instead, the sample is moved away from the focus, the point-spread-function depends on both the position and 3D orientation of the fluorophore, which can be calculated by defocused orientation and position imaging (DOPI). DOPI does not always yield position accurately, but it is possible to switch back and forth between focused and defocused imaging, thereby getting the centroid and the orientation with precision. We have measured the 3D orientation and stepping behavior of single bifunctional rhodamine probes attached to one of the calmodulins of the light-chain domain (LCD) of myosin V as myosin V moves along actin. Concomitant with large and small steps, the LCD rotates and then dwells in the leading and trailing position, respectively. The probe angle relative to the barbed end of the actin (beta) averaged 128 degrees while the LCD was in the leading state and 57 degrees in the trailing state. The angular difference of 71 degrees represents rotation of LCD around the bound motor domain and is consistent with a 37-nm forward step size of myosin V. When beta changes, the probe rotates +/-27 degrees azimuthally around actin and then rotates back again on the next step. Our results remove degeneracy in angles and the appearance of nontilting lever arms that were reported.

| S-EPMC1458912 | biostudies-literature

Simultaneous, accurate measurement of the 3D position and orientation of single molecules.

Project description:Recently, single molecule-based superresolution fluorescence microscopy has surpassed the diffraction limit to improve resolution to the order of 20 nm or better. These methods typically use image fitting that assumes an isotropic emission pattern from the single emitters as well as control of the emitter concentration. However, anisotropic single-molecule emission patterns arise from the transition dipole when it is rotationally immobile, depending highly on the molecule's 3D orientation and z position. Failure to account for this fact can lead to significant lateral (x, y) mislocalizations (up to ∼50-200 nm). This systematic error can cause distortions in the reconstructed images, which can translate into degraded resolution. Using parameters uniquely inherent in the double-lobed nature of the Double-Helix Point Spread Function, we account for such mislocalizations and simultaneously measure 3D molecular orientation and 3D position. Mislocalizations during an axial scan of a single molecule manifest themselves as an apparent lateral shift in its position, which causes the standard deviation (SD) of its lateral position to appear larger than the SD expected from photon shot noise. By correcting each localization based on an estimated orientation, we are able to improve SDs in lateral localization from ∼2× worse than photon-limited precision (48 vs. 25 nm) to within 5 nm of photon-limited precision. Furthermore, by averaging many estimations of orientation over different depths, we are able to improve from a lateral SD of 116 (∼4× worse than the photon-limited precision; 28 nm) to 34 nm (within 6 nm of the photon limit).

| S-EPMC3511094 | biostudies-literature

Fluorescence microscopy for simultaneous observation of 3D orientation and movement and its application to quantum rod-tagged myosin V.

Project description:Single molecule fluorescence polarization techniques have been used for three-dimensional (3D) orientation measurements to observe the dynamic properties of single molecules. However, only few techniques can simultaneously measure 3D orientation and position. Furthermore, these techniques often require complex equipment and cumbersome analysis. We have developed a microscopy system and synthesized highly fluorescent, rod-like shaped quantum dots (Q rods), which have linear polarizations, to simultaneously measure the position and 3D orientation of a single fluorescent probe. The optics splits the fluorescence from the probe into four different spots depending on the polarization angle and projects them onto a CCD camera. These spots are used to determine the 2D position and 3D orientation. Q rod orientations could be determined with better than 10° accuracy at 33 ms time resolution. We applied our microscopy and Q rods to simultaneously measure myosin V movement along an actin filament and rotation around its own axis, finding that myosin V rotates 90° for each step. From this result, we suggest that in the two-headed bound state, myosin V necks are perpendicular to one another, while in the one-headed bound state the detached trailing myosin V head is biased forward in part by rotating its lever arm about its own axis. This microscopy system should be applicable to a wide range of dynamic biological processes that depend on single molecule orientation dynamics.

| S-EPMC3325717 | biostudies-literature

Combining deep learning approaches and point spread function engineering for simultaneous 3D position and 3D orientation measurements of fluorescent single molecules.

Project description:Point Spread Function (PSF) engineering is an effective method to increase the sensitivity of single-molecule fluorescence images to specific parameters. Classical phase mask optimization approaches have enabled the creation of new PSFs that can achieve, for example, localization precision of a few nanometers axially over a capture range of several microns with bright emitters. However, for complex high-dimensional optimization problems, classical approaches are difficult to implement and can be very time-consuming for computation. The advent of deep learning methods and their application to single-molecule imaging has provided a way to solve these problems. Here, we propose to combine PSF engineering and deep learning approaches to obtain both an optimized phase mask and a neural network structure to obtain the 3D position and 3D orientation of fixed fluorescent molecules. Our approach allows us to obtain an axial localization precision around 30 nanometers, as well as an orientation precision around 5 degrees for orientations and positions over a one micron depth range for a signal-to-noise ratio consistent with what is typical in single-molecule cellular imaging experiments.

| S-EPMC10310311 | biostudies-literature

Position and orientation inference via on-board triangulation.

Project description:This work proposes a new approach to determine the spatial location and orientation of an object using measurements performed on the object itself. The on-board triangulation algorithm we outline could be implemented in lieu of, or in addition to, well-known alternatives such as Global Positioning System (GPS) or standard triangulation, since both of these correspond to significantly different geometric pictures and necessitate different hardware and algorithms. We motivate the theory by describing situations in which on-board triangulation would be useful and even preferable to standard methods. The on-board triangulation algorithm we outline involves utilizing dumb beacons which broadcast omnidirectional single frequency radio waves, and smart antenna arrays on the object itself to infer the direction of the beacon signals, which may be used for onboard calculation of the position and orientation of the object. Numerical examples demonstrate the utility of the method and its noise tolerance.

| S-EPMC5482487 | biostudies-literature

Myosin phosphatase Fine-tunes Zebrafish Motoneuron Position during Axonogenesis.

Project description:During embryogenesis the spinal cord shifts position along the anterior-posterior axis relative to adjacent tissues. How motor neurons whose cell bodies are located in the spinal cord while their axons reside in adjacent tissues compensate for such tissue shift is not well understood. Using live cell imaging in zebrafish, we show that as motor axons exit from the spinal cord and extend through extracellular matrix produced by adjacent notochord cells, these cells shift several cell diameters caudally. Despite this pronounced shift, individual motoneuron cell bodies stay aligned with their extending axons. We find that this alignment requires myosin phosphatase activity within motoneurons, and that mutations in the myosin phosphatase subunit mypt1 increase myosin phosphorylation causing a displacement between motoneuron cell bodies and their axons. Thus, we demonstrate that spinal motoneurons fine-tune their position during axonogenesis and we identify the myosin II regulatory network as a key regulator.

| S-EPMC5147773 | biostudies-literature

Acetabular orientation: anterolateral approach in the supine position.

Project description:The anterolateral approach in the supine position provides excellent visualization of the acetabulum. The main advantage of the approach, a low dislocation rate, has been demonstrated in the literature, while the purported disadvantage of abductor dysfunction has not been clearly delineated in the literature. The technique requires meticulous attention to preservation of the gluteus medius and minimus tendons. Impingement and dislocation are avoided by careful attention to the preparation of acetabulum, critical assessment of the implanted components, and intraoperative trialing. Leg lengths are assessed through direct palpation of the malleoli. Routine use of postoperative hip precautions is not necessary when this approach is utilized. The anterolateral approach in the supine position allows for a reproducible result with a low dislocation rate.Level V, therapeutic study. See the Guidelines for Authors for a complete description of levels of evidence.

| S-EPMC2600976 | biostudies-other

Relative Orientation of POTRA Domains from Cyanobacterial Omp85 Studied by Pulsed EPR Spectroscopy.

Project description:Many proteins of the outer membrane of Gram-negative bacteria and of the outer envelope of the endosymbiotically derived organelles mitochondria and plastids have a ?-barrel fold. Their insertion is assisted by membrane proteins of the Omp85-TpsB superfamily. These proteins are composed of a C-terminal ?-barrel and a different number of N-terminal POTRA domains, three in the case of cyanobacterial Omp85. Based on structural studies of Omp85 proteins, including the five POTRA-domain-containing BamA protein of Escherichia coli, it is predicted that anaP2 and anaP3 bear a fixed orientation, whereas anaP1 and anaP2 are connected via a flexible hinge. We challenged this proposal by investigating the conformational space of the N-terminal POTRA domains of Omp85 from the cyanobacterium Anabaena sp. PCC 7120 using pulsed electron-electron double resonance (PELDOR, or DEER) spectroscopy. The pronounced dipolar oscillations observed for most of the double spin-labeled positions indicate a rather rigid orientation of the POTRA domains in frozen liquid solution. Based on the PELDOR distance data, structure refinement of the POTRA domains was performed taking two different approaches: 1) treating the individual POTRA domains as rigid bodies; and 2) using an all-atom refinement of the structure. Both refinement approaches yielded ensembles of model structures that are more restricted compared to the conformational ensemble obtained by molecular dynamics simulations, with only a slightly different orientation of N-terminal POTRA domains anaP1 and anaP2 compared with the x-ray structure. The results are discussed in the context of the native environment of the POTRA domains in the periplasm.

| S-EPMC4881240 | biostudies-literature

Electrostatic interactions and binding orientation of HIV-1 matrix studied by neutron reflectivity.

Project description:The N-terminal matrix (MA) domain of the HIV-1 Gag protein is responsible for binding to the plasma membrane of host cells during viral assembly. The putative membrane-binding interface of MA was previously mapped by means of mutagenesis and analysis of its trimeric crystal structure. However, the orientation of MA on membranes has not been directly determined by experimental measurements. We present neutron reflectivity measurements that resolve the one-dimensional scattering length density profile of MA bound to a biomimetic of the native viral membrane. A molecular refinement procedure was developed using atomic structures of MA to determine the orientation of the protein on the membrane. The orientation defines a lipid-binding interface consistent with previous mutagenesis results. The MA protein maintains this orientation without the presence of a myristate group, driven only by electrostatic interactions. Furthermore, MA is found to penetrate the membrane headgroup region peripherally such that only the side chains of specific Lys and Arg residues interact with the surface. The results suggest that electrostatic interactions are sufficient to favorably orient MA on viral membrane mimics. The spatial determination of the membrane-bound protein demonstrates the ability of neutron reflectivity to discern orientation and penetration under physiologically relevant conditions.

| S-EPMC2955346 | biostudies-literature

Myosin and Other Energy-Transducing ATPases: Structural Dynamics Studied by Electron Paramagnetic Resonance.

Project description:The objective of this article was to document the energy-transducing and regulatory interactions in supramolecular complexes such as motor, pump, and clock ATPases. The dynamics and structural features were characterized by motion and distance measurements using spin-labeling electron paramagnetic resonance (EPR) spectroscopy. In particular, we focused on myosin ATPase with actin-troponin-tropomyosin, neural kinesin ATPase with microtubule, P-type ion-motive ATPase, and cyanobacterial clock ATPase. Finally, we have described the relationships or common principles among the molecular mechanisms of various energy-transducing systems and how the large-scale thermal structural transition of flexible elements from one state to the other precedes the subsequent irreversible chemical reactions.

| S-EPMC7014194 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data