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1.
Proc Natl Acad Sci U S A ; 121(13): e2401625121, 2024 Mar 26.
Article in English | MEDLINE | ID: mdl-38507449

ABSTRACT

Molecular motors employ chemical energy to generate unidirectional mechanical output against a track while navigating a chaotic cellular environment, potential disorder on the track, and against Brownian motion. Nevertheless, decades of nanometer-precise optical studies suggest that myosin-5a, one of the prototypical molecular motors, takes uniform steps spanning 13 subunits (36 nm) along its F-actin track. Here, we use high-resolution interferometric scattering microscopy to reveal that myosin takes strides spanning 22 to 34 actin subunits, despite walking straight along the helical actin filament. We show that cumulative angular disorder in F-actin accounts for the observed proportion of each stride length, akin to crossing a river on variably spaced stepping stones. Electron microscopy revealed the structure of the stepping molecule. Our results indicate that both motor and track are soft materials that can adapt to function in complex cellular conditions.


Subject(s)
Actins , Myosin Type V , Actins/chemistry , Myosins/chemistry , Actin Cytoskeleton/chemistry , Motion , Myosin Type V/chemistry
2.
bioRxiv ; 2023 Jul 16.
Article in English | MEDLINE | ID: mdl-37503193

ABSTRACT

Molecular motors employ chemical energy to generate unidirectional mechanical output against a track. By contrast to the majority of macroscopic machines, they need to navigate a chaotic cellular environment, potential disorder in the track and Brownian motion. Nevertheless, decades of nanometer-precise optical studies suggest that myosin-5a, one of the prototypical molecular motors, takes uniform steps spanning 13 subunits (36 nm) along its F-actin track. Here, we use high-resolution interferometric scattering (iSCAT) microscopy to reveal that myosin takes strides spanning 22 to 34 actin subunits, despite walking straight along the helical actin filament. We show that cumulative angular disorder in F-actin accounts for the observed proportion of each stride length, akin to crossing a river on variably-spaced stepping stones. Electron microscopy revealed the structure of the stepping molecule. Our results indicate that both motor and track are soft materials that can adapt to function in complex cellular conditions.

4.
Mol Cell ; 82(6): 1089-1106.e12, 2022 03 17.
Article in English | MEDLINE | ID: mdl-35231400

ABSTRACT

The recruitment of signaling proteins into activated receptor tyrosine kinases (RTKs) to produce rapid, high-fidelity downstream response is exposed to the ambiguity of random diffusion to the target site. Liquid-liquid phase separation (LLPS) overcomes this by providing elevated, localized concentrations of the required proteins while impeding competitor ligands. Here, we show a subset of phosphorylation-dependent RTK-mediated LLPS states. We then investigate the formation of phase-separated droplets comprising a ternary complex including the RTK, (FGFR2); the phosphatase, SHP2; and the phospholipase, PLCγ1, which assembles in response to receptor phosphorylation. SHP2 and activated PLCγ1 interact through their tandem SH2 domains via a previously undescribed interface. The complex of FGFR2 and SHP2 combines kinase and phosphatase activities to control the phosphorylation state of the assembly while providing a scaffold for active PLCγ1 to facilitate access to its plasma membrane substrate. Thus, LLPS modulates RTK signaling, with potential consequences for therapeutic intervention.


Subject(s)
Protein Tyrosine Phosphatase, Non-Receptor Type 11 , Signal Transduction , Phosphorylation , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Tyrosine/metabolism , src Homology Domains
5.
Nano Lett ; 21(3): 1213-1220, 2021 02 10.
Article in English | MEDLINE | ID: mdl-33253583

ABSTRACT

Inferring the organization of fluorescently labeled nanosized structures from single molecule localization microscopy (SMLM) data, typically obscured by stochastic noise and background, remains challenging. To overcome this, we developed a method to extract high-resolution ordered features from SMLM data that requires only a low fraction of targets to be localized with high precision. First, experimentally measured localizations are analyzed to produce relative position distributions (RPDs). Next, model RPDs are constructed using hypotheses of how the molecule is organized. Finally, a statistical comparison is used to select the most likely model. This approach allows pattern recognition at sub-1% detection efficiencies for target molecules, in large and heterogeneous samples and in 2D and 3D data sets. As a proof-of-concept, we infer ultrastructure of Nup107 within the nuclear pore, DNA origami structures, and α-actinin-2 within the cardiomyocyte Z-disc and assess the quality of images of centrioles to improve the averaged single-particle reconstruction.


Subject(s)
DNA , Single Molecule Imaging
6.
Vision Res ; 92: 75-84, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23994625

ABSTRACT

The accommodative response (AR) to changes in dioptric accommodative stimulus (AS) during the latency period and onset of accommodation was investigated. Participants monocularly observed one period of a square wave in AS, with a 2-D baseline and mean, and amplitude 1 D or 2 D; the period of the square wave ranged from 0.10 s to 1.00 s; both increases and decreases were used for the first step in AS. At periods of 0.30s and longer, accommodation was found to respond to both levels of the stimulus. Rapid retinal monitoring appeared to be taking place for such stimuli. The amplitudes of peaks in AR did not usually depend on whether a particular level of AS occurred first or second, but for 8/40 conditions, a significant difference was found, with a stronger response when the level of AS occurred second. Null or incorrect responses were also observed in many trials, possibly linked with the natural microfluctuations of accommodation. Minimum response times to the changes in AS were observed, which increased with decreasing period of the AS. The time interval between peaks in the AR decreased with decreasing period of the AS. The findings were consistent with a parallel processing model previously proposed for saccades, where input from a later change in stimulus may enter an element of the control system when that element has finished processing an earlier change. More than one change in stimulus may therefore be passing through the multi-element control system at a time.


Subject(s)
Accommodation, Ocular/physiology , Refraction, Ocular/physiology , Adult , Female , Humans , Male , Photic Stimulation/methods , Reaction Time/physiology , Vision, Monocular , Young Adult
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