Biology subjects

Uyeda, T.

Publications and source records attributed to Uyeda, T..

3 recordsLinked to original sources

bioRxiv2025

Atomic Conformational Dynamics and Actin-Crosslinking Function of Alpha-Actinin Revealed by SimHS-AFMfit

Many molecular systems, such as intrinsically disordered proteins and flexible multi-domain complexes, are highly dynamic and often inaccessible to conventional X-ray crystallography or cryo-EM due to their conformational heterogeneity and flexibility. As a result, resolving their atomic-level dynamics remains a significant challenge. In this study, we present SimHS-AFMfit-MD, an integrative framework that combines high-speed atomic force microscopy (HS-AFM), molecular dynamics (MD) simulations, and AFMfit-based structural modeling to reconstruct dynamic protein conformations at atomic resolution. Using alpha-actinin, an actin crosslinking protein, as a challenging test system, we show that AFMfit guided by nonlinear normal mode analysis (AFMfit-NMA) enables accurate structural fitting, while guiding AFMfit with MD trajectories (AFMfit-MD) further enhances the flexible fitting performance, achieving closer agreement with unbiased all-atom MD simulation results. This strategy allows us to convert thousands of three-dimensional HS-AFM images into atomic-scale conformational ensembles, revealing the twisting and bending transitions underlying Ca{superscript 2}-bound and Ca{superscript 2}-unbound alpha-actinin. Together, our results establish a hybrid computational-experimental approach that bridges the spatial and, to some extent, temporal resolution gaps between simulation and imaging, paving the way for real-time visualization of protein conformational dynamics at the atomic scale. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/647477v3_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@147c1aforg.highwire.dtl.DTLVardef@1fd0036org.highwire.dtl.DTLVardef@118da3forg.highwire.dtl.DTLVardef@a062c3_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Actin Painting: a multicolor fluorescence staining method highlighting cell type-specific differences in the composition of filamentous actin architectures.

Actin is a major structural component of the cytoskeleton in eukaryotic cells, and filamentous actin (F-actin) forms a variety of types of cellular structures. Many actin probes have been developed to visualize F-actin architectures in eukaryotic cells. However, it is known that double-stained images of F-actin obtained by two different types of actin probes often show partial inconsistencies. While developing actin probes, we observed that the merged images of three distinct actin probes, each labeled with a different fluorescent dye (red, green and blue), enabled various F-actin architectures to be distinguished based on color. This differentiation arises from slight variations in the distribution of each actin probe, and results in a unique color combination of actin probes that reflects the relative intensities of the three fluorescent dyes. In this report, we introduce a new cell staining method, named Actin Painting, which exploits the unique affinity of actin-binding molecules in each actin probe to F-actin architectures. This technique allows the classification of various cell types based on their specific actin cytoskeleton.

cell biology↗

bioRxiv2020

Actin binding domain of Rng2 strongly inhibits actin movement on myosin II HMM through structural changes of actin filaments

Substoichiometric binding of certain actin-binding proteins induces conformational changes in a disproportionally large number of actin protomers in actin filaments. Here, we report a case in which such conformational changes in actin filaments have profound functional consequences. Rng2 is an IQGAP protein implicated in the assembly and contraction of contractile rings in Schizosaccharomyces pombe. We found that the calponin-homology actin-binding domain of Rng2 (Rng2CHD) strongly inhibits the motility of actin filaments on myosin II in vitro. On skeletal muscle myosin II-coated surfaces, Rng2CHD halved the sliding speed of actin filaments at a binding ratio of 1.3% (=1/77), and virtually stopped movement at a binding ratio of 11% (=1/9). Rng2CHD also inhibited actin movements on Dictyostelium myosin II, but in this case by inducing the detachment of actin filaments from myosin II-coated surfaces. Rng2CHD induced cooperative structural changes of actin filaments accompanied by shortening of the filament helical pitch, and reduced the affinity between actin filaments and subfragment 1 (S1) of muscle myosin II in the presence of ADP. Intriguingly, actin-activated ATPase of S1 was hardly inhibited by Rng2CHD. We suggest that sparsely bound Rng2CHD induces global structural changes of actin filaments and interferes with the force generation by actin-myosin II.

biophysics↗