Biology subjects

Le, N. T. P.

Publications and source records attributed to Le, N. T. P..

3 recordsLinked to original sources

bioRxiv2025

Conformational dynamics of actin filaments crosslinked with alpha-actinin and their roles in suppressing cofilin-induced helical shortening and cluster formation

Actin is a conserved cytoskeletal protein essential for morphogenesis, motility, and division. Its versatility arises from filament assembly and regulation by actin binding proteins. Among these, alpha-actinin organizes filaments into bipolar or unipolar networks, whereas cofilin binds preferentially to ADP-actin regions and forms clusters to shorten the half helical pitch (HHP). Here, we investigated the molecular mechanism of how alpha-actinin alters filament and protomer conformations and influences cofilin binding. Using all-atom molecular dynamics simulations, principal component analysis, and high-speed atomic force microscopy, we show that alpha-actinin crosslinking stabilizes actin filaments in the canonical helical state, thereby preventing the cofilin-induced helical shortening required for cooperative filament decoration. Stabilization occurs without significant changes in protomer twist and rise and subdomain geometry and maintains a flattened protomer conformation that restricts twisting needed for cofilin cooperative binding. By contrast, the isolated alpha-actinin-1 actin binding domain mutant (ABD-E235K), comprising two calponin homology domains (CH1-CH2), transiently binds to actin filaments and induces local transitions from the canonical double-helical filament to the single- and parallel-helical protofilament states, weakly affecting cofilin binding and cluster formation through a distinct structural and binding mechanism. Together, these findings reveal a mechanistically distinct function of full-length alpha-actinin and its isolated ABD and support a stepwise mechanism in which cooperative cofilin binding to double-helical actin filaments requires initial binding to actin regions with shortened HHP, followed by protomer twisting and further helical shortening. By stabilizing the canonical filament architecture or perturbing filament organization, alpha-actinin suppresses these structural transitions and thereby perturbs cofilin cooperativity. TeaserAlpha-actinin stabilizes actin helices, suppressing cofilin cooperative binding through distinct structural mechanisms.

biophysics↗

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

Developing the Computational Image Processing Methodfor Quantitative Analysis of Nanopore Structure Obtained from HS-AFM (AFMnanoQ)

High-Speed Atomic Force Microscopy (HS-AFM) enables imaging of biological structures and dynamics with nanometer spatial and millisecond temporal resolution. AFM images contain three-dimensional (3D) surface information, comprising two-dimensional (2D) lateral (x-y) and one-dimensional (1D) height (z) encoded in pixel intensity. This dynamic structure poses significant challenges for instance boundary detection and morphological analysis. To address this, we develop AFMnanoSALQ, a feature-driven computational framework for semi-automatic labeling and quantitative (SALQ) detection and morphological measurement of HS-AFM data. Unlike conventional methods that rely solely on either visual or geometric features for 2D boundary detection, AFM- nanoSALQ integrates both to extract 3D morphology. It requires neither annotated data nor intensive training, enabling fast deployment at minimal cost. With performance comparable to typical deep-learning models, AFMnanoSALQ facilitates semi-automatic labeling, making it a practical tool for preliminary data inspection and accelerating the creation of training datasets. As a case study, we focus on -hemolysin (HL), a {beta}-barrel pore-forming toxin secreted by Staphylococcus aureus, using both synthetic and experimental AFM data. AFMnanoSALQ provides a foundation for future deep learning studies, enabling both dataset generation and cross-validation between feature-driven and data-driven approaches.

bioinformatics↗