Journal: bioRxiv

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

doi: 10.1101/2025.04.06.647477

Figure Lengend Snippet: Analysis of the dynamic conformations of actin filaments crosslinked by alpha-actinin in unipolar configuration in the presence of ATP, Ca 2+ , and S1 using HS-AFM. (A) A longitudinal section profile illustrating for the analysis of two consecutive HHPs, the number of protomer pairs per HHP, and MADs in various actin filaments captured at different time points. The analytic methods used were developed in our previous study . (B) Still AFM images with time labels were shown for conditions with ATP present and after ATP depletion (primarily converted into ADP and inorganic phosphate in solution) to observe the binding of S1 on actin filaments. As ATP levels gradually declined or nearly depleted due to ATP hydrolysis (ATP → ADP + Pi) during the actomyosin reaction, S1 binding to actin filaments persisted for longer durations, enabling capture by HS-AFM at an imaging rate of 330 ms per frame. All actin filaments in the imaging field were aligned with the pointed end (PE) on the left and the barbed end (BE) on the right, reflecting the actual unipolar crosslinking configuration. As similarly done in our previous study by referring to the EM study , the polarity of actin filaments was confirmed by observing the tilted angles of S1, with its head pointing toward the pointed (minus) end (PE), resembling an arrowhead along the filament. This characteristic orientation allowed differentiation between the PE and the barbed (plus) end (BE). Yellow and cyan arrowheads indicate alpha-actinin and S1 molecules, respectively. Scale bars: 25 nm. Color scale: 0-12 nm. (C) Time-dependent changes in two consecutive HHPs, the number of protomer pairs per HHP, and MADs in actin filaments crosslinked by 50 nM α-actinin in the presence of 1 mM ATP, 0.1 mM CaCl₂, and 1 µM S1, but without MgCl 2 . Related to and Video S2 .

Article Snippet: Video S19 Molecular dynamics of bipolar-flexible crosslinking model obtained from MD simulations.

Techniques: Binding Assay, Imaging

Journal: bioRxiv

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

doi: 10.1101/2025.04.06.647477

Figure Lengend Snippet: A ridgeline plot illustrating the fluctuation of HHP in actin filaments crosslinked by alpha-actinin. (A) Still AFM images of actin filaments crosslinked using 50 nM alpha-actinin and imaged in F-buffer with 0.5 mM MgCl 2 and 0.5 mM ATP, but without CaCl 2 . The polarity of the crosslinking configuration was determined based on a previous study . Accordingly, the crosslinking of actin filaments by alpha-actinin was determined as unipolar configuration in this case. It is because no changes were observed in the preferred attachment angle of the crosslinkers once the filament spacing stabilized, indicating that the filaments were incorporated in the same polarity as previously crosslinked filaments (an unipolar configuration). Scale bars: 25 nm. Color scale: 0-12 nm. Related to Video S3 . ( B ) A histogram of HHP measured for actin filaments crosslinked with alpha-actinin. A total of 4270 HHPs were counted.

Article Snippet: Video S19 Molecular dynamics of bipolar-flexible crosslinking model obtained from MD simulations.

Techniques:

Journal: bioRxiv

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

doi: 10.1101/2025.04.06.647477

Figure Lengend Snippet: The crosslinking of actin filaments by alpha-actinin inhibits both the development of mature cofilin clusters and shortening of HHP in actin filaments. ( A ) Time-dependent changes in the peak heights and HHPs of actin filaments incubated with only 600 nM hs cofilin 1 and 1mM ATP. A ∼25% decrease in HHP and the formation of mature cofilin clusters on actin filaments in the absence of alpha-actinin served as positive controls, providing a basis for comparison with the cofilin clusters and HHP shown in B . Red and green arrowheads indicate the peaks in mature cofilin clusters and bare actin segments (served as negative controls), respectively. Time label indicates the time after adding cofilin. Scale bars: 25 nm. Color scale: 0-12 nm. Related to Video S4A. ( B ) Time-dependent changes in the peak heights and HHPs of actin filaments incubated 400 nM α-actinin and 1 mM ATP, and subsequently added with 600 nM cofilin. The cofilin binding and formation of mature cofilin clusters, and the shortening of HHP were significantly suppressed by crosslinking of alpha-actinin. Red arrowheads denote the peaks in mature cofilin clusters within the supertwisted half helices of actin segments (cofilactin) without alpha-actinin, and blue arrowheads indicate those in immature cofilin clusters within the long half helices of actin segments crosslinked with alpha-actinin (cofilactin-actinin), respectively. Scale bars: 25 nm. Color scale: 0-13 nm. Related to Video S4B. ( C ) Histograms of the peak height and HHP measured from various actin filaments with and without alpha-actinin crosslinking, showing the presence of both mature and immature cofilin clusters. These clusters corresponded to short and long HHPs, respectively. Notably, their categorization as mature or immature clusters was based on their differences in peak heights and length of corresponding HHPs, and their comparisons with bare actin (i.e., peak height of 8.6 ± 0.8 nm, HHP of 36.8 ± 4.3 nm), as reported in our previous studies . The mean ± SD heights and lengths are 10.7 ± 1.0 nm (N = 425) and 27.9 ± 3.0 nm (N= 300) for mature clusters of cofilactin segment without alpha-actinin, and 9.9 ± 1.1 nm (N = 2514) and 33.0 ± 4.6 nm (N = 2003) for immature clusters of cofilactin segment with alpha-actinin crosslinking (cofilactin-actinin). A two-population t-test ( p < 0.05) indicates that the differences in height and HHP distribution between the clusters of cofilactin segments without and with alpha-actinin crosslinking are significant ( p = 2.9x10 -38 for height, p = 8.5x10 -68 for HHP).

Article Snippet: Video S19 Molecular dynamics of bipolar-flexible crosslinking model obtained from MD simulations.

Techniques: Incubation, Comparison, Binding Assay

Journal: bioRxiv

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

doi: 10.1101/2025.04.06.647477

Figure Lengend Snippet: Differences in the conformational changes in the actin filaments crosslinked with alpha-actinin in unipolar and bipolar models, including rise, AD, and residue-level interactions. ( A ) Illustrations of the unipolar and bipolar crosslinking models of the actin filaments with alpha-actinin. The unipolar crosslinking configuration occurs when the actin filaments share the same polarity, whereas the bipolar crosslinking configuration appears when the actin filaments align with opposite polarities. The rise and AD analyses were performed using conformations obtained from MD simulations. See the definitions of rise and AD in the Supplementary Methods. ( B ) Quantitative analysis of the inter-filament lengths and alpha‒actin crosslinking angles relative to the filament axis in the unipolar-flexible, bipolar-flexible, and bipolar-fixed models obtained from the MD simulations. In these models, the flexible condition allowed all Cα atoms in alpha-actinin to vibrate freely, while the fixed condition computationally constrained them. ( C ) Analysis of the HHP, rise, and AD in the bare actin filaments and in the unipolar-flexible, bipolar-flexible, and bipolar-fixed models using the conformations derived from the MD simulations. ( D, E ) Characterization of the residue-level interactions between the alpha-actinin ABDs and actin protomers during crosslinking in the unipolar-flexible and bipolar-flexible models. If an atom of one residue was in contact within 2 Å of an atom of another residue, these two residues were considered to be interacting. The probability of the residue contacts, time averaged contact probability, was calculated as the combined sum of interactions between the two alpha-actinin ABDs (green texts) and actin (red texts) residues, resulting in a maximum value of 2. Related to .

Article Snippet: Video S19 Molecular dynamics of bipolar-flexible crosslinking model obtained from MD simulations.

Techniques: Residue, Derivative Assay

Journal: bioRxiv

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

doi: 10.1101/2025.04.06.647477

Figure Lengend Snippet: PCA of the atomic structures and physical motions of alpha-actinin molecules on the basis of conformational libraries derived from unipolar ( A ) and bipolar ( B ) crosslinking models obtained with MD simulations. The alpha-actinin structures are illustrated in tube conformations, with the ABD, neck, rod, and EF12- EF34 domains represented in red, cyan, green, and yellow, respectively. Eigenvectors indicating the direction of motion are shown with white arrows. The final system of unipolar configurations consisted of two actin fibers (26 protomers), one alpha-actinin molecule, 1032 K + atoms, 718 Cl - atoms, 26 Mg 2+ atoms, 26 ADP molecules, and 1,433,011 water molecules, for a total number of 4,479,739 atoms in the system. The bipolar model was constructed similarly, differing only in the inclusion of 1,432,853 water molecules (see Supplementary Methods). Related to Videos S16-S17 .

Article Snippet: Video S19 Molecular dynamics of bipolar-flexible crosslinking model obtained from MD simulations.

Techniques: Derivative Assay, Construct

Journal: bioRxiv

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

doi: 10.1101/2025.04.06.647477

Figure Lengend Snippet:

Article Snippet: Video S19 Molecular dynamics of bipolar-flexible crosslinking model obtained from MD simulations.

Techniques: Residue, Derivative Assay, Binding Assay

Journal: bioRxiv

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

doi: 10.1101/2025.04.06.647477

Figure Lengend Snippet: Quantitative analysis of spacious inter-filament lengths, HHP, and alpha-actinin crosslinking angles relative to the filament axis, based on experimental HS-AFM data for actin filaments crosslinked by alpha-actinin, both in the presence (A) and absence (B) of S1. In our HS-AFM data, alpha-actinin crosslinked the actin filaments in a unipolar configuration. Two alpha-actinin molecules consistently intercalated between actin filaments at every half-helical turn, forming a parallelogram-like configuration. The segments AB, CD, EF, and HG correspond to HHP, while AD, BC, EG, and FH represent inter-filament spaces or lengths. It is noted that the definition of inter-filament spacing differs slightly from that in the MD simulation data . In the MD simulation, spacing was measured by a vertical line connecting between the two filaments, whereas in the AFM data, it is determined by measuring the parallel sides of a parallelogram. As a result, the inter-filament lengths measured in AFM data were larger than that in MD simulations. Statistical differences were assessed using a two-population t- test, with a significant difference confirmed at p ≤ 0.05. See our analysis in Supplementary Methods for details. Related to , , , , and Videos S2–S3 .

Article Snippet: Video S19 Molecular dynamics of bipolar-flexible crosslinking model obtained from MD simulations.

Techniques:

Journal: bioRxiv

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

doi: 10.1101/2025.04.06.647477

Figure Lengend Snippet: Hypothesis outlining the configurations, functions, and localizations of the actin filaments and their crosslinking with alpha-actinin in both unipolar and bipolar models. ( A ) Summary of our experimental findings on how the actin filaments and crosslinking of the unipolar actin filaments with alpha-actinin regulate the binding of S1 and cofilin. ( B ) Illustration depicting the unipolar and bipolar crosslinking conformations, highlighting their distinct localizations and functions within cells. This figure was created using BioRender.com. Related to .

Article Snippet: Video S19 Molecular dynamics of bipolar-flexible crosslinking model obtained from MD simulations.

Techniques: Binding Assay