Biology subjects

Alushin, G. M.

Publications and source records attributed to Alushin, G. M..

13 recordsLinked to original sources

bioRxiv2026

Structural principles of transcriptional collisions

RNA polymerase (RNAP) must navigate crowded genomic tracks to robustly produce transcripts. DNA-bound proteins act as transcription-impeding roadblocks, which are eventually overcome by RNAPs through unclear mechanisms. Here, we use cryo-electron microscopy to visualize actively transcribing E. coli RNAP upon collision with an inactivated restriction enzyme (EcoRI*) or with another converging RNAP. Both collisions induce RNAP backtracking into an inactive swiveled state. Swiveling is coupled to DNA deformation through a characteristic structural landscape, mediating RNAP pause stabilization across distinct collision geometries. EcoRI* roadblock bypass efficiency is impacted by factors that modulate RNAP swiveling, backtracking rescue, and roadblock stability. In comparison, head-on RNAP-RNAP collisions feature substantial heterogeneity suggestive of sustained dynamics, with variable inter-RNAP distances that are modulated by nascent transcript hairpins which position bidirectional termination sites. By resolving, to our knowledge, novel structures of actively transcribing RNAP undergoing collisions, we provide a mechanistic framework for interpreting mechanical conflicts during transcription.

biophysics↗

bioRxiv2026

Force-modulated structural landscape of the catch bonding F-actin crosslinker α-actinin-4

Catch bonds, noncovalent supramolecular interactions whose lifetimes are increased by force, are ubiquitous in mechanical signaling pathways. The structural mechanisms of catch-bonding proteins remain unclear, hampering efforts to decipher how they are dysregulated in disease and exploit them therapeutically. The crosslinker -actinin-4 (ACTN4) forms catch bonds with actin filaments (F-actin) to support the function of kidney podocytes, and its force-insensitive K255E variant causes autosomal dominant focal segmental glomerulosclerosis (FSGS). Using cryo-electron microscopy (cryo-EM), we find that wild-type ACTN4 engages F-actin in two modes, which biochemical experiments and molecular dynamics simulations assign as strong- and weak-binding states, while K255E ACTN4 only populates the strong binding state. By implementing a cryo-EM platform for applying tension across crosslinker-F-actin interfaces using myosin motors, we find that force promotes a weak-to-strong binding transition for wild-type ACTN4, consistent with a two-state catch bond model. Beyond providing mechanistic insight into how the K255E mutation disrupts ACTN4 F-actin catch-bonding in FSGS, this approach enables structural dissection of force-sensitive actin-binding proteins.

biophysics↗

bioRxiv2024

Afadin mediates cadherin-catenin complex clustering on F-actin linked to cooperative binding and filament curvature

The E-cadherin-{beta}-catenin-E-catenin (cadherin-catenin) complex couples the cytoskeletons of neighboring cells at adherens junctions (AJs) to mediate force transmission across epithelia. Mechanical force and auxiliary binding partners converge to stabilize the cadherin-catenin complexs inherently weak binding to actin filaments (F-actin) through unclear mechanisms. Here we show that afadins coiled-coil (CC) domain and vinculin synergistically enhance the cadherin-catenin complexs F-actin engagement. The cryo-EM structure of an E-cadherin-{beta}-catenin-E-catenin-vinculin-afadin-CC supra-complex bound to F-actin reveals that afadin-CC bridges adjacent E-catenin actin-binding domains along the filament, stabilizing flexible E-catenin segments implicated in mechanical regulation. These cooperative binding contacts promote the formation of supra-complex clusters along F-actin. Additionally, cryo-EM variability analysis links supra-complex binding along individual F-actin strands to nanoscale filament curvature, a deformation mode associated with cytoskeletal forces. Collectively, this work elucidates a mechanistic framework by which vinculin and afadin tune cadherin-catenin complex-cytoskeleton coupling to support AJ function across varying mechanical regimes.

biophysics↗

bioRxiv2024

Myosin forces elicit an F-actin structural landscape that mediates mechanosensitive protein recognition

Cells mechanically interface with their surroundings through cytoskeleton-linked adhesions, allowing them to sense physical cues that instruct development and drive diseases such as cancer. Contractile forces generated by myosin motor proteins mediate these mechanical signal transduction processes through unclear protein structural mechanisms. Here, we show that myosin forces elicit structural changes in actin filaments (F-actin) that modulate binding by the mechanosensitive adhesion protein -catenin. Using correlative cryo-fluorescence microscopy and cryo-electron tomography, we identify F-actin featuring domains of nanoscale oscillating curvature at cytoskeleton-adhesion interfaces enriched in zyxin, a marker of actin-myosin generated traction forces. We next introduce a reconstitution system for visualizing F-actin in the presence of myosin forces with cryo-electron microscopy, which reveals morphologically similar superhelical F-actin spirals. In simulations, transient forces mimicking tugging and release of filaments by motors produce spirals, supporting a mechanistic link to myosins ATPase mechanochemical cycle. Three-dimensional reconstruction of spirals uncovers extensive asymmetric remodeling of F-actins helical lattice. This is recognized by -catenin, which cooperatively binds along individual strands, preferentially engaging interfaces featuring extended inter-subunit distances while simultaneously suppressing rotational deviations to regularize the lattice. Collectively, we find that myosin forces can deform F-actin, generating a conformational landscape that is detected and reciprocally modulated by a mechanosensitive protein, providing a direct structural glimpse at active force transduction through the cytoskeleton.

biophysics↗

bioRxiv2024

Force-activated zyxin assemblies coordinate actin nucleation and crosslinking to orchestrate stress fiber repair

As the cytoskeleton sustains cell and tissue forces, it incurs physical damage that must be repaired to maintain mechanical homeostasis. The LIM-domain protein zyxin detects force-induced ruptures in actin-myosin stress fibers, coordinating downstream repair factors to restore stress fiber integrity through unclear mechanisms. Here, we reconstitute stress fiber repair with purified proteins, uncovering detailed links between zyxins force-regulated binding interactions and cytoskeletal dynamics. In addition to binding individual tensed actin filaments (F-actin), zyxins LIM domains form force-dependent assemblies that bridge broken filament fragments. Zyxin assemblies engage repair factors through multi-valent interactions, coordinating nucleation of new F-actin by VASP and its crosslinking into aligned bundles by [a]-actinin. Through these combined activities, stress fiber repair initiates within the cores of micron-scale damage sites in cells, explaining how these F-actin depleted regions are rapidly restored. Thus, zyxins force-dependent organization of actin repair machinery inherently operates at the network scale to maintain cytoskeletal integrity.

biophysics↗

bioRxiv2024

Fascin structural plasticity mediates flexible actin bundle construction

Fascin crosslinks actin filaments (F-actin) into bundles that support tubular membrane protrusions including filopodia and stereocilia. Fascin dysregulation drives aberrant cell migration during metastasis, and fascin inhibitors are under development as cancer therapeutics. Here, we use cryo-electron microscopy, cryo-electron tomography coupled with custom denoising, and computational modeling to probe fascins F-actin crosslinking mechanisms across spatial scales. Our fascin crossbridge structure reveals an asymmetric F-actin binding conformation that is allosterically blocked by the inhibitor G2. Reconstructions of seven-filament hexagonal bundle elements, variability analysis, and simulations show how structural plasticity enables fascin to bridge varied inter-filament orientations, accommodating mismatches between F-actins helical symmetry and bundle hexagonal packing. Tomography of many-filament bundles and modeling uncovers geometric rules underlying emergent fascin binding patterns, as well as the accumulation of unfavorable crosslinks that limit bundle size. Collectively, this work shows how fascin harnesses fine-tuned nanoscale structural dynamics to build and regulate micron-scale F-actin bundles.

biophysics↗

bioRxiv2023

A platform for dissecting force sensitivity and multivalency in actin networks

The physical structure and dynamics of cells are supported by micron-scale actin networks with diverse geometries, protein compositions, and mechanical properties. These networks are composed of actin filaments and numerous actin binding proteins (ABPs), many of which engage multiple filaments simultaneously to crosslink them into specific functional architectures. Mechanical force has been shown to modulate the interactions between several ABPs and individual actin filaments, but it is unclear how this phenomenon contributes to the emergent force-responsive functional dynamics of actin networks. Here, we engineer filament linker complexes and combine them with photo-micropatterning of myosin motor proteins to produce an in vitro reconstitution platform for examining how force impacts the behavior of ABPs within multi-filament assemblies. Our system enables the monitoring of dozens of actin networks with varying architectures simultaneously using total internal reflection fluorescence microscopy, facilitating detailed dissection of the interplay between force-modulated ABP binding and network geometry. We apply our system to study a dimeric form of the critical cell-cell adhesion protein -catenin, a model force-sensitive ABP. We find that myosin forces increase -catenins engagement of small filament bundles embedded within networks. This activity is absent in a force-sensing deficient mutant, whose binding scales linearly with bundle size in both the presence and absence of force. These data are consistent with filaments in smaller bundles bearing greater per-filament loads that enhance -catenin binding, a mechanism that could equalize -catenins distribution across actin-myosin networks of varying sizes in cells to regularize their stability and composition.

biophysics↗

bioRxiv2022

Actin nucleotide state modulates the F-actin structural landscape evoked by bending forces

ATP hydrolysis-coupled actin polymerization is a fundamental mechanism of cellular force generation. Force and actin filament (F-actin) nucleotide state in turn modulate the engagement of actin binding proteins (ABPs) to regulate actin dynamics through unknown mechanisms. Here, we show that bending forces evoke structural transitions in F-actin which are modulated by actin nucleotide state. Cryo-electron microscopy (cryo-EM) structures of ADP- and ADP-Pi-F-actin with sufficient resolution to visualize bound solvent reveal inter-subunit interactions primarily bridged by waters which could contribute to lattice flexibility. Despite substantial ordered solvent differences in the nucleotide binding cleft, these structures feature essentially indistinguishable protein backbone conformations which are unlikely to be discriminable by ABPs. We next introduce a machine-learning enabled pipeline for reconstructing bent filaments, enabling us to visualize both continuous structural variability and side-chain level detail. Bent F-actin structures reveal major rearrangements at inter-subunit interfaces characterized by striking alterations of helical twist and deformations of individual protomers which are distinct in ADP- and ADP-Pi-F-actin. This suggests phosphate rigidifies actin subunits to alter F-actins bending structural landscape. We therefore propose actin nucleotide state serves as a co-regulator of F-actin mechanical regulation, as bending forces evoke nucleotide-state dependent conformational transitions that could be readily detected by ABPs.

biophysics↗

bioRxiv2021

Structural mechanism for bi-directional actin crosslinking by T-plastin

To fulfill the cytoskeletons diverse functions in cell mechanics and motility, actin networks with specialized architectures are built by crosslinking proteins, which bridge filaments to control micron-scale network geometry through nanoscale binding interactions via poorly defined structural mechanisms. Here, we introduce a machine-learning enabled cryo-EM pipeline for visualizing active crosslinkers, which we use to analyze human T-plastin, a member of the evolutionarily ancient plastin/fimbrin family of tandem calponin-homology domain (CHD) proteins. We define a sequential bundling mechanism which enables T-plastin to bridge filaments in both parallel and anti-parallel orientations. Our structural, biochemical, and cell biological data highlight inter-CHD linkers as key structural elements underlying flexible but stable crosslinking which are likely to be disrupted by mutations causing hereditary bone diseases. Beyond revealing how plastins are evolutionary optimized to crosslink dense actin networks with mixed polarity, our cryo-EM workflow will broadly enable analysis of the structural mechanisms underlying cytoskeletal network construction. One sentence summaryCryo-EM, biochemical, and cellular studies reveal how the crosslinking protein T-plastin bridges actin filaments in two opposing orientations.

biophysics↗

bioRxiv2021

Myosin-driven Nucleation of Actin Filaments Drives Stereocilia Development Critical for Hearing

The assembly and maintenance of actin-based mechanosensitive stereocilia in the cochlea is critical for lifelong hearing. Myosin-15 (MYO15) is hypothesized to modulate stereocilia height by trafficking actin regulatory proteins to their tip compartments, where actin polymerization must be precisely controlled during development. We identified a mutation (p.D1647G) in the MYO15 motor-domain that initially maintained trafficking, but caused progressive hearing loss by stunting stereocilia growth, revealing an additional function for MYO15. Consistent with its maintenance of tip trafficking in vivo, purified p.D1647G MYO15 modestly reduced actin-stimulated ATPase activity in vitro. Using ensemble and single-filament fluorescence in vitro assays, we demonstrated that wild-type MYO15 directly accelerated actin filament polymerization by driving nucleation, whilst p.D1647G MYO15 blocked this activity. Collectively, our studies suggest direct actin nucleation by MYO15 at the stereocilia tip is necessary for elongation in vivo, and that this is a primary mechanism disrupted in DFNB3 hereditary human hearing loss.

cell biology↗

bioRxiv2021

Structural basis for tunable control of actin dynamics by myosin-15 in mechanosensory stereocilia

The motor protein myosin-15 is necessary for the development and maintenance of mechanosensory stereocilia, and myosin-15 mutations cause profound deafness. In a companion study, we report that myosin-15 nucleates actin filament ("F-actin") assembly and identify a progressive hearing loss mutation (p.D1647G, "jordan") which disrupts stereocilia elongation by inhibiting actin polymerization. Here, we present cryo-EM structures of myosin-15 bound to F-actin, providing a framework for interpreting deafness mutations and their impacts on myosin-stimulated actin assembly. Rigor myosin-15 evokes conformational changes in F-actin yet maintains flexibility in actins D-loop, which mediates inter-subunit contacts, while the jordan mutant locks the D-loop in a single conformation. ADP-bound myosin-15 also locks the D-loop, which correspondingly blunts actin-polymerization stimulation. We propose myosin-15 enhances polymerization by bridging actin protomers, regulating nucleation efficiency by modulating actins structural plasticity in a myosin nucleotide-state dependent manner. This tunable regulation of actin polymerization could be harnessed to precisely control stereocilium height.

biophysics↗

bioRxiv2020

Mechanosensing through direct binding of tensed F-actin by LIM domains

Mechanical signals transmitted through the cytoplasmic actin cytoskeleton must be relayed to the nucleus to control gene expression. LIM domains are protein-protein interaction modules found in cytoskeletal proteins and transcriptional regulators; however, it is unclear if there is a direct link between these two functions. Here we identify three LIM protein families (zyxin, paxillin, and FHL) whose members preferentially localize to the actin cytoskeleton in mechanically-stimulated cells through their tandem LIM domains. A minimal actin-myosin reconstitution system reveals that representatives of all three families directly bind F-actin only in the presence of mechanical force. Point mutations at a site conserved in each LIM domain of these proteins selectively disrupt tensed F-actin binding in vitro and cytoskeletal localization in cells, demonstrating a common, avidity-based mechanism. Finally, we find that binding to tensed F-actin in the cytoplasm excludes the cancer-associated transcriptional co-activator FHL2 from the nucleus in stiff microenvironments. This establishes direct force-activated F-actin binding by FHL2 as a mechanosensing mechanism. Our studies suggest that force-dependent sequestration of LIM proteins on the actin cytoskeleton could be a general mechanism for controlling nuclear localization to effect mechanical signaling.

biophysics↗

bioRxiv2020

Molecular mechanism for direct actin force-sensing by α-catenin

The actin cytoskeleton mediates mechanical coupling between cells and their tissue microenvironments. The architecture and composition of actin networks are modulated by force, but it is unclear how interactions between actin filaments (F-actin) and associated proteins are mechanically regulated. Here, we employ both optical trapping and biochemical reconstitution with myosin motor proteins to show force greater than one piconewton applied solely to F-actin enhances binding by the essential cell-cell adhesion protein E-catenin, but not its homolog vinculin. Near atomic-resolution cryo-electron microscopy structures of both proteins bound to F-actin reveal unique rearrangements that facilitate their flexible C-termini refolding to engage distinct interfaces. Truncating -catenins C-terminus eliminates force-activated F-actin binding, and addition of this motif to vinculin confers force-activated binding, demonstrating that -catenins C-terminus is a modular detector of F-actin tension. Our studies establish that piconewton force on F-actin can enhance partner binding, which we propose mechanically regulates cellular adhesion through -catenin.

biophysics↗