Biology subjects

Takagi, Y.

Publications and source records attributed to Takagi, Y..

At least 19 recordsLinked to original sources

bioRxiv2026

Conformational Remodeling Underlies Activity Loss in Disease-Linked Asparagine Synthetase Variant

Asparagine synthetase deficiency (ASNSD) is a devastating congenital disorder characterized by profound neurological impairment and early childhood mortality. It is associated with pathogenic mutations in the asparagine synthetase (ASNS) gene. Despite the critical role of ASNS in the amino acid cycle, the molecular basis by which ASNSD-linked missense mutations impair enzyme function remains poorly understood. Here, we present a comprehensive characterization of a recurrent ASNSD-linked variant, R48Q. Steady-state kinetic assays reveal severe reductions in L-glutamine-dependent catalysis and disrupted product stoichiometry, implicating impaired interdomain communication. Cryogenic electron microscopy (cryo-EM) and 3D variable analysis of the EM map uncovers altered loop conformations at the N-terminal active site and subtle conformational changes at the C-terminal domain. Consistent with the structural data, molecular dynamics simulations support that the local disruption propagates across the protein, thereby decoupling coordinated domain motions essential for catalysis. Additionally, we demonstrate that the flanking arginine and the affected loop are evolutionarily conserved across Class II glutamine amidotransferases, highlighting their shared mechanistic importance. These findings provide the molecular basis of an ASNSD variant and establish a framework for understanding how point mutations disrupt complex enzyme dynamics, with broad implications for precision medicine. SignificanceUnderstanding how mutations affect multidomain enzymes is crucial for elucidating the molecular mechanisms underlying genetic disorders. Here, we examine the molecular consequences of the R48Q variant in human asparagine synthetase (ASNS), the sole enzyme responsible for de novo L-asparagine synthesis; mutations of this enzyme lead to a fatal neurometabolic disorder, asparagine synthetase deficiency (ASNSD). By combining biochemical, cryogenic electron microscopy, and molecular dynamics simulation, we show that a single N-terminal amino acid substitution disrupts both local and global coordination, impairing enzyme activity. Our work provides the first mechanistic blueprint of an ASNSD-linked variant. These findings not only deepen our understanding of ASNS but also offer a generalized framework for studying the dynamic regulation of multidomain enzymes in disease.

biochemistry↗

bioRxiv2026

A PKD-caveolin axis drives secretory carrier biogenesis at the TGN

Constitutive secretion from the trans-Golgi network (TGN) to the cell surface proceeds via carriers thought to form without a canonical cytoplasmic coat, yet how these carriers are generated remains poorly understood. Here, we identify a distinct population of TGN-to-cell surface carriers transporting influenza hemagglutinin (HA) and uncover a coat-like mechanism underlying their formation. HA carrier biogenesis requires non-vesicular lipid transfer at endoplasmic reticulum (ER)-Golgi membrane contact sites (MCSs) and protein kinase D (PKD) activity. We show that caveolin promotes membrane budding by assembling into cholesterol- and PKD-associated oligomers that act as a membrane-embedded, coat-like scaffold at lipid nanodomain-enriched TGN subdomains. These findings establish caveolin as a structural and regulatory component of TGN export and support a model in which a PKD-caveolin axis couples ER-Golgi lipid transfer to cargo sorting, membrane remodeling and fission during secretory carrier biogenesis.

cell biology↗

bioRxiv2025

Cryo-EM reveals how ASX-173 inhibits human asparagine synthetase to activate the integrated stress response

Targeting asparagine metabolism is a promising strategy for treating asparaginase-resistant acute lymphoblastic leukemia (ALL), sarcoma, and potentially other solid tumors. Here, we characterize the molecular mechanism by which a cell-penetrable small molecule, ASX-173, inhibits human asparagine synthetase (ASNS), the enzyme that catalyzes intracellular asparagine biosynthesis. ASX-173 reduces cellular asparagine levels, induces the integrated stress response (ISR), and reduces cell growth in HEK-293A cells. A cryo-EM structure reveals that ASX-173 engages a unique, hydrophobic pocket formed by AMP, Mg2+, and pyrophosphate in the C-terminal synthetase domain of ASNS, thereby enabling multivalent, high-affinity binding. Based on in vitro kinetic and thermal shift assays, we find that ASX-173 binds to the ASNS/Mg2+/ATP complex and is therefore a rare example of an uncompetitive enzyme inhibitor with potential therapeutic use. These findings provide a structural and mechanistic basis for targeting ASNS with small molecules, which have application in treating cancer and other human diseases.

molecular biology↗

bioRxiv2025

High-Density Microdroplet Cultivation Reveals the Essential Role of Microbial Interactions in the Growth of Environmental Microbes

The significance of microbial interactions in nature is not well understood because of the limitations of techniques to assess microbial growth at the single-cell level under conditions of high cell density (more than 107 cells/mL). To address this limitation for further solution of microbial uncultivability, we developed a Gel Microdroplet Aggregate in Oil (GMD-agg) cultivation method. This method provides a high inoculum cell density (>108 cells/mL) but maintains pure cultures of each growth unit. Millions of hydrogel particles (10-30 {micro}m in diameter) entrapping single cells with medium softly aggregated in oil (GMD-oil cultivation) is the key structure of this method. In this study, to assess the effect of microbial interactions on the cultivation of environmental microorganisms, soil and activated sludge samples were cultured, and colony formation and diversity during cultivation in GMDs were investigated. Results showed that the cultivation efficiency of GMD-agg was approximately 10 times higher than that of the method in which microbial interactions were canceled. In addition, the growth of taxonomies containing many uncultured microorganisms, such as Verrucomicrobia, Planctomycetia, Acidobacteria, and Vicinimibacteria, was observed. Furthermore, when isolated strains were co-cultured at high cell densities with either inter-or intra-species, we observed a higher cultivation efficiency than when microbial interactions were canceled. Rebuilding growth-promoting microbial interactions using isolated strains demonstrated that microbial interactions positively influence microbial growth. These findings indicate the effectiveness of this cultivation method, reveal the crucial role of microbial interactions in the growth of environmental microorganisms, and contribute to solving the issue of microbial uncultivability. IMPORTANCEThe overwhelming majority of environmental microbes remain uncultured, limiting our understanding of their physiology and ecological roles. Although microbial interactions have been predicted as one of the key factors for the growth of uncultivable microbial types, the effect of these interactions on cultivability remains poorly understood. In this study, we developed a new droplet-based co-cultivation approach that promotes microbial interactions while maintaining pure cultures and enables growth tracking at the single-cell level. This method significantly improved cultivability (approximately 10 times), including the growth of taxa that are difficult to cultivate. Direct observation of microbial growth in the community using this method clearly demonstrated that microbial interactions are essential for the growth of diverse microbial types. These findings underscore the importance of microbial interactions in cultivation and offer a basis for radically expanding microbial bioresources, manipulating microbial communities, and exploring previously unrecognized microbial interactions.

microbiology↗

bioRxiv2025

Filament transport supports contractile steady states of actin networks.

In all eukaryotic cells, the actin cytoskeleton is maintained in a dynamic steady-state. Actin filaments are continuously displaced from cell periphery, where they assemble, towards the cells center, where they disassemble. Despite this constant flow and turnover, cellular networks maintain their overall architecture constant. How such a flow of material can support dynamic yet steady cellular architectures remains an open question. To investigate the role of myosin-based forces in contractile steady-states of actin networks, we used a reconstituted in vitro system based on a minimal set of purified proteins, namely actin, myosin and actin regulators. We found that, contrary to previous bulk experiments, when confined in microwells, the actin network could self-organize into ordered arrangements of contractile bundles, flowing continuously without collapsing. This was supported by three-dimensional fluxes of actin filaments, spatially separated yet balancing each other. Unexpectedly, maintaining these fluxes did not depend on filament nucleation or elongation, but solely on filament transport. Ablation of the contractile bundles abolished the flux balance and led to network collapse. These findings demonstrate that the dynamic steady state of actin networks can be sustained by filament displacement and recirculation, independently of filament assembly and disassembly. Significance StatementCellular structures continuously self-renew, with new material constantly being added and removed while maintaining overall structural stability. This is particularly true for the actin cytoskeleton, whose components are continuously assembled, displaced, and reassembled. Understanding this process is fundamental to uncovering how cells regulate their architecture and adapt to stimuli. Here, we reconstitute an in vitro actomyosin network capable of contracting steadily over time without collapsing, relying solely on myosin-based transport. These findings demonstrate that a minimal system consisting of actin and molecular motors can effectively recapitulate the ability of actin networks to self-organize into stable yet dynamic architectures.

biophysics↗

bioRxiv2025

Mechanism of counterattack against eIF2α kinase immune signalling by viral pseudo-kinase PK2

Viral infection triggers the activation of host innate immune responses, including the integrated stress response (ISR). The antiviral response is mediated by the phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2) catalyzed by host eIF2 kinase, resulting in suppression of global protein synthesis and induction of selected genes in the cell. To evade and/or subvert the host antiviral signalling pathway, viruses have evolved and harnessed elaborate counter-attack mechanisms to repress the activation of host eIF2 kinase. In insects, eIF2 kinase-mimic protein PK2 encoded in baculovirus directly binds to and inhibits host eIF2 kinase for viral propagation. However, the mechanism underlying how viral PK2 recognizes host eIF2 kinase and interrupts its activity has remained unclear. Here we present a series of crystal structures of apo PK2 and its complex with eIF2 kinase, revealing a conformational transition mechanism of PK2 for eIF2 kinase inhibition. PK2 alone, comprised of an N-terminal extension (NTE) and eIF2 kinase C-lobe mimic (EKCM), adopted a two-fold symmetric homotetramer assembled mainly by the NTE region. Pull-down assay identified a region of Bombyx mori HRI-like kinase (BmHRI) that binds to PK2, and its complex structure revealed that the PK2-binding region of BmHRI, subdomain III-IV, is entrapped in a newly formed groove between the NTE region and EKCM of PK2. The structural data, together with biochemical analyses, indicated that PK2 suppresses eIF2 kinase activity by the pullout and subsequent blockade of the subdomain III-IV, a regulatory element essential for its kinase activity. These results not only provide the molecular mechanisms for the inhibition of host eIF2 kinase by viral pseudo-kinase, but also unveil the viral evolutionary strategy to shut-off host immunity via horizontal gene transfer from host to virus.

biochemistry↗

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

The Brain Tells a Story: Unveiling Distinct Representations of Semantic Content in Speech, Objects, and Stories in the Human Brain with Large Language Models

In recent studies, researchers have used large language models (LLMs) to explore semantic representations in the brain; however, they have typically assessed different levels of semantic content, such as speech, objects, and stories, separately. In this study, we recorded brain activity using functional magnetic resonance imaging (fMRI) while participants viewed 8.3 hours of dramas and movies. We annotated these stimuli at multiple semantic levels, which enabled us to extract latent representations of LLMs for this content. Our findings demonstrate that LLMs predict human brain activity more accurately than traditional language models, particularly for complex background stories. Furthermore, we identify distinct brain regions associated with different semantic representations, including multi-modal vision-semantic representations, which highlights the importance of modeling multi-level and multi-modal semantic representations simultaneously. We will make our fMRI dataset publicly available to facilitate further research on aligning LLMs with human brain function. Please check out our webpage at https://sites.google.com/view/llm-and-brain/.

neuroscience↗

bioRxiv2023

Small-molecule degraders reduce Aβ production through CAPRIN1-mediated lysosomal degradation of APP in Alzheimer iPSC-derived neurons

Overexpression of amyloid precursor protein (APP) is a key driver of amyloid {beta} (A{beta}) pathology, making APP a compelling therapeutic target in Alzheimers disease (AD). Here, we identify small molecules that selectively degrade APP in human neurons through a targeted protein degradation approach. Structural analyses reveal that these compounds act as molecular glues, binding at the interface between cytoplasmic activation/proliferation-associate protein 1 (CAPRIN1) and APP and stabilizing their interaction within a ternary complex. In neurons derived from induced pluripotent stem cells (iPSCs) from sporadic and familial AD patients, these compounds promote CAPRIN1-dependent degradation of both wild-type and mutant APP through the endo-lysosomal pathway. The lead compound, 0152, is blood-brain barrier permeable, and its administration significantly reduces A{beta} production and amyloid burden in the brains of 5xFAD mice. These findings demonstrate that molecular glue degraders harness the CAPRIN1-dependent lysosomal pathway for selective APP degradation, offering a targeted therapeutic strategy for AD.

neuroscience↗

bioRxiv2023

Myosin-5 varies its steps along the irregular F-actin track

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.

biophysics↗

bioRxiv2023

Cryo-EM and Molecular Dynamics Simulations Reveal Hidden Conformational Dynamics Controlling Ammonia Transport in Human Asparagine Synthetase

How motions in enzymes might be linked to catalytic function is of considerable general interest. Advances in X-ray crystallography and cryogenic electron microscopy (cryo-EM) offer the promise of elucidating functionally relevant conformational changes that are not easily studied by other biophysical methods. Here we use 3D variability analysis (3DVA) of the cryo-EM map for wild-type (WT) human asparagine synthetase (ASNS) to identify a functional role for the Arg-142 side chain as a gate that mediates ammonia access to a catalytically relevant intramolecular tunnel. Our 3DVA-derived hypothesis is assessed experimentally, using the R142I variant in which Arg-142 is replaced by isoleucine, and by molecular dynamics (MD) simulations on independent, computational models of the WT human ASNS monomer and its catalytically relevant, ternary complex with {beta}-aspartyl-AMP and MgPPi. Residue fluctuations in the MD trajectories for the human ASNS monomer are consistent with those determined for 3DVA-derived structures. These MD simulations also indicate that the gating function of Arg-142 is separate from the molecular events that form a continuous tunnel linking the two active sites. Experimental support for Arg-142 playing a role in intramolecular ammonia translocation is provided by the glutamine-dependent synthetase activity of the R142 variant relative to WT ASNS. MD simulations of computational models for the R142I variant and the R142I/{beta}-aspartyl-AMP/MgPPi ternary complex provide a possible molecular basis for this observation. Overall, the combination of 3DVA with MD simulations is a generally applicable approach to generate testable hypotheses of how conformational changes in buried side chains might regulate function in enzymes.

molecular biology↗

bioRxiv2022

High-resolution image reconstruction with latent diffusion models from human brain activity

Reconstructing visual experiences from human brain activity offers a unique way to understand how the brain represents the world, and to interpret the connection between computer vision models and our visual system. While deep generative models have recently been employed for this task, reconstructing realistic images with high semantic fidelity is still a challenging problem. Here, we propose a new method based on a diffusion model (DM) to reconstruct images from human brain activity obtained via functional magnetic resonance imaging (fMRI). More specifically, we rely on a latent diffusion model (LDM) termed Stable Diffusion. This model reduces the computational cost of DMs, while preserving their high generative performance. We also characterize the inner mechanisms of the LDM by studying how its different components (such as the latent vector of image Z, conditioning inputs C, and different elements of the denoising U-Net) relate to distinct brain functions. We show that our proposed method can reconstruct high-resolution images with high fidelity in straight-forward fashion, without the need for any additional training and fine-tuning of complex deep-learning models. We also provide a quantitative interpretation of different LDM components from a neuroscientific perspective. Overall, our study proposes a promising method for reconstructing images from human brain activity, and provides a new framework for understanding DMs. Please check out our webpage at https://sites.google.com/view/stablediffusion-with-brain/.

neuroscience↗

bioRxiv2022

A global atlas of substrate specificities for the human serine/threonine kinome

Protein phosphorylation is one of the most widespread post-translational modifications in biology. With the advent of mass spectrometry-based phosphoproteomics, more than 200,000 sites of serine and threonine phosphorylation have been reported, of which several thousand have been associated with human diseases and biological processes. For the vast majority of phosphorylation events, it is not yet known which of the more than 300 protein Ser/Thr kinases encoded in the human genome is responsible. Here, we utilize synthetic peptide libraries to profile the substrate sequence specificity of nearly every functional human Ser/Thr kinase. Viewed in its entirety, the substrate specificity of the kinome was substantially more diverse than expected and was driven extensively by negative selectivity. Our kinome-wide dataset was used to computationally annotate and identify the most likely protein kinases for every reported phosphorylation site in the human Ser/Thr phosphoproteome. For the small minority of phosphosites where the protein kinases involved have been previously identified, our predictions were in excellent agreement. When this approach was applied to examine the signaling response of tissues and cell lines to hormones, growth factors, targeted inhibitors, and environmental or genetic perturbations, it revealed unexpected insights into pathway complexity and compensation. Overall, these studies reveal the full extent of substrate specificity of the human Ser/Thr kinome, illuminate cellular signaling responses, and provide a rich resource to link unannotated phosphorylation events to biological pathways.

systems biology↗

bioRxiv2021

Structure and RNA template requirements of Arabidopsis RNA-DEPENDENT RNA POLYMERASE 2

RNA-dependent RNA polymerases play essential roles in RNA-mediated gene silencing in eukaryotes. In Arabidopsis, RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) physically interacts with DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and their activities are tightly coupled, with Pol IV transcriptional arrest or termination, involving the nontemplate DNA strand, somehow enabling RDR2 to engage Pol IV transcripts and generate double-stranded RNAs. The dsRNAs are then released from the Pol IV-RDR2 complex and diced into siRNAs that guide RNA-directed DNA methylation and silencing. Here we report the structure of full-length RDR2, at an overall resolution of 3.1 [A], determined by cryo-electron microscopy. The N-terminal region contains an RNA-recognition motif (RRM) adjacent to a positively charged channel that leads to a catalytic center with striking structural homology to the catalytic centers of multisubunit DNA-dependent RNA polymerases. We show that RDR2 initiates 1-2 nucleotides (nt) internal to the 3 ends of its templates and can transcribe the RNA of an RNA-DNA hybrid provided that 9 or more nucleotides at the RNAs 3 end is unpaired. Using a nucleic acid configuration that mimics the arrangement of RNA and DNA strands upon Pol IV transcriptional arrest, we show that displacement of the RNA 3 end occurs as the DNA template and non-template strands reanneal, enabling RDR2 transcription. These results suggest a model in which Pol IV arrest and backtracking displaces the RNA 3 end as the DNA strands reanneal, allowing RDR2 to engage the RNA and transcribe the second strand. SignificanceRDR2 is critical for siRNA-directed DNA methylation in Arabidopsis, functioning in physical association with DNA-dependent Pol IV to synthesize the second strands of double-stranded siRNA precursors. Basepairing between the DNA template strand transcribed by Pol IV and the nontemplate DNA strand is known to induce Pol IV arrest and Pol IV-RDR2 transcriptional coupling, but how this occurs is unknown. We report the structure of RDR2 and experimental evidence for how RDR2 engages its RNA templates and initiates transcription. RDR2 engages the ends of RNAs displaced from RNA-DNA hybrids, suggesting a model in which Pol IV arrest and backtracking, accompanied by DNA strand reannealing, extrudes the 3 end of the Pol IV transcript, allowing RNA engagement and second-strand synthesis.

molecular biology↗

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↗

bioRxiv2020

Assembly of a double-stranded RNA synthesizing complex: RNA-DEPENDENT RNA POLYMERASE 2 docks with NUCLEAR RNA POLYMERASE IV at the clamp domain

In plants, transcription of selfish genetic elements such as transposons and DNA viruses is suppressed by RNA-directed DNA methylation. This process is guided by 24 nt short-interfering RNAs (siRNAs) whose double-stranded precursors are synthesized by DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2). Pol IV and RDR2 co-immunoprecipitate, and their activities are tightly coupled, yet the basis for their association is unknown. Here, we show that RDR2 stably associates with Pol IVs largest catalytic subunit, NRPD1 at three sites, all within the clamp module. The clamp is a ubiquitous feature of DNA-dependent RNA polymerases that opens to allow DNA template entry and closes to encase the DNA-RNA hybrid adjacent to the RNA exit channel. The clamp also provides binding sites for polymerase-specific subunits or regulatory proteins, thus RDR2 binding to the Pol IV clamp is consistent with this theme. Within RDR2, the site of interaction with NRPD1 is very near the catalytic center. The locations of the NRPD1-RDR2 contact sites suggest a model in which transcripts emanating from Pol IVs RNA exit channel align with the template cleft of RDR2, facilitating rapid conversion of terminated Pol IV transcripts into double-stranded RNAs. Significance StatementShort interfering RNAs (siRNAs) play important roles in gene regulation by inhibiting mRNA translation into proteins or by guiding chromatin modifications that inhibit gene transcription. In plants, transcriptional gene silencing is guided by siRNAs derived from double-stranded (ds) RNAs generated by coupling the activities of DNA-dependent NUCLEAR RNA POLYMERASE IV and RNA-DEPENDENT RNA POLYMERASE 2. We show that the physical basis for Pol IV-RDR2 coupling is RDR2 binding to the clamp domain of Pol IVs largest subunit. The positions of the protein docking sites suggest that nascent Pol IV transcripts are generated in close proximity to RDR2s catalytic site, enabling rapid conversion of Pol IV transcripts into dsRNAs.

plant biology↗

bioRxiv2020

The Formin Inhibitor, SMIFH2, Inhibits Members of the Myosin Superfamily

The small molecular inhibitor of formin FH2 domains, SMIFH2, is widely used in cell biological studies. It was selected in a chemical screen as a compound inhibiting formin-driven actin polymerization in vitro, but not polymerization of pure actin, and found to be active against several types of formins from different species (Rizvi et al., 2009). Here, in experiments with cultured fibroblasts, we found that SMIFH2 inhibits retrograde flow of myosin 2 filaments and contraction of stress fibers. We further checked the effect of SMIFH2 on non-muscle myosin 2A and skeletal muscle myosin 2 in vitro and found that SMIFH2 inhibits myosin ATPase activity and ability to translocate actin filaments in the in vitro motility assay. While inhibition of myosin 2A in vitro required somewhat higher concentration of SMIFH2 than inhibition of retrograde flow and stress fiber contraction in cells, inhibition of several other non-muscle myosin types, e.g. mammalian myosin 10, Drosophila myosin 7a and Drosophila myosin 5 by SMIFH2, was equally or more efficient than inhibition of formins. Since actin polymerization and myosin contractility are linked in many cytoskeleton processes, additional careful analysis is needed in each case when function of formins was proposed solely on the basis of experiment with SMIFH2.

cell biology↗

bioRxiv2020

The ATPase mechanism of myosin 15, the molecular motor mutated in DFNB3 deafness

Cochlear hair cells possess an exquisite bundle of actin-based stereocilia that detect sound. Unconventional myosin 15 (MYO15A) traffics and delivers critical molecules required for stereocilia development and is essential for building the mechanosensory hair bundle. Mutations in the human MYO15A gene interfere with stereocilia trafficking and cause hereditary hearing loss, DFNB3. To understand the molecular mechanism of how MYO15A delivers proteins within stereocilia, we performed a kinetic study of the ATPase motor domain to characterize its mechano-chemical cycle. Using the baculovirus-Sf9 system, we purified a recombinant minimal motor domain (S1) by co-expressing the mouse MYO15 ATPase, essential and regulatory light chains that bind its IQ domains, and UNC45 and HSP90A chaperones required for correct folding of the ATPase. MYO15 purified with either UNC45A or UNC45B co-expression had similar ATPase activities (kcat = ~ 6 s-1 at 20{degrees}C). Using stopped-flow and quenched-flow transient kinetic analyses, we measured the major rate constants describing the ATPase cycle, including ATP, ADP and actin binding, hydrolysis and phosphate release. Actin-attached ADP release was the slowest measured transition (~ 12 s-1 at 20{degrees}C), although this did not rate-limit the ATPase cycle. The kinetic analysis shows the MYO15 motor domain has a moderate duty ratio (~ 0.5) and weak thermodynamic coupling between ADP and actin binding. This is consistent with MYO15 being adapted for strain sensing as a monomer, or processive motility if oligomerized into ensembles. Our kinetic characterization enables future studies into how deafness-causing mutations affect MYO15 and ultimately disrupt stereocilia trafficking necessary for normal hearing.

biochemistry↗