Pub Date : 2024-09-08DOI: 10.1101/2024.09.05.611571
Ayan Roychowdhury, Saptarshi Dasgupta, Madan Rao
Material renewability in active living systems, such as in cells and tissues, can drive the large-scale patterning of forces, with distinctive phenotypic consequences. This is especially significant in the cell cytoskeleton, where multiple species of myosin bound to actin, apply contractile stresses and undergo continual turnover, that result in patterned force channeling. Here we study the dynamical patterning of stresses that emerge in a hydrodynamic model of a renewable active actomyosin elastomer comprising two myosin species. We find that a uniform active contractile elastomer spontaneously segregates into spinodal stress patterns, followed by a finite-time collapse into tension carrying singular structures that display self-similar scaling and caustics. These singular structures move and merge, and gradually result in a slow coarsening dynamics in one dimension. In addition, the nonreciprocal nature of the underlying dynamics gives rise to exceptional points that are associated with a variety of travelling states - from peristalsis to swap and trains of regular and singular stress patterns, that may coexist with each other. Both the novel segregation and excitability are consequences of time reversal symmetry breaking of the underlying active dynamics. We discuss the implications of our findings to the emergence of stress fibers and the spatial patterning of myosin.
{"title":"Segregation, Finite Time Elastic Singularities and Coarsening in Renewable Active Matter","authors":"Ayan Roychowdhury, Saptarshi Dasgupta, Madan Rao","doi":"10.1101/2024.09.05.611571","DOIUrl":"https://doi.org/10.1101/2024.09.05.611571","url":null,"abstract":"Material renewability in active living systems, such as in cells and tissues, can drive the large-scale patterning of forces, with distinctive phenotypic consequences. This is especially significant in the cell cytoskeleton, where multiple species of myosin bound to actin, apply contractile stresses and undergo continual turnover, that result in patterned force channeling. Here we study the dynamical patterning of stresses that emerge in a hydrodynamic model of a renewable active actomyosin elastomer comprising two myosin species. We find that a uniform active contractile elastomer spontaneously segregates into spinodal stress patterns, followed by a finite-time collapse into tension carrying singular structures that display self-similar scaling and caustics. These singular structures move and merge, and gradually result in a slow coarsening dynamics in one dimension. In addition, the nonreciprocal nature of the underlying dynamics gives rise to exceptional points that are associated with a variety of travelling states - from peristalsis to swap and trains of regular and singular stress patterns, that may coexist with each other. Both the novel segregation and excitability are consequences of time reversal symmetry breaking of the underlying active dynamics. We discuss the implications of our findings to the emergence of stress fibers and the spatial patterning of myosin.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"723 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178244","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-08DOI: 10.1101/2024.09.04.609659
Inna Rozman Grinberg, Ornella Bimai, Saher Shahid, Lena Philipp, Markel Martinez-Carranza, Ipsita Banerjee, Daniel Lundin, Pal Stenmark, Britt-Marie Sjoberg, Derek T. Logan
NrdR is a bacterial transcriptional repressor consisting of a Zn-ribbon domain followed by an ATP-cone domain. Understanding its mechanism of action could aid the design of novel antibacterials. NrdR binds specifically to two "NrdR boxes" upstream of ribonucleotide reductase operons, of which Escherichia coli has three: nrdHIEF, nrdDG and nrdAB, where we identified a new box. We show that E. coli NrdR (EcoNrdR) has similar binding strength to all three sites when loaded with ATP plus dATP or equivalent diphosphate combinations. No other combination of nucleotides promotes binding to DNA. We present crystal structures of EcoNrdR-ATP-dATP and EcoNrdR-ADP-dATP, which are the first high resolution crystal structures of an NrdR. We have also determined cryo-EM structures of DNA-bound EcoNrdRATP-dATP and novel filaments of EcoNrdR-ATP. Tetrameric forms of EcoNrdR involve alternating interactions between pairs of Zn-ribbon domains and ATP-cones. The structures reveal considerable flexibility in relative orientation of ATP-cones vs Zn-ribbon domains. The structure of DNA-bound EcoNrdR-ATP-dATP shows that significant conformational rearrangements between ATP-cones and Zn-ribbons accompany DNA binding while the ATPcones retain the same relative orientation. In contrast, ATP-loaded EcoNrdR filaments show rearrangements of the ATP-cone pairs and sequester the DNA-binding residues of NrdR such that they are unable to bind to DNA. Our results, in combination with a previous structural and biochemical study, point to highly flexible EcoNrdR structures that when loaded with the correct nucleotides adapt to an optimal promoter binding conformation.
NrdR 是一种细菌转录抑制因子，由一个 Zn-ribbon 结构域和一个 ATP-cone 结构域组成。了解其作用机制有助于设计新型抗菌药物。NrdR 与核糖核苷酸还原酶操作子上游的两个 "NrdR 框 "特异性结合，大肠杆菌有三个 "NrdR 框"：nrdHIEF、nrdDG 和 nrdAB。我们的研究表明，大肠杆菌 NrdR（EcoNrdR）与 ATP 加 dATP 或等效二磷酸组合时，与所有三个位点的结合强度相似。没有其他核苷酸组合能促进与 DNA 的结合。我们展示了 EcoNrdR-ATP-dATP 和 EcoNrdR-ADP-dATP 的晶体结构，这是 NrdR 的首个高分辨率晶体结构。我们还测定了与 DNA 结合的 EcoNrdRATP-dATP 和新型 EcoNrdR-ATP 细丝的冷冻电镜结构。四聚体形式的 EcoNrdR 涉及成对的 Zn-ribbon 结构域和 ATP-cones 之间的交替相互作用。这些结构揭示了 ATP-cones 与 Zn-ribbon domains 的相对取向具有相当大的灵活性。与 DNA 结合的 EcoNrdR-ATP-dATP 的结构表明，在与 DNA 结合的同时，ATP-锥体和 Zn-ribbon 之间发生了显著的构象重排，而 ATP 锥体则保持相同的相对方向。与此相反，ATP 负载的 EcoNrdR 细丝显示出 ATP-锥对的重排，并封存了 NrdR 的 DNA 结合残基，使其无法与 DNA 结合。我们的研究结果与之前的结构和生化研究相结合，表明 EcoNrdR 结构具有高度灵活性，当负载正确的核苷酸时，可适应最佳的启动子结合构象。
{"title":"Bacterial transcriptional repressor NrdR - a flexible multifactorial nucleotide sensor","authors":"Inna Rozman Grinberg, Ornella Bimai, Saher Shahid, Lena Philipp, Markel Martinez-Carranza, Ipsita Banerjee, Daniel Lundin, Pal Stenmark, Britt-Marie Sjoberg, Derek T. Logan","doi":"10.1101/2024.09.04.609659","DOIUrl":"https://doi.org/10.1101/2024.09.04.609659","url":null,"abstract":"NrdR is a bacterial transcriptional repressor consisting of a Zn-ribbon domain followed by an ATP-cone domain. Understanding its mechanism of action could aid the design of novel antibacterials. NrdR binds specifically to two \"NrdR boxes\" upstream of ribonucleotide reductase operons, of which Escherichia coli has three: nrdHIEF, nrdDG and nrdAB, where we identified a new box. We show that E. coli NrdR (EcoNrdR) has similar binding strength to all three sites when loaded with ATP plus dATP or equivalent diphosphate combinations. No other combination of nucleotides promotes binding to DNA. We present crystal structures of EcoNrdR-ATP-dATP and EcoNrdR-ADP-dATP, which are the first high resolution crystal structures of an NrdR. We have also determined cryo-EM structures of DNA-bound EcoNrdRATP-dATP and novel filaments of EcoNrdR-ATP. Tetrameric forms of EcoNrdR involve alternating interactions between pairs of Zn-ribbon domains and ATP-cones. The structures reveal considerable flexibility in relative orientation of ATP-cones vs Zn-ribbon domains. The structure of DNA-bound EcoNrdR-ATP-dATP shows that significant conformational rearrangements between ATP-cones and Zn-ribbons accompany DNA binding while the ATPcones retain the same relative orientation. In contrast, ATP-loaded EcoNrdR filaments show rearrangements of the ATP-cone pairs and sequester the DNA-binding residues of NrdR such that they are unable to bind to DNA. Our results, in combination with a previous structural and biochemical study, point to highly flexible EcoNrdR structures that when loaded with the correct nucleotides adapt to an optimal promoter binding conformation.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"9 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178223","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-08DOI: 10.1101/2024.09.06.611766
Johannes Heyn, Miguel Atienza Juanatey, Martin Falcke, Joachim Raedler
Trajectories of motile cells represent a rich source of data that provide insights into the mechanisms of cell migration via mathematical modeling and statistical analysis. However, mechanistic models require cell type dependent parameter estimation, which in case of computational simulation is technically challenging due to the nonlinear and inherently stochastic nature of the models. Here, we employ simulation-based inference (SBI) to estimate cell specific model parameters from cell trajectories based on Bayesian inference. Using automated time-lapse image acquisition and image recognition large sets of 1D single cell trajectories are recorded from cells migrating on microfabricated lanes. A deep neural density estimator is trained via simulated trajectories generated from a previously published mechanical model of cell migration. The trained neural network in turn is used to infer the probability distribution of a limited number of model parameters that correspond to the experimental trajectories. Our results demonstrate the efficacy of SBI in discerning properties specific to non-cancerous breast epithelial cell line MCF-10A and cancerous breast epithelial cell line MDA-MB-231. Moreover, SBI is capable of unveiling the impact of inhibitors Latrunculin A and Y-27632 on the relevant elements in the model without prior knowledge of the effect of inhibitors. The proposed approach of SBI based data analysis combined with a standardized migration platform opens new avenues for the installation of cell motility libraries, including cytoskeleton drug efficacies,and may play a role in the evaluation of refined models.
{"title":"Cell-mechanical parameter estimation from 1D cell trajectories using simulation-based inference","authors":"Johannes Heyn, Miguel Atienza Juanatey, Martin Falcke, Joachim Raedler","doi":"10.1101/2024.09.06.611766","DOIUrl":"https://doi.org/10.1101/2024.09.06.611766","url":null,"abstract":"Trajectories of motile cells represent a rich source of data that provide insights into the mechanisms of cell migration via mathematical modeling and statistical analysis. However, mechanistic models require cell type dependent parameter estimation, which in case of computational simulation is technically challenging due to the nonlinear and inherently stochastic nature of the models. Here, we employ simulation-based inference (SBI) to estimate cell specific model parameters from cell trajectories based on Bayesian inference. Using automated time-lapse image acquisition and image recognition large sets of 1D single cell trajectories are recorded from cells migrating on microfabricated lanes. A deep neural density estimator is trained via simulated trajectories generated from a previously published mechanical model of cell migration. The trained neural network in turn is used to infer the probability distribution of a limited number of model parameters that correspond to the experimental trajectories. Our results demonstrate the efficacy of SBI in discerning properties specific to non-cancerous breast epithelial cell line MCF-10A and cancerous breast epithelial cell line MDA-MB-231. Moreover, SBI is capable of unveiling the impact of inhibitors Latrunculin A and Y-27632 on the relevant elements in the model without prior knowledge of the effect of inhibitors. The proposed approach of SBI based data analysis combined with a standardized migration platform opens new avenues for the installation of cell motility libraries, including cytoskeleton drug efficacies,and may play a role in the evaluation of refined models.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"59 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178242","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Biomolecular condensates formed via phase separation of proteins and nucleic acids are crucial for the spatiotemporal regulation of a diverse array of essential cellular functions and the maintenance of cellular homeostasis. However, aberrant liquid-to-solid phase transitions of such condensates are associated with several fatal human diseases. Such dynamic membraneless compartments can contain a range of molecular chaperones that can regulate the phase behavior of proteins involved in the formation of these biological condensates. Here, we show that a heat shock protein 40 (Hsp40), Ydj1, exhibits a holdase activity by potentiating the phase separation of a disease-associated stop codon mutant of the prion protein (Y145Stop) either by recruitment into Y145Stop condensates or via Y145Stop-Ydj1 two-component heterotypic phase separation that prevents the conformational conversion of Y145Stop into amyloid fibrils. Utilizing site-directed mutagenesis, multicolor fluorescence imaging, single-droplet steady-state and picosecond time-resolved fluorescence anisotropy, fluorescence recovery after photobleaching, and fluorescence correlation spectroscopy, we delineate the complex network of interactions that govern the heterotypic phase separation of Y145Stop and Ydj1. We also show that the properties of such heterotypic condensates can further be tuned by RNA that promotes the formation of multicomponent multiphasic protein-RNA condensates. Our vibrational Raman spectroscopy results in conjunction with atomic force microscopy imaging reveal that Ydj1 effectively redirects the self-assembly of Y145Stop towards a dynamically-arrested non-amyloidogenic pathway, preventing the formation of typical amyloid fibrils. Our findings underscore the importance of chaperone-mediated heterotypic phase separation in regulating aberrant phase transitions and amyloid formation associated with a wide range of deadly neurodegenerative diseases.
{"title":"Chaperone-mediated heterotypic phase separation prevents the amyloid formation of the pathological Y145Stop variant of the prion protein","authors":"Lisha Arora, Dipankar Bhowmik, Snehasis Sarkar, Anusha Sarbahi, Sandeep K Rai, Samrat Mukhopadhyay","doi":"10.1101/2024.09.05.611572","DOIUrl":"https://doi.org/10.1101/2024.09.05.611572","url":null,"abstract":"Biomolecular condensates formed via phase separation of proteins and nucleic acids are crucial for the spatiotemporal regulation of a diverse array of essential cellular functions and the maintenance of cellular homeostasis. However, aberrant liquid-to-solid phase transitions of such condensates are associated with several fatal human diseases. Such dynamic membraneless compartments can contain a range of molecular chaperones that can regulate the phase behavior of proteins involved in the formation of these biological condensates. Here, we show that a heat shock protein 40 (Hsp40), Ydj1, exhibits a holdase activity by potentiating the phase separation of a disease-associated stop codon mutant of the prion protein (Y145Stop) either by recruitment into Y145Stop condensates or via Y145Stop-Ydj1 two-component heterotypic phase separation that prevents the conformational conversion of Y145Stop into amyloid fibrils. Utilizing site-directed mutagenesis, multicolor fluorescence imaging, single-droplet steady-state and picosecond time-resolved fluorescence anisotropy, fluorescence recovery after photobleaching, and fluorescence correlation spectroscopy, we delineate the complex network of interactions that govern the heterotypic phase separation of Y145Stop and Ydj1. We also show that the properties of such heterotypic condensates can further be tuned by RNA that promotes the formation of multicomponent multiphasic protein-RNA condensates. Our vibrational Raman spectroscopy results in conjunction with atomic force microscopy imaging reveal that Ydj1 effectively redirects the self-assembly of Y145Stop towards a dynamically-arrested non-amyloidogenic pathway, preventing the formation of typical amyloid fibrils. Our findings underscore the importance of chaperone-mediated heterotypic phase separation in regulating aberrant phase transitions and amyloid formation associated with a wide range of deadly neurodegenerative diseases.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"11 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178100","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-08DOI: 10.1101/2024.09.05.611541
Biao Qiu, Olga Boudker
Excitatory amino acid transporters (EAATs) reside on cell surfaces and uptake substrates, including L-glutamate, L-aspartate, and D-aspartate, using ion gradients. Among five EAATs, EAAT3 is the only isoform that can efficiently transport L-cysteine, a substrate for glutathione synthesis. Recent work suggests that EAAT3 also transports the oncometabolite R-2-hydroxyglutarate (R-2HG). Here, we examined the structural basis of substrate promiscuity by determining the cryo-EM structures of EAAT3 bound to different substrates. We found that L-cysteine binds to EAAT3 in thiolate form, and EAAT3 recognizes different substrates by fine-tuning local conformations of the coordinating residues. However, using purified human EAAT3, we could not observe R-2HG binding or transport. Imaging of EAAT3 bound to L-cysteine revealed several conformational states, including an outward-facing state with a semi-open gate and a disrupted sodium-binding site. These structures illustrate that the full gate closure, coupled with the binding of the last sodium ion, occurs after substrate binding. Furthermore, we observed that different substrates affect how the transporter distributes between a fully outward-facing conformation and intermediate occluded states on a path to the inward-facing conformation, suggesting that translocation rates are substrate-dependent.
{"title":"Structural basis of the excitatory amino acid transporter 3 substrate recognition","authors":"Biao Qiu, Olga Boudker","doi":"10.1101/2024.09.05.611541","DOIUrl":"https://doi.org/10.1101/2024.09.05.611541","url":null,"abstract":"Excitatory amino acid transporters (EAATs) reside on cell surfaces and uptake substrates, including L-glutamate, L-aspartate, and D-aspartate, using ion gradients. Among five EAATs, EAAT3 is the only isoform that can efficiently transport L-cysteine, a substrate for glutathione synthesis. Recent work suggests that EAAT3 also transports the oncometabolite R-2-hydroxyglutarate (R-2HG). Here, we examined the structural basis of substrate promiscuity by determining the cryo-EM structures of EAAT3 bound to different substrates. We found that L-cysteine binds to EAAT3 in thiolate form, and EAAT3 recognizes different substrates by fine-tuning local conformations of the coordinating residues. However, using purified human EAAT3, we could not observe R-2HG binding or transport. Imaging of EAAT3 bound to L-cysteine revealed several conformational states, including an outward-facing state with a semi-open gate and a disrupted sodium-binding site. These structures illustrate that the full gate closure, coupled with the binding of the last sodium ion, occurs after substrate binding. Furthermore, we observed that different substrates affect how the transporter distributes between a fully outward-facing conformation and intermediate occluded states on a path to the inward-facing conformation, suggesting that translocation rates are substrate-dependent.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"10 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178249","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-08DOI: 10.1101/2024.09.04.611123
Raymond Chia, Chin-Teng Lin
Presynaptic inhibition after spinal cord injury (SCI) has been hypothesised to disproportionately affect flexion reflex loops in locomotor spinal circuitry. Reducing gamma-aminobutyric acid (GABA) inhibitory activity increases the excitation of flexion circuits, restoring muscle activation, and stepping ability. Conversely, nociceptive sensitisation and muscular spasticity can emerge from insufficient GABAergic inhibition. To investigate the effects of neuromodulation and proprioceptive sensory afferents in the spinal cord, a biologically constrained spiking neural network (SNN) was developed. The network describes the flexor motoneuron (MN) reflex loop with inputs from ipsilateral Ia- and II-fibres and tonically firing interneurons. The model was tuned to a baseline level of locomotive activity before simulating an inhibitory-dominant and body-weight supported (BWS) SCI state. Electrical stimulation (ES) and serotonergic agonists were simulated by the excitation of dorsal fibres and reduced conductance in excitatory neurons. ES was applied across all afferent fibres without phase- or muscle-specific protocols. The present study describes, for the first time, the release of GABAergic inhibition on flexor MNs as a potential mechanism underlying BWS treadmill training. The results demonstrate the synaptic mechanisms by which neuromodulatory therapy tunes the excitation and inhibition of ankle flexor MNs during locomotion for smoother and more coordinated movement.
{"title":"Balancing Excitation and Inhibition in the Locomotor Spinal Circuits","authors":"Raymond Chia, Chin-Teng Lin","doi":"10.1101/2024.09.04.611123","DOIUrl":"https://doi.org/10.1101/2024.09.04.611123","url":null,"abstract":"Presynaptic inhibition after spinal cord injury (SCI) has been hypothesised to disproportionately affect flexion reflex loops in locomotor spinal circuitry. Reducing gamma-aminobutyric acid (GABA) inhibitory activity increases the excitation of flexion circuits, restoring muscle activation, and stepping ability. Conversely, nociceptive sensitisation and muscular spasticity can emerge from insufficient GABAergic inhibition. To investigate the effects of neuromodulation and proprioceptive sensory afferents in the spinal cord, a biologically constrained spiking neural network (SNN) was developed. The network describes the flexor motoneuron (MN) reflex loop with inputs from ipsilateral Ia- and II-fibres and tonically firing interneurons. The model was tuned to a baseline level of locomotive activity before simulating an inhibitory-dominant and body-weight supported (BWS) SCI state. Electrical stimulation (ES) and serotonergic agonists were simulated by the excitation of dorsal fibres and reduced conductance in excitatory neurons. ES was applied across all afferent fibres without phase- or muscle-specific protocols. The present study describes, for the first time, the release of GABAergic inhibition on flexor MNs as a potential mechanism underlying BWS treadmill training. The results demonstrate the synaptic mechanisms by which neuromodulatory therapy tunes the excitation and inhibition of ankle flexor MNs during locomotion for smoother and more coordinated movement.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"16 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178218","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-08DOI: 10.1101/2024.09.05.611413
Zainab M. Rashid, Salina Quack, Misha Klein, Quinte Smitskamp, Pim America, Marvin Albers, Jannik Paulus, Tom Grossmann, David Dulin
Double-stranded RNA (dsRNA) has evolved into a key tool in understanding and regulating biological processes, with promising implications in therapeutics. However, its efficacy is often limited due to instability in biological settings. Recently, the development of peptidic dsRNA binders derived from naturally occurring RNA-binding proteins has emerged as a favorable starting point to address this limitation. Nevertheless, it remains unclear how these high affinity dsRNA binders alter the structure and flexibility of dsRNA. To this end, we employed single-molecule magnetic tweezers experiments to investigate the effects of TAV2b-derived peptidic dsRNA binders on the mechanical properties of dsRNA. Torque spectroscopy assays demonstrated that these peptides underwind dsRNA, while also stabilizing the duplex. Additionally, force spectroscopy experiments demonstrate that a wild type TAV2b peptide derivative extends the contour length and lowers the bending rigidity of dsRNA, while a homodimeric version triggers the formation of higher order complexes at forces below 1 pN. Our study presents a quantitative approach to investigate how these peptides alter the structure of dsRNA, and whether peptide structural design alters the affinity to dsRNA and its stability. This approach could inform the design of more potent and effective dsRNA binders in the efforts to advance RNA therapeutics.
{"title":"Single-molecule magnetic tweezers reveals that TAV2b-derived peptides underwind and stabilize double-stranded RNA","authors":"Zainab M. Rashid, Salina Quack, Misha Klein, Quinte Smitskamp, Pim America, Marvin Albers, Jannik Paulus, Tom Grossmann, David Dulin","doi":"10.1101/2024.09.05.611413","DOIUrl":"https://doi.org/10.1101/2024.09.05.611413","url":null,"abstract":"Double-stranded RNA (dsRNA) has evolved into a key tool in understanding and regulating biological processes, with promising implications in therapeutics. However, its efficacy is often limited due to instability in biological settings. Recently, the development of peptidic dsRNA binders derived from naturally occurring RNA-binding proteins has emerged as a favorable starting point to address this limitation. Nevertheless, it remains unclear how these high affinity dsRNA binders alter the structure and flexibility of dsRNA. To this end, we employed single-molecule magnetic tweezers experiments to investigate the effects of TAV2b-derived peptidic dsRNA binders on the mechanical properties of dsRNA. Torque spectroscopy assays demonstrated that these peptides underwind dsRNA, while also stabilizing the duplex. Additionally, force spectroscopy experiments demonstrate that a wild type TAV2b peptide derivative extends the contour length and lowers the bending rigidity of dsRNA, while a homodimeric version triggers the formation of higher order complexes at forces below 1 pN. Our study presents a quantitative approach to investigate how these peptides alter the structure of dsRNA, and whether peptide structural design alters the affinity to dsRNA and its stability. This approach could inform the design of more potent and effective dsRNA binders in the efforts to advance RNA therapeutics.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"36 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178215","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-08DOI: 10.1101/2024.09.06.611699
Stanisław Niziński, Elisabeth Hartmann, Robert L. Shoeman, Adjélé Wilson, Jochen Reinstein, Diana Kirilovsky, Michel Sliwa, Gotard Burdziński, Ilme Schlichting
Orange carotenoid protein (OCP) is a photoactive protein that mediates photoprotection in cyanobacteria. OCP binds different ketocarotenoid chromophores such as echinenone (ECN), 3′- hydroxyechinenone (hECN) and canthaxanthin (CAN). In the dark, OCP is in an inactive orange form known as OCPO; upon illumination, a red active state is formed, referred to as OCPR, that can interact with the phycobilisome. Large gaps still exist in the mechanistic understanding of the events between photon absorption and formation of the OCPR state. Recent studies suggested that more than one photon may be absorbed during the photocycle. Using a two-pulse excitation setup with variable time delays we demonstrate that canthaxanthin-functionalized OCPO forms the OCPR signature after absorption of a single photon. By contrast, OCPO complexed with hECN or ECN does not photoconvert to OCPR upon single photon absorption. Instead, OCPR is formed only upon absorption of a second photon, arriving roughly one second after the first one, implying the existence of a metastable light-sensitive OCP1hv intermediate. To the best of our knowledge, a sequential 2-photon absorption mechanism in a single biological photoreceptor chromophore is unique. It results in a non-linear response function with respect to light intensity, effectively generating a threshold switch. In the case of OCP, this prevents down regulation of photosynthesis at low light irradiance.
{"title":"Two-photon driven photoprotection mechanism in echinenone-functionalized Orange Carotenoid Protein","authors":"Stanisław Niziński, Elisabeth Hartmann, Robert L. Shoeman, Adjélé Wilson, Jochen Reinstein, Diana Kirilovsky, Michel Sliwa, Gotard Burdziński, Ilme Schlichting","doi":"10.1101/2024.09.06.611699","DOIUrl":"https://doi.org/10.1101/2024.09.06.611699","url":null,"abstract":"Orange carotenoid protein (OCP) is a photoactive protein that mediates photoprotection in cyanobacteria. OCP binds different ketocarotenoid chromophores such as echinenone (ECN), 3′- hydroxyechinenone (hECN) and canthaxanthin (CAN). In the dark, OCP is in an inactive orange form known as OCP<sup>O</sup>; upon illumination, a red active state is formed, referred to as OCP<sup>R</sup>, that can interact with the phycobilisome. Large gaps still exist in the mechanistic understanding of the events between photon absorption and formation of the OCP<sup>R</sup> state. Recent studies suggested that more than one photon may be absorbed during the photocycle. Using a two-pulse excitation setup with variable time delays we demonstrate that canthaxanthin-functionalized OCP<sup>O</sup> forms the OCP<sup>R</sup> signature after absorption of a single photon. By contrast, OCP<sup>O</sup> complexed with hECN or ECN does not photoconvert to OCP<sup>R</sup> upon single photon absorption. Instead, OCP<sup>R</sup> is formed only upon absorption of a second photon, arriving roughly one second after the first one, implying the existence of a metastable light-sensitive OCP<sup>1hv</sup> intermediate. To the best of our knowledge, a sequential 2-photon absorption mechanism in a single biological photoreceptor chromophore is unique. It results in a non-linear response function with respect to light intensity, effectively generating a threshold switch. In the case of OCP, this prevents down regulation of photosynthesis at low light irradiance.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"7 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178252","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-08DOI: 10.1101/2024.09.05.611526
Albert Kong, Alessia Pallaoro, Donald Yapp, Gwynn J Elfring, Mattia Bacca
Cells rely on contractility to proliferate, and cancerous ones exhibit an increased glucose dependence. It is therefore hypothesized that glucose restriction can mitigate cancer cell proliferation by stunting their contractility. However, glucose-restriction studies have mostly been based on experiments that have yielded conflicting results; some cells become less contractile under glucose-restriction, intuitively, while, others become surprisingly more contractile. Active mechanistic modeling may prove fruitful in resolving these conflicts. In this study, we develop a model for glucose-mediated cell contractility to capture the mechanical implications of glucose restriction. The model is calibrated on cell contraction data taken from 2D-cultured glioma cells, laying on a collagen substrate. The model predicts the existence of a critical level of glucose restriction that must be exceeded for contractility to be affected, and this is validated by our experiments. Our model provides an initial step toward a fundamental understanding of the metabolic implications of cell contractility, particularly in the context of glucose restriction: an essential step in cancer studies.
{"title":"The effect of glucose restriction on cancer cell contractility: A threshold response in U-87 glioma","authors":"Albert Kong, Alessia Pallaoro, Donald Yapp, Gwynn J Elfring, Mattia Bacca","doi":"10.1101/2024.09.05.611526","DOIUrl":"https://doi.org/10.1101/2024.09.05.611526","url":null,"abstract":"Cells rely on contractility to proliferate, and cancerous ones exhibit an increased glucose dependence. It is therefore hypothesized that glucose restriction can mitigate cancer cell proliferation by stunting their contractility. However, glucose-restriction studies have mostly been based on experiments that have yielded conflicting results; some cells become less contractile under glucose-restriction, intuitively, while, others become surprisingly more contractile. Active mechanistic modeling may prove fruitful in resolving these conflicts. In this study, we develop a model for glucose-mediated cell contractility to capture the mechanical implications of glucose restriction. The model is calibrated on cell contraction data taken from 2D-cultured glioma cells, laying on a collagen substrate. The model predicts the existence of a critical level of glucose restriction that must be exceeded for contractility to be affected, and this is validated by our experiments. Our model provides an initial step toward a fundamental understanding of the metabolic implications of cell contractility, particularly in the context of glucose restriction: an essential step in cancer studies.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"47 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178256","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-08DOI: 10.1101/2024.09.05.611417
Dimple Karia, Adrian F Koh, Wen Yang, Victoria I Cushing, Benjamin Basanta, Daniel B Mihaylov, Sagar Khavnekar, Ondřej Vyroubal, Miloš Malínský, Ondřej Sháněl, Vojtëch Doležal, Juergen M Plitzko, Lingbo Yu, Gabriel C Lander, A. Radu C Aricescu, Basil J Greber, Abhay Kotecha
Cryo-electron microscopy (cryo-EM) has revolutionized structural biology by providing high-resolution insights into biological macromolecules. Here, we present sub-3 Å resolution structures determined using the 100 keV Tundra cryogenic transmission electron microscope (cryo-TEM), equipped with the newly developed Falcon C direct electron detector (DED). Our results demonstrate that this lower voltage microscope, when combined with advanced electron optics and detectors, can achieve high-resolution reconstructions that were previously only attainable with higher voltage systems. The implementation of an extreme-brightness field emission gun (XFEG) and an SP-TWIN objective lens significantly enhanced the spatial and temporal coherence of the system. Furthermore, the semi-automated sample loader minimized contamination and drift, allowing extended data collection sessions without manual intervention. The high detective quantum efficiency (DQE) of Falcon C further improved the signal-to-noise ratio, which is critical for achieving high-resolution structures. We validated the performance of this microscope by determining the structures of various biological samples, including apoferritin, T20S proteasome, GABAA receptor, haemoglobin, and human transthyretin ranging in size from 440 kDa to 50 kDa. The highest resolutions achieved were 2.1 Å for apoferritin, 2.7 Å for the 20S proteasome, 2.8 Å for the GABAA receptor, 5.0 Å for haemoglobin, and 3.5 Å for transthyretin. We also explored a larger specimen, a 3.9 MDa Adeno-associated virus (AAV9) capsid and resolved it a 2.8 Å. This work highlights the potential of 100 keV TEMs to make high-resolution cryo-EM more accessible to the structural biology community. Furthermore, it sets a precedent for the use of lower voltage TEMs in routine cryo-EM studies, not only for screening grids for single particle analysis but also for achieving high-resolution structures of protein samples.
低温电子显微镜（cryo-EM）通过提供高分辨率的生物大分子洞察力，彻底改变了结构生物学。在这里，我们展示了使用 100 keV Tundra 低温透射电子显微镜（冷冻电子显微镜）测定的亚 3 Å 分辨率结构，该显微镜配备了新开发的 Falcon C 直接电子探测器（DED）。我们的研究结果表明，这种低电压显微镜与先进的电子光学和探测器相结合，可以实现高分辨率重构，而这在以前只能通过更高的电压系统才能实现。极亮度场发射枪（XFEG）和 SP-TWIN 物镜的应用大大提高了系统的空间和时间一致性。此外，半自动样品装载器最大限度地减少了污染和漂移，无需人工干预即可进行长时间的数据采集。Falcon C 的高探测量子效率（DQE）进一步提高了信噪比，这对实现高分辨率结构至关重要。我们通过测定各种生物样本的结构验证了这台显微镜的性能，包括从 440 kDa 到 50 kDa 大小不等的载脂蛋白、T20S 蛋白酶体、GABAA 受体、血红蛋白和人类转甲状腺素。所达到的最高分辨率分别为：apoferritin 2.1 Å、20S 蛋白酶体 2.7 Å、GABAA 受体 2.8 Å、血红蛋白 5.0 Å 和转甲状腺素 3.5 Å。我们还研究了一个更大的样本，即 3.9 MDa 的腺相关病毒 (AAV9) 荚膜，并将其解析为 2.8 Å。这项工作突出了 100 keV TEM 在使结构生物学界更容易获得高分辨率冷冻电镜方面的潜力。此外，它还开创了在常规冷冻电镜研究中使用低电压 TEM 的先例，不仅可用于筛选网格进行单颗粒分析，还可用于实现蛋白质样本的高分辨率结构。
{"title":"Sub-3 Å resolution protein structure determination by single-particle cryo-EM at 100 keV","authors":"Dimple Karia, Adrian F Koh, Wen Yang, Victoria I Cushing, Benjamin Basanta, Daniel B Mihaylov, Sagar Khavnekar, Ondřej Vyroubal, Miloš Malínský, Ondřej Sháněl, Vojtëch Doležal, Juergen M Plitzko, Lingbo Yu, Gabriel C Lander, A. Radu C Aricescu, Basil J Greber, Abhay Kotecha","doi":"10.1101/2024.09.05.611417","DOIUrl":"https://doi.org/10.1101/2024.09.05.611417","url":null,"abstract":"Cryo-electron microscopy (cryo-EM) has revolutionized structural biology by providing high-resolution insights into biological macromolecules. Here, we present sub-3 Å resolution structures determined using the 100 keV Tundra cryogenic transmission electron microscope (cryo-TEM), equipped with the newly developed Falcon C direct electron detector (DED). Our results demonstrate that this lower voltage microscope, when combined with advanced electron optics and detectors, can achieve high-resolution reconstructions that were previously only attainable with higher voltage systems. The implementation of an extreme-brightness field emission gun (XFEG) and an SP-TWIN objective lens significantly enhanced the spatial and temporal coherence of the system. Furthermore, the semi-automated sample loader minimized contamination and drift, allowing extended data collection sessions without manual intervention. The high detective quantum efficiency (DQE) of Falcon C further improved the signal-to-noise ratio, which is critical for achieving high-resolution structures. We validated the performance of this microscope by determining the structures of various biological samples, including apoferritin, T20S proteasome, GABAA receptor, haemoglobin, and human transthyretin ranging in size from 440 kDa to 50 kDa. The highest resolutions achieved were 2.1 Å for apoferritin, 2.7 Å for the 20S proteasome, 2.8 Å for the GABAA receptor, 5.0 Å for haemoglobin, and 3.5 Å for transthyretin. We also explored a larger specimen, a 3.9 MDa Adeno-associated virus (AAV9) capsid and resolved it a 2.8 Å. This work highlights the potential of 100 keV TEMs to make high-resolution cryo-EM more accessible to the structural biology community. Furthermore, it sets a precedent for the use of lower voltage TEMs in routine cryo-EM studies, not only for screening grids for single particle analysis but also for achieving high-resolution structures of protein samples.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"7 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178253","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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