Tumor Necrosis Factor Receptor 1 (TNFR1) signaling determines cell fate during inflammation, immunopathogenesis, and tumorigenesis. TNFR1 proteins homo-oligomerize into clusters on the plasma membrane. The potential impact of TNFR1 clustering on downstream signaling remains unexplored. Homo-FRET measurements elucidate that alterations in intra-cluster receptor density (IRD) dictate the outcomes of downstream TNFR1 signaling. Soluble TNF-α (sTNF-α) elevates IRD within the TNFR1 clusters core while diminishing it in the rim, through intra-cluster dynamic reorganization of TNFR1. Decreasing TNFR1 IRD through increasing membrane tension, administering TNFR1 antagonist zafirlukast, actin depolymerization, or depleting cholesterol impedes sTNF-α-mediated stimulation. Conversely, increasing IRD by reducing membrane tension or exposing cells to 3D gel-like microenvironment induces ligand-independent TNFR1 signaling. These findings suggest a broader applicability of IRD in modulating signaling pathways across other receptor families, offering insights for innovative strategies in TNFR1 signaling modulation.
{"title":"Intra-cluster receptor density (IRD) dictates TNFR1 clusters' signaling efficacy","authors":"Subhamoy Jana, Priyanka Roy, Jibitesh Das, Parijat Biswas, Nandana Nanda, Bidisha Sinha, Deepak Sinha","doi":"10.1101/2024.08.09.607302","DOIUrl":"https://doi.org/10.1101/2024.08.09.607302","url":null,"abstract":"Tumor Necrosis Factor Receptor 1 (TNFR1) signaling determines cell fate during inflammation, immunopathogenesis, and tumorigenesis. TNFR1 proteins homo-oligomerize into clusters on the plasma membrane. The potential impact of TNFR1 clustering on downstream signaling remains unexplored. Homo-FRET measurements elucidate that alterations in intra-cluster receptor density (IRD) dictate the outcomes of downstream TNFR1 signaling. Soluble TNF-α (sTNF-α) elevates IRD within the TNFR1 clusters core while diminishing it in the rim, through intra-cluster dynamic reorganization of TNFR1. Decreasing TNFR1 IRD through increasing membrane tension, administering TNFR1 antagonist zafirlukast, actin depolymerization, or depleting cholesterol impedes sTNF-α-mediated stimulation. Conversely, increasing IRD by reducing membrane tension or exposing cells to 3D gel-like microenvironment induces ligand-independent TNFR1 signaling. These findings suggest a broader applicability of IRD in modulating signaling pathways across other receptor families, offering insights for innovative strategies in TNFR1 signaling modulation.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"11 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141945062","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 : 2024-08-09DOI: 10.1101/2024.08.08.607202
Bernardo I Pinto-Anwandter, Carlos A Z Bassetto, Ramon Latorre, Francisco Bezanilla
Voltage-dependent potassium channels (Kv) are extremely sensitive to membrane voltage and play a crucial role in membrane repolarization during action potentials. Kv channels undergo voltage-dependent transitions between closed states before opening. Despite all we have learned using electrophysiological methods and structural studies, we still lack a detailed picture of the energetics of the activation process. We show here that even a single mutation can drastically modify the temperature response of the Shaker Kv channel. Using rapid cell membrane temperature steps (Tsteps), we explored the effects of temperature on the ILT mutant (V369I, I372L, and S376T) and the I384N mutant. The ILT mutant produces a significant separation between the transitions of the voltage sensor domain (VSD) activation and the I384N uncouples its movement from the opening of the domain (PD). ILT and I384N respond to temperature in drastically different ways. In ILT, temperature facilitates the opening of the channel akin to a ″hot″ receptor, reflecting the temperature dependence of the voltage sensor ′s last transition and facilitating VSD to PD coupling (electromechanical coupling). In I384N, temperature stabilizes the channel closed configuration analogous to a ″cold″ receptor. Since I384N drastically uncouples the VSD from the pore opening, we reveal the intrinsic temperature dependence of the PD itself. Here, we propose that the electromechanical coupling has either a ″loose″ or ″tight″ conformation. In the loose conformation, the movement of the VSD is necessary but not sufficient to efficiently propagate the electromechanical energy to the S6 gate. In the tight conformation the VSD activation is more effectively translated into the opening of the PD. This conformational switch can be tuned by temperature and modifications of the S4 and S4-S5 linker. Our results show that we can modulate the temperature dependence of Kv channels by affecting its electromechanical coupling.
{"title":"Turning a Kv channel into hot and cold receptor by perturbing its electromechanical coupling.","authors":"Bernardo I Pinto-Anwandter, Carlos A Z Bassetto, Ramon Latorre, Francisco Bezanilla","doi":"10.1101/2024.08.08.607202","DOIUrl":"https://doi.org/10.1101/2024.08.08.607202","url":null,"abstract":"Voltage-dependent potassium channels (Kv) are extremely sensitive to membrane voltage and play a crucial role in membrane repolarization during action potentials. Kv channels undergo voltage-dependent transitions between closed states before opening. Despite all we have learned using electrophysiological methods and structural studies, we still lack a detailed picture of the energetics of the activation process. We show here that even a single mutation can drastically modify the temperature response of the Shaker Kv channel. Using rapid cell membrane temperature steps (Tsteps), we explored the effects of temperature on the ILT mutant (V369I, I372L, and S376T) and the I384N mutant. The ILT mutant produces a significant separation between the transitions of the voltage sensor domain (VSD) activation and the I384N uncouples its movement from the opening of the domain (PD). ILT and I384N respond to temperature in drastically different ways. In ILT, temperature facilitates the opening of the channel akin to a ″hot″ receptor, reflecting the temperature dependence of the voltage sensor\t′s last transition and facilitating VSD to PD coupling (electromechanical coupling). In I384N, temperature stabilizes the channel closed configuration analogous to a ″cold″ receptor. Since I384N drastically uncouples the VSD from the pore opening, we reveal the intrinsic temperature dependence of the PD itself. Here, we propose that the electromechanical coupling has either a ″loose″ or ″tight″ conformation. In the loose conformation, the movement of the VSD is necessary but not sufficient to efficiently propagate the electromechanical energy to the S6 gate. In the tight conformation the VSD activation is more effectively translated into the opening of the PD. This conformational switch can be tuned by temperature and modifications of the S4 and S4-S5 linker. Our results show that we can modulate the temperature dependence of Kv channels by affecting its electromechanical coupling.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"199 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141945304","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}
Recent research in biogeomorphology has shown that many macroscale systems exhibit spatiotemporal self-organized patterns with coarsening behaviors and also phase separation behaviors, successfully described by a mass-conserving dynamical model. Also recently, macromolecules, such as nucleic acids and proteins, have been found to assemble mesoscale biomolecular condensates inside living cells. Despite their significance, the fundamental biophysical properties of these biomolecular condensates remain poorly understood. Here, we selected DNA and the human transcription factor p53 as a model system to form a specific type of biomolecular condensate, DNA-protein interactive co-condensates (DPICs). We developed a mass-conserving dynamical model, with all parameters derived from direct experimental measurements. This model successfully reproduces the spatiotemporal dynamics of DPICs. Our findings reveal that both mesoscale biomolecular condensates and macroscale biogeomorphological systems exhibit cross-scale spatiotemporal self-organized patterns with coarsening behaviors, and cross-scale phase separation behavior. Both systems also exhibit emergent properties. Our theoretical framework offers a deeper understanding of the mechanisms underlying these phase-separation systems.
最近的生物地貌学研究表明，许多宏观系统表现出具有粗化行为和相分离行为的时空自组织模式，并成功地用质量保证动力学模型进行了描述。最近，人们还发现核酸和蛋白质等大分子在活细胞内聚集成中尺度生物分子凝聚体。尽管这些生物分子凝聚体具有重要意义，但其基本生物物理特性仍鲜为人知。在这里，我们选择 DNA 和人类转录因子 p53 作为模型系统，以形成一种特定类型的生物分子凝聚物--DNA-蛋白质交互共凝聚物（DPICs）。我们建立了一个质量守恒动力学模型，所有参数都来自直接的实验测量。该模型成功地再现了 DPIC 的时空动态。我们的研究结果表明，中尺度生物分子凝聚物和宏观生物地貌系统都表现出具有粗化行为的跨尺度时空自组织模式和跨尺度相分离行为。这两个系统还表现出突现特性。我们的理论框架有助于深入理解这些相分离系统的内在机制。
{"title":"Biomolecular condensates and biogeomorphological systems exhibit a phase-separation behavior unified by a mass-conserving model","authors":"Cheng Li, Man-Ting Guo, Xiaoqing He, Quan-Xing Liu, Zhi Qi","doi":"10.1101/2024.08.08.607271","DOIUrl":"https://doi.org/10.1101/2024.08.08.607271","url":null,"abstract":"Recent research in biogeomorphology has shown that many macroscale systems exhibit spatiotemporal self-organized patterns with coarsening behaviors and also phase separation behaviors, successfully described by a mass-conserving dynamical model. Also recently, macromolecules, such as nucleic acids and proteins, have been found to assemble mesoscale biomolecular condensates inside living cells. Despite their significance, the fundamental biophysical properties of these biomolecular condensates remain poorly understood. Here, we selected DNA and the human transcription factor p53 as a model system to form a specific type of biomolecular condensate, DNA-protein interactive co-condensates (DPICs). We developed a mass-conserving dynamical model, with all parameters derived from direct experimental measurements. This model successfully reproduces the spatiotemporal dynamics of DPICs. Our findings reveal that both mesoscale biomolecular condensates and macroscale biogeomorphological systems exhibit cross-scale spatiotemporal self-organized patterns with coarsening behaviors, and cross-scale phase separation behavior. Both systems also exhibit emergent properties. Our theoretical framework offers a deeper understanding of the mechanisms underlying these phase-separation systems.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"14 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141945065","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 : 2024-08-09DOI: 10.1101/2024.08.07.606954
David Krause, John Boehm, Leon Liebig, Nektarios Koukourakis, Juergen Czarske
Brillouin microscopy has become an important tool for investigating the mechanical properties of tissue. The recently developed Impulsive stimulated Brillouin Scattering (ISBS) promises a label-free, non-invasive measurements of viscoelastic properties of transparent samples and offers the potential for a high temporal resolution. However, the spatial resolution of ISBS is currently limited, which hinders its transfer to real-world applications. Increasing the spatial resolution of ISBS leads to an increase in the energy density of the pump beams, which requires a balancing of the excitation parameters to stay below the phototoxic threshold. This paper focuses on the influences of different excitation parameters on the spatial, temporal and spectral resolution and their optimal values. Combined with the adoption of a noise suppressing window function, a measurement rate of 20μs/pixel in hydrogel is achieved, which is promising for fast 3D imaging. The presented advanced impulsive stimulated Brillouin microscopy can be applied for fast tissue elastography toward disease studies.
{"title":"Single-shot impulsive stimulated Brillouin microscopy by tailored ultrashort pulses","authors":"David Krause, John Boehm, Leon Liebig, Nektarios Koukourakis, Juergen Czarske","doi":"10.1101/2024.08.07.606954","DOIUrl":"https://doi.org/10.1101/2024.08.07.606954","url":null,"abstract":"Brillouin microscopy has become an important tool for investigating the mechanical properties of tissue. The recently developed Impulsive stimulated Brillouin Scattering (ISBS) promises a label-free, non-invasive measurements of viscoelastic properties of transparent samples and offers the potential for a high temporal resolution. However, the spatial resolution of ISBS is currently limited, which hinders its transfer to real-world applications. Increasing the spatial resolution of ISBS leads to an increase in the energy density of the pump beams, which requires a balancing of the excitation parameters to stay below the phototoxic threshold. This paper focuses on the influences of different excitation parameters on the spatial, temporal and spectral resolution and their optimal values. Combined with the adoption of a noise suppressing window function, a measurement rate of 20μs/pixel in hydrogel is achieved, which is promising for fast 3D imaging. The presented advanced impulsive stimulated Brillouin microscopy can be applied for fast tissue elastography toward disease studies.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"84 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141945087","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 : 2024-08-09DOI: 10.1101/2024.08.08.607183
Nicky W Tam, Rumiana Dimova, Amaia Cipitria
Extracellular vesicle (EV) and nanoparticle interactions with extracellular matrix (ECM) environments are often studied through a paradigm whereby particles are a passive element whose diffusion and behaviour are subject to the composition and structure of the environment they are in. While EV diffusion and distribution in tissues are indeed governed by matrix interactions, accumulating evidence suggests that EVs contain much of the cellular machinery required for actively remodeling ECM as well. Using rheology and confocal reflectance microscopy to investigate the gelation of collagen I hydrogels formed in the presence of EVs, we show that EVs can play an active role in the formation of new ECM. EVs appear to nucleate new fibrils, recruiting collagen molecules from solution and accelerating their polymerization. Trypsinization of EVs to digest their surface proteins shows that proteins are primarily responsible for this phenomenon. The use of extruded plasma membrane vesicles shows that membrane composition plays an important role in determining final fibril length and matrix structure. EVs also become integrated into the fibril structures that they help form, reminiscent of matrix vesicles found in situ within tissues. This represents a plausible way by which EVs are deposited into the extracellular environment, becoming important contextual signaling cues for resident cells. Our data show that EV-matrix interactions are dynamic and reciprocal, contributing to the remodeling of tissue microenvironments.
{"title":"Breast cancer cell-derived extracellular vesicles accelerate collagen fibrillogenesis and integrate into the matrix","authors":"Nicky W Tam, Rumiana Dimova, Amaia Cipitria","doi":"10.1101/2024.08.08.607183","DOIUrl":"https://doi.org/10.1101/2024.08.08.607183","url":null,"abstract":"Extracellular vesicle (EV) and nanoparticle interactions with extracellular matrix (ECM) environments are often studied through a paradigm whereby particles are a passive element whose diffusion and behaviour are subject to the composition and structure of the environment they are in. While EV diffusion and distribution in tissues are indeed governed by matrix interactions, accumulating evidence suggests that EVs contain much of the cellular machinery required for actively remodeling ECM as well. Using rheology and confocal reflectance microscopy to investigate the gelation of collagen I hydrogels formed in the presence of EVs, we show that EVs can play an active role in the formation of new ECM. EVs appear to nucleate new fibrils, recruiting collagen molecules from solution and accelerating their polymerization. Trypsinization of EVs to digest their surface proteins shows that proteins are primarily responsible for this phenomenon. The use of extruded plasma membrane vesicles shows that membrane composition plays an important role in determining final fibril length and matrix structure. EVs also become integrated into the fibril structures that they help form, reminiscent of matrix vesicles found in situ within tissues. This represents a plausible way by which EVs are deposited into the extracellular environment, becoming important contextual signaling cues for resident cells. Our data show that EV-matrix interactions are dynamic and reciprocal, contributing to the remodeling of tissue microenvironments.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"12 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141945084","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 : 2024-08-09DOI: 10.1101/2024.08.09.607224
Laura M Weber, Koen J.A. Martens, Clément Cabriel, Joel J. Gates, Manon Albecq, Fredrik Vermeulen, Katharina Hein, Ignacio Izeddin, Ulrike Endesfelder
Event-based sensors (EBS), or neuromorphic vision sensors, offer a novel approach to imaging by recording light intensity changes asynchronously, unlike conventional cameras that capture light over fixed exposure times. This capability results in high temporal resolution, reduced data redundancy, and a wide dynamic range. This makes EBS ideal for Single-Molecule Localization Microscopy (SMLM) as SMLM relies on the sequential imaging of sparse, blinking fluorescent emitters to achieve super-resolution. Recent studies have shown that EBS can effectively capture these emitters, achieving spatial resolution comparable to traditional cameras. However, existing analyses of event-based SMLM (eveSMLM) data have relied on converting event lists into image frames for conventional analysis, limiting the full potential of the technology. To overcome this limitation, we developed EVE, a specialized software for analyzing eveSMLM data. EVE offers an integrated platform for detection, localization, and post-processing, with various algorithmic options tailored for the unique structure of eveSMLM data. EVE is user-friendly and features an open, modular infrastructure that supports ongoing development and optimization. EVE is the first dedicated tool for event-based SMLM, transforming the analysis process to fully utilize the spatiotemporal data generated by EBS. This allows researchers to explore the full potential of eveSMLM and encourages the development of new analytical methods and experimental improvements.
{"title":"EVE is an open modular data analysis software for event-based localization microscopy","authors":"Laura M Weber, Koen J.A. Martens, Clément Cabriel, Joel J. Gates, Manon Albecq, Fredrik Vermeulen, Katharina Hein, Ignacio Izeddin, Ulrike Endesfelder","doi":"10.1101/2024.08.09.607224","DOIUrl":"https://doi.org/10.1101/2024.08.09.607224","url":null,"abstract":"Event-based sensors (EBS), or neuromorphic vision sensors, offer a novel approach to imaging by recording light intensity changes asynchronously, unlike conventional cameras that capture light over fixed exposure times. This capability results in high temporal resolution, reduced data redundancy, and a wide dynamic range. This makes EBS ideal for Single-Molecule Localization Microscopy (SMLM) as SMLM relies on the sequential imaging of sparse, blinking fluorescent emitters to achieve super-resolution. Recent studies have shown that EBS can effectively capture these emitters, achieving spatial resolution comparable to traditional cameras. However, existing analyses of event-based SMLM (eveSMLM) data have relied on converting event lists into image frames for conventional analysis, limiting the full potential of the technology. To overcome this limitation, we developed EVE, a specialized software for analyzing eveSMLM data. EVE offers an integrated platform for detection, localization, and post-processing, with various algorithmic options tailored for the unique structure of eveSMLM data. EVE is user-friendly and features an open, modular infrastructure that supports ongoing development and optimization. EVE is the first dedicated tool for event-based SMLM, transforming the analysis process to fully utilize the spatiotemporal data generated by EBS. This allows researchers to explore the full potential of eveSMLM and encourages the development of new analytical methods and experimental improvements.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"26 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141945064","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 : 2024-08-08DOI: 10.1101/2024.08.07.607093
Toshi Parmar, Liam P. Dow, Beth L. Pruitt, M. Cristina Marchetti
The feedback between mechanical and chemical signals plays a key role in controlling many bisological processes and collective cell behavior. Here we focus on the emergence of spatiotemporal density waves in a one-dimensional “cell train.” Combining a minimal theoretical model with observations in an in vitro experimental system of MDCK epithelial cells confined to a linear pattern, we examine the spontaneous oscillations driven by the feedback between myosin activation and mechanical deformations and their effect on the response of the tissue to externally applied deformations. We show that the nature and frequency of spontaneous oscillations is controlled by the size of the cell train, with a transition from size-dependent standing waves to intrinsic spontaneous waves at the natural frequency of the tissue. The response to external boundary perturbations exhibit a resonance at this natural frequency, providing a possible venue for inferring the mechanochemical couplings that control the tissue behavior from rheological experiments.
{"title":"Spontaneous and Induced Oscillations in Confined Epithelia","authors":"Toshi Parmar, Liam P. Dow, Beth L. Pruitt, M. Cristina Marchetti","doi":"10.1101/2024.08.07.607093","DOIUrl":"https://doi.org/10.1101/2024.08.07.607093","url":null,"abstract":"The feedback between mechanical and chemical signals plays a key role in controlling many bisological processes and collective cell behavior. Here we focus on the emergence of spatiotemporal density waves in a one-dimensional “cell train.” Combining a minimal theoretical model with observations in an <em>in vitro</em> experimental system of MDCK epithelial cells confined to a linear pattern, we examine the spontaneous oscillations driven by the feedback between myosin activation and mechanical deformations and their effect on the response of the tissue to externally applied deformations. We show that the nature and frequency of spontaneous oscillations is controlled by the size of the cell train, with a transition from size-dependent standing waves to intrinsic spontaneous waves at the natural frequency of the tissue. The response to external boundary perturbations exhibit a resonance at this natural frequency, providing a possible venue for inferring the mechanochemical couplings that control the tissue behavior from rheological experiments.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"41 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141945205","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 : 2024-08-07DOI: 10.1101/2024.08.05.603709
Aliki Perdikari, Virgil A. Woods, Ali Ebrahim, Katherine Lawler, Rebecca Bounds, Nathanael I. Singh, Tamar (Skaist) Mehlman, Blake T. Riley, Shivani Sharma, Jackson W. Morris, Julia M. Keogh, Elana Henning, Miriam Smith, I. Sadaf Farooqi, Daniel A. Keedy
Protein Tyrosine Phosphatase 1B (PTP1B) is a negative regulator of leptin signaling whose disruption protects against diet-induced obesity in mice. We investigated whether structural characterization of human PTP1B variant proteins might reveal precise mechanisms to target for weight loss therapy. We selected 12 rare variants for functional characterization from exomes from 997 people with persistent thinness and 200,000 people from UK Biobank. Seven of 12 variants impaired PTP1B function by increasing leptin-stimulated STAT3 phosphorylation in cells. Using room-temperature X-ray crystallography, hydrogen-deuterium exchange mass spectrometry, and computational modeling, we determined that human variants modulate the 3-dimensional structure of PTP1B through distinct allosteric conduits that energetically link distal, highly ligandable structural regions to the active site. These studies inform the design of allosteric PTP1B inhibitors for the treatment of obesity.
{"title":"Structures of human PTP1B variants reveal allosteric sites to target for weight loss therapy","authors":"Aliki Perdikari, Virgil A. Woods, Ali Ebrahim, Katherine Lawler, Rebecca Bounds, Nathanael I. Singh, Tamar (Skaist) Mehlman, Blake T. Riley, Shivani Sharma, Jackson W. Morris, Julia M. Keogh, Elana Henning, Miriam Smith, I. Sadaf Farooqi, Daniel A. Keedy","doi":"10.1101/2024.08.05.603709","DOIUrl":"https://doi.org/10.1101/2024.08.05.603709","url":null,"abstract":"Protein Tyrosine Phosphatase 1B (PTP1B) is a negative regulator of leptin signaling whose disruption protects against diet-induced obesity in mice. We investigated whether structural characterization of human PTP1B variant proteins might reveal precise mechanisms to target for weight loss therapy. We selected 12 rare variants for functional characterization from exomes from 997 people with persistent thinness and 200,000 people from UK Biobank. Seven of 12 variants impaired PTP1B function by increasing leptin-stimulated STAT3 phosphorylation in cells. Using room-temperature X-ray crystallography, hydrogen-deuterium exchange mass spectrometry, and computational modeling, we determined that human variants modulate the 3-dimensional structure of PTP1B through distinct allosteric conduits that energetically link distal, highly ligandable structural regions to the active site. These studies inform the design of allosteric PTP1B inhibitors for the treatment of obesity.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"41 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141945206","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 : 2024-08-07DOI: 10.1101/2024.03.29.585638
Jiayi Liu, Javier Boix-Campos, Jonathan E. Ron, Johan M. Kux, Magdalena E.M. Oremek, Adriano G. Rossi, Nir S. Gov, Pablo J. Sáez
Migrating cells often face microenvironmental constraints that force them to extend multiple, often highly dynamic, protrusions, that compete to choose the new direction. However, the analy-sis of how cells coordinate shape dynamics during this directional decision-making process has been restricted to single junctions. Here, we present a theoretical model and the corresponding experimen-tal proof of concept using in vivo and in vitro live-cell microscopy and a neuronal network-based image analysis pipeline, to explore the shape and migration dynamics of highly bifurcated cells during spontaneous random migration. We found that macrophages and endothelial cells display different migration regimes in a hexagonal adhesive network, despite sharing a mesenchymal migra-tory strategy. Macrophages moved faster and presented larger changes in cell length in comparison to endothelial cells. The theoretical model describes the behavior of both cells during directional decision-making, and it reveals a trade-off between exploration for directional cues and long-range migration efficiency, showing the fine tune regulation of shape dynamics in complex geometries.
{"title":"Shape dynamics and migration of branched cells on complex networks","authors":"Jiayi Liu, Javier Boix-Campos, Jonathan E. Ron, Johan M. Kux, Magdalena E.M. Oremek, Adriano G. Rossi, Nir S. Gov, Pablo J. Sáez","doi":"10.1101/2024.03.29.585638","DOIUrl":"https://doi.org/10.1101/2024.03.29.585638","url":null,"abstract":"Migrating cells often face microenvironmental constraints that force them to extend multiple, often highly dynamic, protrusions, that compete to choose the new direction. However, the analy-sis of how cells coordinate shape dynamics during this directional decision-making process has been restricted to single junctions. Here, we present a theoretical model and the corresponding experimen-tal proof of concept using <em>in vivo</em> and <em>in vitro</em> live-cell microscopy and a neuronal network-based image analysis pipeline, to explore the shape and migration dynamics of highly bifurcated cells during spontaneous random migration. We found that macrophages and endothelial cells display different migration regimes in a hexagonal adhesive network, despite sharing a mesenchymal migra-tory strategy. Macrophages moved faster and presented larger changes in cell length in comparison to endothelial cells. The theoretical model describes the behavior of both cells during directional decision-making, and it reveals a trade-off between exploration for directional cues and long-range migration efficiency, showing the fine tune regulation of shape dynamics in complex geometries.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"199 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141945207","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 : 2024-08-06DOI: 10.1101/2024.08.05.606620
Teddy X Cai, Nathan H Williamson, Rea Ravin, Peter Basser
Water exchange is increasingly recognized as an important biological process that can affect the study of biological tissue using diffusion MR. Methods to measure exchange, however, remain immature as opposed to those used to characterize restriction, with no consensus on the optimal pulse sequence(s) or signal model(s). In general, the trend has been towards data-intensive fitting of highly parameterized models. We take the opposite approach and show that a judicious sub-sample of diffusion exchange spectroscopy (DEXSY) data can be used to robustly quantify exchange, as well as restriction, in a data-efficient manner. This sampling produces a ratio of two points per mixing time: (i) one point with equal diffusion weighting in both encoding periods, which gives maximal exchange contrast, and (ii) one point with the same total diffusion weighting in just the first encoding period, for normalization. We call this quotient the Diffusion EXchange Ratio (DEXR). Furthermore, we show that it can be used to probe time-dependent diffusion by estimating the velocity autocorrelation function (VACF) over intermediate to long times (~ 2-500 ms). We provide a comprehensive theoretical framework for the design of DEXR experiments in the case of static or constant gradients. Data from Monte Carlo simulations and experiments acquired in fixed and viable ex vivo neonatal mouse spinal cord using a permanent magnet system are presented to test and validate this approach. In viable spinal cord, we report the following apparent parameters from just 6 data points: τk = 17 ± 4 ms, fNG = 0.71 ± 0.01, Reff = 1.10 ± 0.01 μm, and κeff = 0.21 ± 0.06 μm/ms, which correspond to the exchange time, restricted or non-Gaussian signal fraction, an effective spherical radius, and permeability, respectively. For the VACF, we report a long-time, power-law scaling with ≈ t-2.4, which is approximately consistent with disordered domains in 3-D. Overall, the DEXR method is shown to be highly efficient, capable of providing valuable quantitative diffusion metrics using minimal MR data.
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