Pub Date : 2024-10-01Epub Date: 2024-07-31DOI: 10.1016/j.bpj.2024.07.040
Carey E Dougan, Brandon L Roberts, Alfred J Crosby, Ilia N Karatsoreos, Shelly R Peyton
Traumatic brain injury (TBI) is an established risk factor for developing neurodegenerative disease. However, how TBI leads from acute injury to chronic neurodegeneration is limited to postmortem models. There is a lack of connections between in vitro and in vivo TBI models that can relate injury forces to both macroscale tissue damage and brain function at the cellular level. Needle-induced cavitation (NIC) is a technique that can produce small cavitation bubbles in soft tissues, which allows us to relate small strains and strain rates in living tissue to ensuing acute cell death, tissue damage, and tissue remodeling. Here, we applied NIC to mouse brain slices to create a new model of TBI with high spatial and temporal resolution. We specifically targeted the hippocampus, which is a brain region critical for learning and memory and an area in which injury causes cognitive pathologies in humans and rodent models. By combining NIC with patch-clamp electrophysiology, we demonstrate that NIC in the cornu ammonis 3 region of the hippocampus dynamically alters synaptic release onto cornu ammonis 1 pyramidal neurons in a cannabinoid 1 receptor-dependent manner. Further, we show that NIC induces an increase in extracellular matrix protein GFAP associated with neural repair that is mitigated by cannabinoid 1 receptor antagonism. Together, these data lay the groundwork for advanced approaches in understanding how TBI impacts neural function at the cellular level and the development of treatments that promote neural repair in response to brain injury.
创伤性脑损伤(TBI)是神经退行性疾病的既定风险因素。然而,创伤性脑损伤如何导致从急性损伤到慢性神经退行性病变的研究仅限于尸体模型。体外和体内创伤性脑损伤模型之间缺乏联系,无法在细胞水平上将损伤力与宏观组织损伤和大脑功能联系起来。针诱导空化(NIC)是一种能在软组织中产生小空化泡的技术,它能让我们将活组织中的小应变和应变率与随后的急性细胞死亡、组织损伤和组织重塑联系起来。在这里,我们将 NIC 应用于小鼠脑片,创建了一个具有高空间和时间分辨率的创伤性脑损伤新模型。我们特别针对海马区进行了研究,海马区是学习和记忆的关键脑区,在人类和啮齿类动物模型中,海马区的损伤会导致认知病变。通过将 NIC 与贴片钳电生理学相结合,我们证明了海马 Cornu Ammonis(CA)3 区的 NIC 以大麻素 1 受体(CB1R)依赖的方式动态改变了 CA1 锥体神经元的突触释放。此外,我们还发现,NIC 会诱导与神经修复相关的细胞外基质蛋白 GFAP 的增加,而 CB1R 拮抗剂会减轻这种增加。这些数据为了解创伤性脑损伤如何在细胞水平影响神经功能以及开发促进脑损伤后神经修复的治疗方法奠定了基础。
{"title":"Short-term neural and glial response to mild traumatic brain injury in the hippocampus.","authors":"Carey E Dougan, Brandon L Roberts, Alfred J Crosby, Ilia N Karatsoreos, Shelly R Peyton","doi":"10.1016/j.bpj.2024.07.040","DOIUrl":"10.1016/j.bpj.2024.07.040","url":null,"abstract":"<p><p>Traumatic brain injury (TBI) is an established risk factor for developing neurodegenerative disease. However, how TBI leads from acute injury to chronic neurodegeneration is limited to postmortem models. There is a lack of connections between in vitro and in vivo TBI models that can relate injury forces to both macroscale tissue damage and brain function at the cellular level. Needle-induced cavitation (NIC) is a technique that can produce small cavitation bubbles in soft tissues, which allows us to relate small strains and strain rates in living tissue to ensuing acute cell death, tissue damage, and tissue remodeling. Here, we applied NIC to mouse brain slices to create a new model of TBI with high spatial and temporal resolution. We specifically targeted the hippocampus, which is a brain region critical for learning and memory and an area in which injury causes cognitive pathologies in humans and rodent models. By combining NIC with patch-clamp electrophysiology, we demonstrate that NIC in the cornu ammonis 3 region of the hippocampus dynamically alters synaptic release onto cornu ammonis 1 pyramidal neurons in a cannabinoid 1 receptor-dependent manner. Further, we show that NIC induces an increase in extracellular matrix protein GFAP associated with neural repair that is mitigated by cannabinoid 1 receptor antagonism. Together, these data lay the groundwork for advanced approaches in understanding how TBI impacts neural function at the cellular level and the development of treatments that promote neural repair in response to brain injury.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11480756/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141874104","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-30DOI: 10.1016/j.bpj.2024.09.031
Busra Ozguney, Priyesh Mohanty, Jeetain Mittal
TAR DNA binding protein 43 (TDP-43) is a nuclear RNA/DNA-binding protein with pivotal roles in RNA-related processes such as splicing, transcription, transport, and stability. The high binding affinity and specificity of TDP-43 toward its cognate RNA sequences (GU-rich) is mediated by highly conserved residues in its tandem RNA recognition motif (RRM) domains (aa: 104-263). Importantly, the loss of RNA binding to the tandem RRMs caused by physiological stressors and chemical modifications promotes cytoplasmic mislocalization and pathological aggregation of TDP-43. Despite the substantial implications of RNA binding in TDP-43 function and pathology, its precise effects on the intradomain stability, and conformational dynamics of the tandem RRMs is not properly understood. Here, we employed all-atom molecular dynamics (MD) simulations to assess the effect of RNA binding on the conformational landscape and intradomain stability of TDP-43 tandem RRMs. RNA limits the overall conformational space of the tandem RRMs and promotes intradomain stability through a combination of specific base stacking interactions and transient electrostatic interactions. In contrast, tandem RRMs exhibit a high intrinsic conformational plasticity in the absence of RNA, which, surprisingly, is accompanied by a tendency of RRM1 to adopt partially unfolded conformations. Overall, our simulations reveal how RNA binding dynamically tunes the structural and conformational landscape of TDP-43 tandem RRMs, contributing to physiological function and mitigating pathological aggregation.
{"title":"RNA binding tunes the conformational plasticity and intradomain stability of TDP-43 tandem RNA recognition motifs.","authors":"Busra Ozguney, Priyesh Mohanty, Jeetain Mittal","doi":"10.1016/j.bpj.2024.09.031","DOIUrl":"10.1016/j.bpj.2024.09.031","url":null,"abstract":"<p><p>TAR DNA binding protein 43 (TDP-43) is a nuclear RNA/DNA-binding protein with pivotal roles in RNA-related processes such as splicing, transcription, transport, and stability. The high binding affinity and specificity of TDP-43 toward its cognate RNA sequences (GU-rich) is mediated by highly conserved residues in its tandem RNA recognition motif (RRM) domains (aa: 104-263). Importantly, the loss of RNA binding to the tandem RRMs caused by physiological stressors and chemical modifications promotes cytoplasmic mislocalization and pathological aggregation of TDP-43. Despite the substantial implications of RNA binding in TDP-43 function and pathology, its precise effects on the intradomain stability, and conformational dynamics of the tandem RRMs is not properly understood. Here, we employed all-atom molecular dynamics (MD) simulations to assess the effect of RNA binding on the conformational landscape and intradomain stability of TDP-43 tandem RRMs. RNA limits the overall conformational space of the tandem RRMs and promotes intradomain stability through a combination of specific base stacking interactions and transient electrostatic interactions. In contrast, tandem RRMs exhibit a high intrinsic conformational plasticity in the absence of RNA, which, surprisingly, is accompanied by a tendency of RRM1 to adopt partially unfolded conformations. Overall, our simulations reveal how RNA binding dynamically tunes the structural and conformational landscape of TDP-43 tandem RRMs, contributing to physiological function and mitigating pathological aggregation.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142360989","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-28DOI: 10.1016/j.bpj.2024.09.030
Luigi Catacuzzeno, Maria Vittoria Leonardi, Fabio Franciolini, Carmen Domene, Antonio Michelucci, Simone Furini
Molecular dynamics (MD) simulation of biological processes has always been a challenging task due to the long timescales of the processes involved and the large amount of output data to handle. Markov state models (MSMs) have been introduced as a powerful tool in this area of research, as they provide a mechanistically comprehensible synthesis of the large amount of MD data and, at the same time, can be used to rapidly estimate experimental properties of biological processes. Herein, we propose a method for building MSMs of ion channel permeation from MD trajectories, which directly evaluates the current flowing through the channel from the model's transition matrix (T), which is crucial for comparing simulations and experimental data. This is achieved by including in the model a flux matrix that summarizes information on the charge moving across the channel between each pair of states of the MSM and can be used in conjunction with T to predict the ion current. A procedure to drastically reduce the number of states in the MSM while preserving the estimated ion current is also proposed. Applying the method to the KcsA channel returned an MSM with five states with significant equilibrium occupancy, capable of accurately reproducing the single-channel ion current from microsecond MD trajectories.
{"title":"Building predictive Markov models of ion channel permeation from molecular dynamics simulations.","authors":"Luigi Catacuzzeno, Maria Vittoria Leonardi, Fabio Franciolini, Carmen Domene, Antonio Michelucci, Simone Furini","doi":"10.1016/j.bpj.2024.09.030","DOIUrl":"10.1016/j.bpj.2024.09.030","url":null,"abstract":"<p><p>Molecular dynamics (MD) simulation of biological processes has always been a challenging task due to the long timescales of the processes involved and the large amount of output data to handle. Markov state models (MSMs) have been introduced as a powerful tool in this area of research, as they provide a mechanistically comprehensible synthesis of the large amount of MD data and, at the same time, can be used to rapidly estimate experimental properties of biological processes. Herein, we propose a method for building MSMs of ion channel permeation from MD trajectories, which directly evaluates the current flowing through the channel from the model's transition matrix (T), which is crucial for comparing simulations and experimental data. This is achieved by including in the model a flux matrix that summarizes information on the charge moving across the channel between each pair of states of the MSM and can be used in conjunction with T to predict the ion current. A procedure to drastically reduce the number of states in the MSM while preserving the estimated ion current is also proposed. Applying the method to the KcsA channel returned an MSM with five states with significant equilibrium occupancy, capable of accurately reproducing the single-channel ion current from microsecond MD trajectories.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-09-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142340645","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-28DOI: 10.1016/j.bpj.2024.09.026
Isaac Pincus, Alison Rodger, J Ravi Prakash
We demonstrate the use of multiscale polymer modeling to quantitatively predict DNA linear dichroism (LD) in shear flow. LD is the difference in absorption of light polarized along two perpendicular axes and has long been applied to study biopolymer structure and drug-biopolymer interactions. As LD is orientation dependent, the sample must be aligned in order to measure a signal. Shear flow via a Couette cell can generate the required orientation; however, it is challenging to separate the LD due to changes in polymer conformation from specific interactions, e.g., drug-biopolymer. In this study, we have applied a combination of Brownian dynamics and equilibrium Monte Carlo simulations to accurately predict polymer alignment, and hence flow LD, at modest computational cost. As the optical and conformational contributions to the LD can be explicitly separated, our findings allow for enhanced quantitative interpretation of LD spectra through the use of an in silico model to capture conformational changes. Our model requires no fitting and only five input parameters: the DNA contour length, persistence length, optical factor, solvent quality, and relaxation time, all of which have been well characterized in prior literature. The method is sufficiently general to apply to a wide range of biopolymers beyond DNA, and our findings could help guide the search for new pharmaceutical drug targets via flow LD.
我们展示了利用多尺度聚合物建模定量预测剪切流中 DNA 线性二色性(LD)的方法。线性二色性是沿两个垂直轴偏振光的吸收差异,长期以来一直被用于研究生物聚合物结构和药物与生物聚合物之间的相互作用。由于 LD 与方向有关,因此必须对齐样品才能测量信号。通过 Couette 单元产生的剪切流可以产生所需的取向,但要将聚合物构象变化引起的 LD 与特定的相互作用(如药物-生物聚合物)区分开来则具有挑战性。在这项研究中,我们将布朗动力学和平衡蒙特卡洛模拟相结合,以适度的计算成本准确预测聚合物的配向,进而预测流动 LD。由于可以明确区分光学和构象对 LD 的贡献,我们的研究结果通过使用捕捉构象变化的室内模型,增强了对 LD 光谱的定量解释。我们的模型无需拟合,只需五个输入参数,即 DNA 轮廓长度、持久长度、光学因子、溶剂质量和弛豫时间,所有这些参数在之前的文献中都有很好的描述。该方法具有足够的通用性,可应用于 DNA 之外的多种生物聚合物,我们的发现有助于指导通过流动 LD 寻找新的药物靶点。
{"title":"Flow dichroism of DNA can be quantitatively predicted via coarse-grained molecular simulations.","authors":"Isaac Pincus, Alison Rodger, J Ravi Prakash","doi":"10.1016/j.bpj.2024.09.026","DOIUrl":"10.1016/j.bpj.2024.09.026","url":null,"abstract":"<p><p>We demonstrate the use of multiscale polymer modeling to quantitatively predict DNA linear dichroism (LD) in shear flow. LD is the difference in absorption of light polarized along two perpendicular axes and has long been applied to study biopolymer structure and drug-biopolymer interactions. As LD is orientation dependent, the sample must be aligned in order to measure a signal. Shear flow via a Couette cell can generate the required orientation; however, it is challenging to separate the LD due to changes in polymer conformation from specific interactions, e.g., drug-biopolymer. In this study, we have applied a combination of Brownian dynamics and equilibrium Monte Carlo simulations to accurately predict polymer alignment, and hence flow LD, at modest computational cost. As the optical and conformational contributions to the LD can be explicitly separated, our findings allow for enhanced quantitative interpretation of LD spectra through the use of an in silico model to capture conformational changes. Our model requires no fitting and only five input parameters: the DNA contour length, persistence length, optical factor, solvent quality, and relaxation time, all of which have been well characterized in prior literature. The method is sufficiently general to apply to a wide range of biopolymers beyond DNA, and our findings could help guide the search for new pharmaceutical drug targets via flow LD.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-09-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142340658","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-27DOI: 10.1016/j.bpj.2024.09.028
Alice J Pettitt, Vaibhav Kumar Shukla, Angelo Miguel Figueiredo, Lydia S Newton, Stephen McCarthy, Alethea B Tabor, Gabriella T Heller, Christian D Lorenz, D Flemming Hansen
Intrinsically disordered proteins (IDPs) often contain proline residues that undergo cis/trans isomerization. While molecular dynamics (MD) simulations have the potential to fully characterize the proline cis and trans subensembles, they are limited by the slow timescales of isomerization and force field inaccuracies. NMR spectroscopy can report on ensemble-averaged observables for both the cis-proline and trans-proline states, but a full atomistic characterization of these conformers is challenging. Given the importance of proline cis/trans isomerization for influencing the conformational sampling of disordered proteins, we employed a combination of all-atom MD simulations with enhanced sampling (metadynamics), NMR, and small-angle x-ray scattering (SAXS) to characterize the two subensembles of the ORF6 C-terminal region (ORF6CTR) from SARS-CoV-2 corresponding to the proline-57 (P57) cis and trans states. We performed MD simulations in three distinct force fields: AMBER03ws, AMBER99SB-disp, and CHARMM36m, which are all optimized for disordered proteins. Each simulation was run for an accumulated time of 180-220 μs until convergence was reached, as assessed by blocking analysis. A good agreement between the cis-P57 populations predicted from metadynamic simulations in AMBER03ws was observed with populations obtained from experimental NMR data. Moreover, we observed good agreement between the radius of gyration predicted from the metadynamic simulations in AMBER03ws and that measured using SAXS. Our findings suggest that both the cis-P57 and trans-P57 conformations of ORF6CTR are extremely dynamic and that interdisciplinary approaches combining both multiscale computations and experiments offer avenues to explore highly dynamic states that cannot be reliably characterized by either approach in isolation.
{"title":"An integrative characterization of proline cis and trans conformers in a disordered peptide.","authors":"Alice J Pettitt, Vaibhav Kumar Shukla, Angelo Miguel Figueiredo, Lydia S Newton, Stephen McCarthy, Alethea B Tabor, Gabriella T Heller, Christian D Lorenz, D Flemming Hansen","doi":"10.1016/j.bpj.2024.09.028","DOIUrl":"10.1016/j.bpj.2024.09.028","url":null,"abstract":"<p><p>Intrinsically disordered proteins (IDPs) often contain proline residues that undergo cis/trans isomerization. While molecular dynamics (MD) simulations have the potential to fully characterize the proline cis and trans subensembles, they are limited by the slow timescales of isomerization and force field inaccuracies. NMR spectroscopy can report on ensemble-averaged observables for both the cis-proline and trans-proline states, but a full atomistic characterization of these conformers is challenging. Given the importance of proline cis/trans isomerization for influencing the conformational sampling of disordered proteins, we employed a combination of all-atom MD simulations with enhanced sampling (metadynamics), NMR, and small-angle x-ray scattering (SAXS) to characterize the two subensembles of the ORF6 C-terminal region (ORF6<sub>CTR</sub>) from SARS-CoV-2 corresponding to the proline-57 (P57) cis and trans states. We performed MD simulations in three distinct force fields: AMBER03ws, AMBER99SB-disp, and CHARMM36m, which are all optimized for disordered proteins. Each simulation was run for an accumulated time of 180-220 μs until convergence was reached, as assessed by blocking analysis. A good agreement between the cis-P57 populations predicted from metadynamic simulations in AMBER03ws was observed with populations obtained from experimental NMR data. Moreover, we observed good agreement between the radius of gyration predicted from the metadynamic simulations in AMBER03ws and that measured using SAXS. Our findings suggest that both the cis-P57 and trans-P57 conformations of ORF6<sub>CTR</sub> are extremely dynamic and that interdisciplinary approaches combining both multiscale computations and experiments offer avenues to explore highly dynamic states that cannot be reliably characterized by either approach in isolation.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-09-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142340644","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-26DOI: 10.1016/j.bpj.2024.09.022
Shambhavi Pandey, Thorsten Wohland
The epidermal growth factor receptor (EGFR) governs pivotal signaling pathways in cell proliferation and survival, with mutations implicated in numerous cancers. The organization of EGFR on the plasma membrane (PM) is influenced by the lipids and the cortical actin (CA) cytoskeleton. Despite the presence of a putative actin-binding domain (ABD) spanning 13 residues, a direct interaction between EGFR and CA has not been definitively established. While disrupting the cytoskeleton can impact EGFR behavior, suggesting a connection, the influence of the static actin cytoskeleton has been found to be indirect. Here, we investigate the potential interaction between EGFR and CA, as well as the extent to which CA regulates EGFR's distribution on the PM using SRRF'n'TIRF, a spatiotemporal super-resolution microscopy technique that provides sub-100 nm resolution and ms-scale dynamics from the same data set. To label CA, we constructed PMT-mEGFP-F-tractin, which combines an inner leaflet targeting domain PMT, fluorescent probe mEGFP, and the actin-binding protein F-tractin. In addition to EGFR-mEGFP, we included two control constructs: 1) an ABD deletion mutant, EGFRΔABD-mEGFP serving as a negative control and 2) EGFR-mApple-F-tractin, where F-tractin is fused to the C-terminus of EGFR-mApple, serving as the positive control. We find that EGFR-mEGFP and EGFRΔABD-mEGFP show similar membrane dynamics, implying that EGFR-mEGFP dynamics and organization are independent of CA. EGFR dynamics show CA dependence when F-tractin is anchored to the cytoplasmic tail. Together, our results demonstrate that EGFR does not directly interact with the CA in its resting and activated state.
表皮生长因子受体(EGFR)是细胞增殖和存活的关键信号通路,其突变与多种癌症有关。表皮生长因子受体在质膜(PM)上的组织受脂类和皮质肌动蛋白(CA)细胞骨架的影响。尽管表皮生长因子受体存在一个跨越 13 个残基的推测肌动蛋白结合域 (ABD),但表皮生长因子受体与肌动蛋白之间的直接相互作用尚未得到明确证实。虽然破坏细胞骨架会影响表皮生长因子受体的行为,这表明两者之间存在联系,但已发现静态肌动蛋白细胞骨架的影响是间接的。在这里,我们利用 SRRF'n'TIRF 研究了表皮生长因子受体和 CA 之间的潜在相互作用,以及 CA 在多大程度上调节表皮生长因子受体在 PM 上的分布,SRRF'n'TIRF 是一种时空超分辨显微镜技术,可从同一数据集提供亚 100 纳米分辨率和毫秒级动态。为了标记 CA,我们构建了 PMT-mEGFP-F-tractin,它结合了内叶靶向结构域 PMT、荧光探针 mEGFP 和肌动蛋白结合蛋白 F-tractin。除 EGFR-mEGFP 外,我们还加入了两个对照构建物:a)ABD 缺失突变体 EGFR ΔABD-mEGFP 作为阴性对照;b)EGFR-mApple-F-tractin,其中 F-tractin 与 EGFR-mApple 的 C 端融合,作为阳性对照。我们发现 EGFR-mEGFP 和 EGFR Δ ABD-mEGFP 表现出相似的膜动态,这意味着 EGFR-mEGFP 的动态和组织与 CA 无关。当F-tractin锚定在胞质尾部时,表皮生长因子受体的动态表现出对CA的依赖性。总之,我们的研究结果表明,表皮生长因子受体在静止和活化状态下并不直接与 CA 相互作用。
{"title":"EGFR does not directly interact with cortical actin: A SRRF'n'TIRF study.","authors":"Shambhavi Pandey, Thorsten Wohland","doi":"10.1016/j.bpj.2024.09.022","DOIUrl":"10.1016/j.bpj.2024.09.022","url":null,"abstract":"<p><p>The epidermal growth factor receptor (EGFR) governs pivotal signaling pathways in cell proliferation and survival, with mutations implicated in numerous cancers. The organization of EGFR on the plasma membrane (PM) is influenced by the lipids and the cortical actin (CA) cytoskeleton. Despite the presence of a putative actin-binding domain (ABD) spanning 13 residues, a direct interaction between EGFR and CA has not been definitively established. While disrupting the cytoskeleton can impact EGFR behavior, suggesting a connection, the influence of the static actin cytoskeleton has been found to be indirect. Here, we investigate the potential interaction between EGFR and CA, as well as the extent to which CA regulates EGFR's distribution on the PM using SRRF'n'TIRF, a spatiotemporal super-resolution microscopy technique that provides sub-100 nm resolution and ms-scale dynamics from the same data set. To label CA, we constructed PMT-mEGFP-F-tractin, which combines an inner leaflet targeting domain PMT, fluorescent probe mEGFP, and the actin-binding protein F-tractin. In addition to EGFR-mEGFP, we included two control constructs: 1) an ABD deletion mutant, EGFRΔ<sup>ABD</sup>-mEGFP serving as a negative control and 2) EGFR-mApple-F-tractin, where F-tractin is fused to the C-terminus of EGFR-mApple, serving as the positive control. We find that EGFR-mEGFP and EGFRΔ<sup>ABD</sup>-mEGFP show similar membrane dynamics, implying that EGFR-mEGFP dynamics and organization are independent of CA. EGFR dynamics show CA dependence when F-tractin is anchored to the cytoplasmic tail. Together, our results demonstrate that EGFR does not directly interact with the CA in its resting and activated state.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142340646","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Many animal cells that crawl on extracellular substrates exhibit durotaxis, i.e., directed migration toward stiffer substrate regions. This has implications in several biological processes including tissue development and tumor progression. Here, we introduce a phenomenological model for single-cell durotaxis that incorporates both elastic deformation-mediated cell-substrate interactions and the stochasticity of cell migration. Our model is motivated by a key observation in an early demonstration of durotaxis: a single, contractile cell at a sharp interface between a softer and a stiffer region of an elastic substrate reorients and migrates toward the stiffer region. We model migrating cells as self-propelling, persistently motile agents that exert contractile traction forces on their elastic substrate. The resulting substrate deformations induce elastic interactions with mechanical boundaries, captured by an elastic potential. The dynamics is determined by two crucial parameters: the strength of the cellular traction-induced boundary elastic interaction (A), and the persistence of cell motility (Pe). Elastic forces and torques resulting from the potential orient cells perpendicular (parallel) to the boundary and accumulate (deplete) them at the clamped (free) boundary. Thus, a clamped boundary induces an attractive potential that drives durotaxis, while a free boundary induces a repulsive potential that prevents antidurotaxis. By quantifying the steady-state position and orientation probability densities, we show how the extent of accumulation (depletion) depends on the strength of the elastic potential and motility. We compare and contrast crawling cells with biological microswimmers and other synthetic active particles, where accumulation at confining boundaries is well known. We define metrics quantifying boundary accumulation and durotaxis, and present a phase diagram that identifies three possible regimes: durotaxis, and adurotaxis with and without motility-induced accumulation at the boundary. Overall, our model predicts how durotaxis depends on cell contractility and motility, successfully explains some previous observations, and provides testable predictions to guide future experiments.
{"title":"Elastic interactions compete with persistent cell motility to drive durotaxis.","authors":"Subhaya Bose, Haiqin Wang, Xinpeng Xu, Arvind Gopinath, Kinjal Dasbiswas","doi":"10.1016/j.bpj.2024.09.021","DOIUrl":"10.1016/j.bpj.2024.09.021","url":null,"abstract":"<p><p>Many animal cells that crawl on extracellular substrates exhibit durotaxis, i.e., directed migration toward stiffer substrate regions. This has implications in several biological processes including tissue development and tumor progression. Here, we introduce a phenomenological model for single-cell durotaxis that incorporates both elastic deformation-mediated cell-substrate interactions and the stochasticity of cell migration. Our model is motivated by a key observation in an early demonstration of durotaxis: a single, contractile cell at a sharp interface between a softer and a stiffer region of an elastic substrate reorients and migrates toward the stiffer region. We model migrating cells as self-propelling, persistently motile agents that exert contractile traction forces on their elastic substrate. The resulting substrate deformations induce elastic interactions with mechanical boundaries, captured by an elastic potential. The dynamics is determined by two crucial parameters: the strength of the cellular traction-induced boundary elastic interaction (A), and the persistence of cell motility (Pe). Elastic forces and torques resulting from the potential orient cells perpendicular (parallel) to the boundary and accumulate (deplete) them at the clamped (free) boundary. Thus, a clamped boundary induces an attractive potential that drives durotaxis, while a free boundary induces a repulsive potential that prevents antidurotaxis. By quantifying the steady-state position and orientation probability densities, we show how the extent of accumulation (depletion) depends on the strength of the elastic potential and motility. We compare and contrast crawling cells with biological microswimmers and other synthetic active particles, where accumulation at confining boundaries is well known. We define metrics quantifying boundary accumulation and durotaxis, and present a phase diagram that identifies three possible regimes: durotaxis, and adurotaxis with and without motility-induced accumulation at the boundary. Overall, our model predicts how durotaxis depends on cell contractility and motility, successfully explains some previous observations, and provides testable predictions to guide future experiments.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142340647","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-26DOI: 10.1016/j.bpj.2024.09.027
Kuilin Meng, Haosheng Chen, Yunfan Pan, Yongjian Li
Hemolysis, including subclinical hemolysis, is a potentially severe complications of mechanical heart valves (MHVs), which leads to shortened red blood cell (RBC) lifespan and hemolytic anemia. Serious hemolysis is usually associated with structural deterioration and regurgitation. However, the shear stress in MHVs' narrow leakage slits is much lower than the shear stress threshold causing hemolysis and the mechanisms in this context remain largely unclear. This study investigated the hemolysis mechanism of RBCs in cell-size slits under high shear rates by establishing in vitro microfluidic devices and a coarse-grained molecular dynamics (CGMD) model, considering both fluid and structural effects simultaneously. Microfluidic experiments and computational simulation revealed six distinct dynamic states of RBC traversal through MHVs' microscale slits under various shear rates and slit sizes. It elucidated that RBC dynamic states were influenced by not only by fluid forces but significantly by the compressive force of slit walls. The variation of the potential energy of the cell membrane indicated its stretching, deformation, and rupture during traversal, corresponding to the six dynamic states. The maximum forces exerted on membrane by water particles and slit walls directly determined membrane rupture, serving as a critical determinant. This analysis helps in understanding the contribution of the slit walls to membrane rupture and identifying the threshold force that leads to membrane rupture. The hemolysis mechanism of traversing microscale slits is revealed to effectively explain the occurrences of hemolysis and subclinical hemolysis.
{"title":"The dynamics of red blood cells traversing slits of mechanical heart valves under high shear.","authors":"Kuilin Meng, Haosheng Chen, Yunfan Pan, Yongjian Li","doi":"10.1016/j.bpj.2024.09.027","DOIUrl":"10.1016/j.bpj.2024.09.027","url":null,"abstract":"<p><p>Hemolysis, including subclinical hemolysis, is a potentially severe complications of mechanical heart valves (MHVs), which leads to shortened red blood cell (RBC) lifespan and hemolytic anemia. Serious hemolysis is usually associated with structural deterioration and regurgitation. However, the shear stress in MHVs' narrow leakage slits is much lower than the shear stress threshold causing hemolysis and the mechanisms in this context remain largely unclear. This study investigated the hemolysis mechanism of RBCs in cell-size slits under high shear rates by establishing in vitro microfluidic devices and a coarse-grained molecular dynamics (CGMD) model, considering both fluid and structural effects simultaneously. Microfluidic experiments and computational simulation revealed six distinct dynamic states of RBC traversal through MHVs' microscale slits under various shear rates and slit sizes. It elucidated that RBC dynamic states were influenced by not only by fluid forces but significantly by the compressive force of slit walls. The variation of the potential energy of the cell membrane indicated its stretching, deformation, and rupture during traversal, corresponding to the six dynamic states. The maximum forces exerted on membrane by water particles and slit walls directly determined membrane rupture, serving as a critical determinant. This analysis helps in understanding the contribution of the slit walls to membrane rupture and identifying the threshold force that leads to membrane rupture. The hemolysis mechanism of traversing microscale slits is revealed to effectively explain the occurrences of hemolysis and subclinical hemolysis.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142340660","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-26DOI: 10.1016/j.bpj.2024.09.025
Emil E Tranchant, Francesco Pesce, Nina L Jacobsen, Catarina B Fernandes, Birthe B Kragelund, Kresten Lindorff-Larsen
Measuring the compaction of a protein or complex is key to our understanding of the interactions within and between biomolecules. Experimentally, protein compaction is often probed either by estimating the radius of gyration (Rg) obtained from small-angle x-ray scattering (SAXS) experiments or the hydrodynamic radius (Rh) obtained, for example, by pulsed field gradient NMR (PFG NMR) spectroscopy. PFG NMR experiments generally report on the translational diffusion coefficient, which in turn can be used to estimate Rh using an internal standard to account for sample viscosity and uncertainty about the gradient strength. 1,4-Dioxane is one such commonly used internal standard, and the reference value of Rh is therefore important. We have revisited the basis for the commonly used reference value for the Rh of dioxane (2.12 Å) that is used to convert measured diffusion coefficients into a hydrodynamic radius. We followed the same approach that was used to establish the current reference value by measuring SAXS and PFG NMR data for a set of seven different proteins and using these as standards. Our analysis shows that the current Rh reference value for dioxane Rh is underestimated, and we instead suggest a new value of 2.27 ± 0.04 Å. Using this updated reference value results in a ∼7% increase in Rh values for proteins whose hydrodynamic radii have been measured by PFG NMR. These results are particularly important when the absolute value of Rh is of interest such as when determining or validating ensemble descriptions of intrinsically disordered proteins.
{"title":"On the use of dioxane as reference for determination of the hydrodynamic radius by NMR spectroscopy.","authors":"Emil E Tranchant, Francesco Pesce, Nina L Jacobsen, Catarina B Fernandes, Birthe B Kragelund, Kresten Lindorff-Larsen","doi":"10.1016/j.bpj.2024.09.025","DOIUrl":"10.1016/j.bpj.2024.09.025","url":null,"abstract":"<p><p>Measuring the compaction of a protein or complex is key to our understanding of the interactions within and between biomolecules. Experimentally, protein compaction is often probed either by estimating the radius of gyration (R<sub>g</sub>) obtained from small-angle x-ray scattering (SAXS) experiments or the hydrodynamic radius (R<sub>h</sub>) obtained, for example, by pulsed field gradient NMR (PFG NMR) spectroscopy. PFG NMR experiments generally report on the translational diffusion coefficient, which in turn can be used to estimate R<sub>h</sub> using an internal standard to account for sample viscosity and uncertainty about the gradient strength. 1,4-Dioxane is one such commonly used internal standard, and the reference value of R<sub>h</sub> is therefore important. We have revisited the basis for the commonly used reference value for the R<sub>h</sub> of dioxane (2.12 Å) that is used to convert measured diffusion coefficients into a hydrodynamic radius. We followed the same approach that was used to establish the current reference value by measuring SAXS and PFG NMR data for a set of seven different proteins and using these as standards. Our analysis shows that the current R<sub>h</sub> reference value for dioxane R<sub>h</sub> is underestimated, and we instead suggest a new value of 2.27 ± 0.04 Å. Using this updated reference value results in a ∼7% increase in R<sub>h</sub> values for proteins whose hydrodynamic radii have been measured by PFG NMR. These results are particularly important when the absolute value of R<sub>h</sub> is of interest such as when determining or validating ensemble descriptions of intrinsically disordered proteins.</p>","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":null,"pages":null},"PeriodicalIF":3.2,"publicationDate":"2024-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142340659","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-25DOI: 10.1016/j.bpj.2024.09.023
Gregg A Duncan
{"title":"Mind the gap: Exploring extracellular spaces in the brain with particle tracking and AI.","authors":"Gregg A Duncan","doi":"10.1016/j.bpj.2024.09.023","DOIUrl":"https://doi.org/10.1016/j.bpj.2024.09.023","url":null,"abstract":"","PeriodicalId":8922,"journal":{"name":"Biophysical journal","volume":null,"pages":null},"PeriodicalIF":3.4,"publicationDate":"2024-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142328640","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}