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}
Pub Date : 2024-09-08DOI: 10.1101/2024.09.07.611813
Ke Xiao, Sujeong Park, Jeanne Stachowiak, Padmini Rangamani
Generation of membrane curvature is fundamental to cellular function. Recent studies have established that the glycocalyx, a sugar-rich polymer layer at the cell surface, can generate membrane curvature. While there have been some theoretical efforts to understand the interplay between the glycocalyx and membrane bending, there remain open questions about how the properties of the glycocalyx affect membrane bending. For example, the relationship between membrane curvature and the density of glycosylated proteins on its surface remains unclear. In this work, we use polymer brush theory to develop a detailed biophysical model of the energetic interactions of the glycocalyx with the membrane. Using this model, we identify the conditions under which the glycocalyx can both generate and sense curvature. Our model predicts that the extent of membrane curvature generated depends on the grafting density of the glycocalyx and the length of the polymers constituting the glycocalyx. Furthermore, when coupled with the intrinsic membrane properties such as spontaneous curvature and a line tension along the membrane, the curvature generation properties of the glycocalyx are enhanced. These predictions were tested experimentally by examining the propensity of glycosylated transmembrane proteins to drive the assembly of highly-curved filopodial protrusions at the plasma membrane of adherent mammalian cells. Our model also predicts that the glycocalyx has curvature-sensing capabilities, in agreement with the results of our experiments. Thus, our study develops a quantitative framework for mapping the properties of the glycocalyx to the curvature generation capability of the membrane.
{"title":"Biophysical modeling of membrane curvature generation and curvature sensing by the glycocalyx","authors":"Ke Xiao, Sujeong Park, Jeanne Stachowiak, Padmini Rangamani","doi":"10.1101/2024.09.07.611813","DOIUrl":"https://doi.org/10.1101/2024.09.07.611813","url":null,"abstract":"Generation of membrane curvature is fundamental to cellular function. Recent studies have established that the glycocalyx, a sugar-rich polymer layer at the cell surface, can generate membrane curvature. While there have been some theoretical efforts to understand the interplay between the glycocalyx and membrane bending, there remain open questions about how the properties of the glycocalyx affect membrane bending. For example, the relationship between membrane curvature and the density of glycosylated proteins on its surface remains unclear. In this work, we use polymer brush theory to develop a detailed biophysical model of the energetic interactions of the glycocalyx with the membrane. Using this model, we identify the conditions under which the glycocalyx can both generate and sense curvature. Our model predicts that the extent of membrane curvature generated depends on the grafting density of the glycocalyx and the length of the polymers constituting the glycocalyx. Furthermore, when coupled with the intrinsic membrane properties such as spontaneous curvature and a line tension along the membrane, the curvature generation properties of the glycocalyx are enhanced. These predictions were tested experimentally by examining the propensity of glycosylated transmembrane proteins to drive the assembly of highly-curved filopodial protrusions at the plasma membrane of adherent mammalian cells. Our model also predicts that the glycocalyx has curvature-sensing capabilities, in agreement with the results of our experiments.\u0000Thus, our study develops a quantitative framework for mapping the properties of the glycocalyx to the curvature generation capability of the membrane.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"13 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178221","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: 10.1101/2024.09.04.611338
Hiroyuki Iwamoto
The contractile machinery of muscle, especially that of skeletal muscle, has a very regular array of contractile protein filaments, and gives rise to a very complex and informative diffraction pattern when irradiated with X-rays. However, the analysis of the diffraction patterns is often difficult, because (1) only rotationally averaged diffraction patterns can be obtained, resulting in substantial loss of information, and (2) the contractile machinery contains two different sets of protein filaments (actin and myosin) with different helical symmetries, and the reflections originating from them are often overlapped. These problems may be solved if the real-space 3-D structure of the contractile machinery is directly calculated from the diffraction pattern. Here we demonstrate that, by using the conventional phase-retrieval algorithm (hybrid input-output), the real-space 3-D structure of the contractile machinery can be well restored from a single rotationally averaged 2-D diffraction pattern. In this calculation, we used a model structure of insect flight muscle, known to have a very regular structure. Possibilities of extending this technique to the actual muscle diffraction patterns is discussed.
肌肉（尤其是骨骼肌）的收缩机制具有非常规则的收缩蛋白丝阵列，用 X 射线照射时会产生非常复杂且信息丰富的衍射图样。然而，对衍射图样的分析往往很困难，因为：（1）只能获得旋转平均衍射图样，导致大量信息丢失；（2）收缩机械包含两组不同螺旋对称性的不同蛋白丝（肌动蛋白和肌球蛋白），它们产生的反射常常重叠。如果能根据衍射图样直接计算出收缩机械的实空间三维结构，这些问题就能迎刃而解。在这里，我们证明了通过使用传统的相位检索算法（混合输入输出），可以从单一的旋转平均二维衍射图样中很好地还原出收缩机械的实空间三维结构。在计算中，我们使用了昆虫飞行肌肉的模型结构，已知其结构非常规则。我们还讨论了将这一技术扩展到实际肌肉衍射图样的可能性。
{"title":"Restoration of 3-D structure of insect flight muscle from a rotationally averaged 2-D X-ray diffraction pattern","authors":"Hiroyuki Iwamoto","doi":"10.1101/2024.09.04.611338","DOIUrl":"https://doi.org/10.1101/2024.09.04.611338","url":null,"abstract":"The contractile machinery of muscle, especially that of skeletal muscle, has a very regular array of contractile protein filaments, and gives rise to a very complex and informative diffraction pattern when irradiated with X-rays. However, the analysis of the diffraction patterns is often difficult, because (1) only rotationally averaged diffraction patterns can be obtained, resulting in substantial loss of information, and (2) the contractile machinery contains two different sets of protein filaments (actin and myosin) with different helical symmetries, and the reflections originating from them are often overlapped. These problems may be solved if the real-space 3-D structure of the contractile machinery is directly calculated from the diffraction pattern. Here we demonstrate that, by using the conventional phase-retrieval algorithm (hybrid input-output), the real-space 3-D structure of the contractile machinery can be well restored from a single rotationally averaged 2-D diffraction pattern. In this calculation, we used a model structure of insect flight muscle, known to have a very regular structure. Possibilities of extending this technique to the actual muscle diffraction patterns is discussed.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"48 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178220","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}
Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) is widely used in the design of genetically encoded fluorescent biosensors, which are powerful tools for monitoring the dynamics of biochemical activities in live cells. FRET ratio, defined as the ratio between acceptor and donor signals, is often used as a proxy for the actual FRET efficiency, which must be corrected for signal crosstalk using donor-only and acceptor-only samples. However, the FRET ratio is highly sensitive to imaging conditions, making direct comparisons across different experiments and over time challenging. Inspired by a method for multiplexed biosensor imaging using barcoded cells, we reasoned that calibration standards with fixed FRET efficiency can be introduced into a subset of cells for normalization of biosensor signals. Our theoretical analysis indicated that the FRET ratio of high-FRET species relative to non-FRET species slightly decreases at high excitation intensity, suggesting the need for calibration using both high and low FRET standards. To test these predictions, we created FRET donor-acceptor pairs locked in "FRET-ON" and "FRET-OFF" conformations and introduced them into a subset of cells using the cell barcoding strategy. Our results confirmed the theoretical predictions and showed that the calibrated FRET ratio is independent of imaging settings. We also provided a strategy for calculating the FRET efficiency. Together, our study presents a simple strategy for calibrated and highly multiplexed imaging of FRET biosensors, facilitating reliable comparisons across experiments and supporting long-term imaging applications.
{"title":"Calibration of FRET-based biosensors using multiplexed biosensor barcoding","authors":"Jhen-Wei Wu, Jr-Ming Yang, Chao-Cheng Chen, Gabriel Au, Suyang Wang, Gia-Wei Chern, Chuan-Hsiang Huang","doi":"10.1101/2024.09.04.610346","DOIUrl":"https://doi.org/10.1101/2024.09.04.610346","url":null,"abstract":"Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) is widely used in the design of genetically encoded fluorescent biosensors, which are powerful tools for monitoring the dynamics of biochemical activities in live cells. FRET ratio, defined as the ratio between acceptor and donor signals, is often used as a proxy for the actual FRET efficiency, which must be corrected for signal crosstalk using donor-only and acceptor-only samples. However, the FRET ratio is highly sensitive to imaging conditions, making direct comparisons across different experiments and over time challenging. Inspired by a method for multiplexed biosensor imaging using barcoded cells, we reasoned that calibration standards with fixed FRET efficiency can be introduced into a subset of cells for normalization of biosensor signals. Our theoretical analysis indicated that the FRET ratio of high-FRET species relative to non-FRET species slightly decreases at high excitation intensity, suggesting the need for calibration using both high and low FRET standards. To test these predictions, we created FRET donor-acceptor pairs locked in \"FRET-ON\" and \"FRET-OFF\" conformations and introduced them into a subset of cells using the cell barcoding strategy. Our results confirmed the theoretical predictions and showed that the calibrated FRET ratio is independent of imaging settings. We also provided a strategy for calculating the FRET efficiency. Together, our study presents a simple strategy for calibrated and highly multiplexed imaging of FRET biosensors, facilitating reliable comparisons across experiments and supporting long-term imaging applications.","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":"142178243","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: 10.1101/2024.09.04.611326
Daichi Takahashi, Hana Kiyama, Hideaki Matsubayashi, Ikuko Fujiwara, Makoto Miyata
Spiroplasma is a wall−less helical bacterium possessing five isoforms of bacterial actin MreBs (SMreB1−5) for its swimming, speculated to be the sole motility system driven by endogenous bacterial actin proteins. Its detailed molecular mechanism remains elusive due to the lack of soluble constructs of SMreB1 essential for Spiroplasma swimming. Here, we isolated soluble SMreB1 of Spiroplasma eriocheiris (SpeMreB1) and evaluated its activity. The phosphate release rate and fold changes of polymerization−critical concentrations over the nucleotide states of SpeMreB1 are the highest among the MreB family proteins. SpeMreB1 interacts with polymerized SpeMreB5, another SMreB essential for Spiroplasma swimming, and decreases SpeMreB5 filament amount depending on the nucleotide state. A decrease in SpeMreB5 filament amount is independent of SpeMreB1 polymerization, although it is essential for swimming motility. SpeMreB1 binds to negatively charged lipids, regardless of their nucleotide state. These results suggest that SpeMreB1 manages SpeMreB5 filaments to drive Spiroplasma swimming.
{"title":"A highly active bacterial actin actuates the polymerization of another isoform essential for swimming motility of Spiroplasma","authors":"Daichi Takahashi, Hana Kiyama, Hideaki Matsubayashi, Ikuko Fujiwara, Makoto Miyata","doi":"10.1101/2024.09.04.611326","DOIUrl":"https://doi.org/10.1101/2024.09.04.611326","url":null,"abstract":"<em>Spiroplasma</em> is a wall−less helical bacterium possessing five isoforms of bacterial actin MreBs (SMreB1−5) for its swimming, speculated to be the sole motility system driven by endogenous bacterial actin proteins. Its detailed molecular mechanism remains elusive due to the lack of soluble constructs of SMreB1 essential for <em>Spiroplasma</em> swimming. Here, we isolated soluble SMreB1 of <em>Spiroplasma eriocheiris </em>(SpeMreB1) and evaluated its activity. The phosphate release rate and fold changes of polymerization−critical concentrations over the nucleotide states of SpeMreB1 are the highest among the MreB family proteins. SpeMreB1 interacts with polymerized SpeMreB5, another SMreB essential for <em>Spiroplasma</em> swimming, and decreases SpeMreB5 filament amount depending on the nucleotide state. A decrease in SpeMreB5 filament amount is independent of SpeMreB1 polymerization, although it is essential for swimming motility. SpeMreB1 binds to negatively charged lipids, regardless of their nucleotide state. These results suggest that SpeMreB1 manages SpeMreB5 filaments to drive <em>Spiroplasma</em> swimming.","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":"142178251","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: 10.1101/2024.09.04.611260
Aher Jayesh Bhausaheb, Aniruddha N, Debraj Koiri, Jafarulla Shaikh, Sandeep Choubey, Mohammed Saleem
Pathogenic bacteria evade host defense by rupturing the phagosomal compartment, enabling their escape into the host cytosol. The bacteria is known to establish direct physical contact with the host compartment prior to phagosome disruption. However, the impact of direct-physical contact on the phagosome remodeling and deformation remains elusive. To probe this, we first developed a method wherein we reconstitute a phagosome-like giant compartment encapsulating Mycobacterium smegmatis, a non-motile opportunistic bacterium. We discover that the direct contact between the bacteria and the encapsulating host membrane induces membrane bending, lipid wrapping, and local lipid phase separation at the contact site. The degree of phase separation is driven by the bacterial load leading to fluidization of the membrane, as evident from the decreased area stretch and bending modulus, making the host compartment more deformable. Surprisingly, for saturating bacterial load the fluid host membrane transforms into a scaffold-like rigid layer. We also find that the direct contact of the bacteria enhances the membranolytic potential of ESAT-6 thus contributing to its virulence. Together our findings provide mechanistic insights into the role of direct physical contact of the bacteria during phagosome disruption.
{"title":"Direct contact of the bacterial surface induces phase separation in the host phagosome membrane","authors":"Aher Jayesh Bhausaheb, Aniruddha N, Debraj Koiri, Jafarulla Shaikh, Sandeep Choubey, Mohammed Saleem","doi":"10.1101/2024.09.04.611260","DOIUrl":"https://doi.org/10.1101/2024.09.04.611260","url":null,"abstract":"Pathogenic bacteria evade host defense by rupturing the phagosomal compartment, enabling their escape into the host cytosol. The bacteria is known to establish direct physical contact with the host compartment prior to phagosome disruption. However, the impact of direct-physical contact on the phagosome remodeling and deformation remains elusive. To probe this, we first developed a method wherein we reconstitute a phagosome-like giant compartment encapsulating Mycobacterium smegmatis, a non-motile opportunistic bacterium. We discover that the direct contact between the bacteria and the encapsulating host membrane induces membrane bending, lipid wrapping, and local lipid phase separation at the contact site. The degree of phase separation is driven by the bacterial load leading to fluidization of the membrane, as evident from the decreased area stretch and bending modulus, making the host compartment more deformable. Surprisingly, for saturating bacterial load the fluid host membrane transforms into a scaffold-like rigid layer. We also find that the direct contact of the bacteria enhances the membranolytic potential of ESAT-6 thus contributing to its virulence. Together our findings provide mechanistic insights into the role of direct physical contact of the bacteria during phagosome disruption.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"25 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178216","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}
Alzheimer's disease (AD) is caused by the assembly of amyloid-beta (Aβ) peptides into oligomers and fibrils. Endogenous Aβ aggregation may be assisted by cell membranes, which can accelerate the nucleation step enormously, but knowledge of membrane-assisted aggregation is still very limited. Here we used extensive MD simulations to structurally and energetically characterize key intermediates along the membrane-assisted aggregation pathways of Aβ40. Reinforcing experimental observations, the simulations reveal unique roles of GM1 ganglioside and cholesterol in stabilizing membrane-embedded β-sheets and of Y10 and K28 in the ordered release of a small oligomeric seed into solution. The same seed leads to either an open-shaped or R-shaped fibril, with significant stabilization provided by inter- or intra-subunit interfaces between a straight β-sheet (residues Q15-D23) and a bent β-sheet (residues A30-V36). This work presents the first comprehensive picture of membrane-assisted aggregation of Aβ40, with broad implications for developing AD therapies and rationalizing disease-specific polymorphisms of amyloidogenic proteins.
{"title":"Membrane-assisted Aβ40 aggregation pathways","authors":"Fidha Nazreen Kunnath Muhammedkutty, Huan-Xiang Zhou","doi":"10.1101/2024.09.05.611426","DOIUrl":"https://doi.org/10.1101/2024.09.05.611426","url":null,"abstract":"Alzheimer's disease (AD) is caused by the assembly of amyloid-beta (Aβ) peptides into oligomers and fibrils. Endogenous Aβ aggregation may be assisted by cell membranes, which can accelerate the nucleation step enormously, but knowledge of membrane-assisted aggregation is still very limited. Here we used extensive MD simulations to structurally and energetically characterize key intermediates along the membrane-assisted aggregation pathways of Aβ40. Reinforcing experimental observations, the simulations reveal unique roles of GM1 ganglioside and cholesterol in stabilizing membrane-embedded β-sheets and of Y10 and K28 in the ordered release of a small oligomeric seed into solution. The same seed leads to either an open-shaped or R-shaped fibril, with significant stabilization provided by inter- or intra-subunit interfaces between a straight β-sheet (residues Q15-D23) and a bent β-sheet (residues A30-V36). This work presents the first comprehensive picture of membrane-assisted aggregation of Aβ40, with broad implications for developing AD therapies and rationalizing disease-specific polymorphisms of amyloidogenic proteins.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"4 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178240","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 : 2024-09-07DOI: 10.1101/2024.09.05.611544
Henry T Phan, Chun-Wei Lin, Brittany L. Stinger, Joseph B. DeGrandchamp, L.J. Nugent Lew, Serena J. Huang, Jay T Groves
Upon ligand binding, the kinase domain of EGFR phosphorylates multiple tyrosine residues on the receptor cytoplasmic tail through a trans-autophosphorylation process. Phosphotyrosine sites on activated receptors recruit Grb2, which further recruits SOS to initiate downstream signaling by activating Ras. Multivalent binding between SOS and Grb2, as well as direct Grb2:Grb2 interactions, contribute to formation of a protein condensate of activated EGFR. The condensed state of EGFR facilitates autoinhibition release in SOS and exerts regulatory control over signal propagation from activated EGFR to Ras. While kinase activity of EGFR is an essential driver of this signaling process, phosphorylation at residue Y160 on Grb2 blocks Grb2:Grb2 binding and can interfere with EGFR condensation. Here, using a reconstituted system, we examine how titrating kinase activity in the EGFR system can both promote and inhibit signal output to Ras. The results reveal how effects of tyrosine kinase inhibition can, under some circumstances, promote Ras activation by inhibiting negative feedback through Grb2 phosphorylation and disruption of a Grb2 SH2/SH3 dimer interface.
{"title":"Grb2 Phosphorylation Antagonizes EGFR-driven Ras Activation by Interfering with Condensate Assembly","authors":"Henry T Phan, Chun-Wei Lin, Brittany L. Stinger, Joseph B. DeGrandchamp, L.J. Nugent Lew, Serena J. Huang, Jay T Groves","doi":"10.1101/2024.09.05.611544","DOIUrl":"https://doi.org/10.1101/2024.09.05.611544","url":null,"abstract":"Upon ligand binding, the kinase domain of EGFR phosphorylates multiple tyrosine residues on the receptor cytoplasmic tail through a trans-autophosphorylation process. Phosphotyrosine sites on activated receptors recruit Grb2, which further recruits SOS to initiate downstream signaling by activating Ras. Multivalent binding between SOS and Grb2, as well as direct Grb2:Grb2 interactions, contribute to formation of a protein condensate of activated EGFR. The condensed state of EGFR facilitates autoinhibition release in SOS and exerts regulatory control over signal propagation from activated EGFR to Ras. While kinase activity of EGFR is an essential driver of this signaling process, phosphorylation at residue Y160 on Grb2 blocks Grb2:Grb2 binding and can interfere with EGFR condensation. Here, using a reconstituted system, we examine how titrating kinase activity in the EGFR system can both promote and inhibit signal output to Ras. The results reveal how effects of tyrosine kinase inhibition can, under some circumstances, promote Ras activation by inhibiting negative feedback through Grb2 phosphorylation and disruption of a Grb2 SH2/SH3 dimer interface.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"15 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178254","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 : 2024-09-07DOI: 10.1101/2024.09.06.611667
Shubhadeep Sadhukhan, Cristina Martinez-Torres, Samo Penic, Carsten Beta, Ales Iglic, Nir S. gov
Cell motility is fundamental to many biological processes, and cells exhibit a variety of migration patterns. Many motile cell types follow a universal law that connects their speed and persistency, a property that can originate from the intracellular transport of polarity cues due to the global actin retrograde flow. This mechanism was termed the ``Universal Coupling between cell Speed and Persistency"(UCSP). Here we implemented a simplified version of the UCSP mechanism in a coarse-grained ``minimal-cell" model, which is composed of a three-dimensional vesicle that contains curved active proteins. This model spontaneously forms a lamellipodia-like motile cell shape, which is however sensitive and can depolarize into a non-motile form due to random fluctuations or when interacting with external obstacles. The UCSP implementation introduces long-range inhibition, which stabilizes the motile phenotype. This allows our model to describe the robust polarity observed in cells and explain a large variety of cellular dynamics, such as the relation between cell speed and aspect ratio, cell-barrier scattering, and cellular oscillations in different types of geometric confinements.
{"title":"Modelling how lamellipodia-driven cells maintain persistent migration and interact with external barriers","authors":"Shubhadeep Sadhukhan, Cristina Martinez-Torres, Samo Penic, Carsten Beta, Ales Iglic, Nir S. gov","doi":"10.1101/2024.09.06.611667","DOIUrl":"https://doi.org/10.1101/2024.09.06.611667","url":null,"abstract":"Cell motility is fundamental to many biological processes, and cells exhibit a variety of migration patterns. Many motile cell types follow a universal law that connects their speed and persistency, a property that can originate from the intracellular transport of polarity cues due to the global actin retrograde flow. This mechanism was termed the ``Universal Coupling between cell Speed and Persistency\"(UCSP). Here we implemented a simplified version of the UCSP mechanism in a coarse-grained ``minimal-cell\" model, which is composed of a three-dimensional vesicle that contains curved active proteins. This model spontaneously forms a lamellipodia-like motile cell shape, which is however sensitive and can depolarize into a non-motile form due to random fluctuations or when interacting with external obstacles. The UCSP implementation introduces long-range inhibition, which stabilizes the motile phenotype. This allows our model to describe the robust polarity observed in cells and explain a large variety of cellular dynamics, such as the relation between cell speed and aspect ratio, cell-barrier scattering, and cellular oscillations in different types of geometric confinements.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"38 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142178255","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 : 2024-08-11DOI: 10.1101/2024.08.11.607499
Sebastian T Coupe, Nikta Fakhri
Biomolecular condensates represent a frontier in cellular organization, existing as dynamic materials driven out of equilibrium by active cellular processes. Here we explore active mechanisms of condensate regulation by examining the interplay between DEAD-box helicase activity and RNA base-pairing interactions within ribonucleoprotein condensates. We demonstrate how the ATP-dependent activity of DEAD-box helicases—a key class of enzymes in condensate regulation—acts as a nonequilibrium driver of condensate properties through the continuous remodeling of RNA interactions. By combining the LAF-1 DEAD-box helicase with a designer RNA hairpin concatemer, we unveil a complex landscape of dynamic behaviors, including time-dependent alterations in RNA partitioning, evolving condensate morphologies, and shifting condensate dynamics. Importantly, we reveal an antagonistic relationship between RNA secondary structure and helicase activity which promotes condensate homogeneity via a nonequilibrium steady state. By elucidating these nonequilibrium mechanisms, we gain a deeper understanding of cellular organization and expand the potential for active synthetic condensate systems.
{"title":"Nonequilibrium phases of a biomolecular condensate facilitated by enzyme activity","authors":"Sebastian T Coupe, Nikta Fakhri","doi":"10.1101/2024.08.11.607499","DOIUrl":"https://doi.org/10.1101/2024.08.11.607499","url":null,"abstract":"Biomolecular condensates represent a frontier in cellular organization, existing as dynamic materials driven out of equilibrium by active cellular processes. Here we explore active mechanisms of condensate regulation by examining the interplay between DEAD-box helicase activity and RNA base-pairing interactions within ribonucleoprotein condensates. We demonstrate how the ATP-dependent activity of DEAD-box helicases—a key class of enzymes in condensate regulation—acts as a nonequilibrium driver of condensate properties through the continuous remodeling of RNA interactions. By combining the LAF-1 DEAD-box helicase with a designer RNA hairpin concatemer, we unveil a complex landscape of dynamic behaviors, including time-dependent alterations in RNA partitioning, evolving condensate morphologies, and shifting condensate dynamics. Importantly, we reveal an antagonistic relationship between RNA secondary structure and helicase activity which promotes condensate homogeneity via a nonequilibrium steady state. By elucidating these nonequilibrium mechanisms, we gain a deeper understanding of cellular organization and expand the potential for active synthetic condensate systems.","PeriodicalId":501048,"journal":{"name":"bioRxiv - Biophysics","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141945053","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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