Nature Protocols最新文献_第2页

Author Correction: Sequential orthogonal assays for longitudinal and endpoint characterization of three-dimensional spheroids. 作者更正：三维球体纵向和终点特征的顺序正交试验。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-06-09 DOI: 10.1038/s41596-025-01223-y

Eva Blondeel, Sam Ernst, Felix De Vuyst, Ákos Diósdi, Cláudio Pinheiro, Diogo Estêvão, Pekka Rappu, Robin Boiy, Sándor Dedeyne, Ligia Craciun, Vera Goossens, Jonas Dehairs, Tânia Cruz, Dominique Audenaert, Wim Ceelen, Michael Linnebacher, Tom Boterberg, Jo Vandesompele, Pieter Mestdagh, Johan Swinnen, Jyrki Heino, Peter Horvath, Maria José Oliveira, An Hendrix, Pieter Demetter, Olivier De Wever

引用次数: 0

Single-molecule parallel analysis for rapid exploration of sequence space. 用于序列空间快速探索的单分子平行分析。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-06-04 DOI: 10.1038/s41596-025-01196-y

Carolien Bastiaanssen, Ivo Severins, John van Noort, Chirlmin Joo

{"title":"Single-molecule parallel analysis for rapid exploration of sequence space.","authors":"Carolien Bastiaanssen, Ivo Severins, John van Noort, Chirlmin Joo","doi":"10.1038/s41596-025-01196-y","DOIUrl":"https://doi.org/10.1038/s41596-025-01196-y","url":null,"abstract":"Single-molecule fluorescence techniques have been successfully applied to uncover the structure, dynamics and interactions of DNA, RNA and proteins at the molecular scale. While the structure and function of these biomolecules are imposed by their sequences, single-molecule studies have been limited to a small number of sequences due to constraints in time and cost. To gain a comprehensive understanding on how sequence influences these essential biomolecules and the processes in which they act, a vast number of sequences have to be probed, requiring a high-throughput parallel approach. To address this need, we developed SPARXS: single-molecule parallel analysis for rapid exploration of sequence space. This platform enables simultaneous profiling of millions of molecules, covering thousands of distinct sequences, at the single-molecule level by coupling single-molecule fluorescence microscopy with next-generation high-throughput sequencing. Here we describe how to implement SPARXS and give examples from our study into the effect of sequence on Holliday junction kinetics. We provide a detailed description of sample and library design, single-molecule measurement, sequencing, coupling of sequencing and single-molecule fluorescence data, and data analysis. The protocol requires experience with single-molecule fluorescence microscopy and a basic command of Python to use our Papylio package for SPARXS data analysis. Familiarity with the underlying principles of Illumina sequencing is also beneficial. The entire process takes ~1-2 weeks and provides a detailed quantitative picture of the effect of sequence on the studied process.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-06-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144226012","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Multiplexed single-molecule characterization at the library scale. 在文库规模上的多路单分子表征。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-06-04 DOI: 10.1038/s41596-025-01198-w

M Panfilov, G Mao, J Guo, J Aguirre Rivera, A Sabantsev, S Deindl

{"title":"Multiplexed single-molecule characterization at the library scale.","authors":"M Panfilov, G Mao, J Guo, J Aguirre Rivera, A Sabantsev, S Deindl","doi":"10.1038/s41596-025-01198-w","DOIUrl":"https://doi.org/10.1038/s41596-025-01198-w","url":null,"abstract":"Single-molecule techniques are exceptionally well suited for analyzing the complex dynamic behavior of macromolecules involved in fundamental biological processes. Nevertheless, time and cost usually restrict current single-molecule methods to examining a limited number of different samples. At the same time, a broad sequence or chemical space often needs to be investigated to gain a thorough understanding of complex biological phenomena. To address this urgent need, we have developed multiplexed single-molecule characterization at the library scale (MUSCLE), a method that combines single-molecule fluorescence microscopy with next-generation sequencing to enable highly multiplexed observations of complex dynamics on millions of individual molecules spanning thousands of distinct sequences or barcoded entities. In this protocol, we outline the implementation of MUSCLE and present examples from our recent research, such as the sequence-dependent dynamics of Cas9-induced target DNA unwinding and rewinding. This example demonstrates that MUSCLE can be applied to study protein-nucleic acid interactions, going beyond nucleic-acid-only model systems. We detail the sample and library design, high-throughput single-molecule data acquisition, next-generation sequencing, spatial registration of single-molecule fluorescence and sequencing data and downstream data analysis. The ligation-based surface immobilization approach of MUSCLE ensures high clustering efficiency (>40%), increasing throughput and simplifying registration. In addition, MUSCLE includes a 3D-printed flow cell adapter that enables liquid exchange during single-molecule fluorescence microscopy. The complete procedure typically spans 3-4 days and yields a dataset that comprehensively characterizes the dynamic behavior of a library of constructs.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-06-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144226011","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Highly stable planar asymmetric suspended membranes for investigating protein dynamics and membrane fusion. 用于研究蛋白质动力学和膜融合的高度稳定的平面不对称悬浮膜。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-06-03 DOI: 10.1038/s41596-025-01192-2

Manindra Bera, Ramalingam Venkat Kalyana Sundaram, Jeff Coleman, Atrouli Chatterjee, Sikha Thoduvayil, Frederic Pincet, Sathish Ramakrishnan

{"title":"Highly stable planar asymmetric suspended membranes for investigating protein dynamics and membrane fusion.","authors":"Manindra Bera, Ramalingam Venkat Kalyana Sundaram, Jeff Coleman, Atrouli Chatterjee, Sikha Thoduvayil, Frederic Pincet, Sathish Ramakrishnan","doi":"10.1038/s41596-025-01192-2","DOIUrl":"https://doi.org/10.1038/s41596-025-01192-2","url":null,"abstract":"Membrane fusion is central to cellular signaling and trafficking, requiring a detailed understanding of protein-lipid interactions. Studying these dynamic events in live cells presents challenges due to their complexity and heterogeneity. To address this, we developed a reductionist in vitro membrane model system that enables the controlled investigation of individual molecular components. This approach begins with a minimal membrane environment, with the opportunity for the stepwise addition of specific components to incrementally increase complexity achieving a level of experimental precision often unattainable in cellular studies. We developed suspended lipid membranes, a platform that uses pore-spanning lipid bilayers formed on microfabricated silicon chips with micrometer-sized holes. These membranes closely mimic native cellular architecture by maintaining aqueous compartments on both sides, providing a solvent-free, near-native environment with exceptional lateral diffusion properties. Their high stability makes them ideal for time-lapse imaging and dynamic process analysis using total internal reflection fluorescence and confocal microscopy. Here we present a detailed protocol for generating pore-spanning, planar suspended lipid membranes from native and synthetic reconstituted lipids using our silicon chip platform. Using SNARE proteins and molecular chaperones, we demonstrate the system's ability to capture ultrafast membrane fusion events. Additionally, we demonstrate single-molecule protein counting, protein dynamics analysis and single-vesicle fusion assays using fluorescently labeled proteins and vesicles. The ability to preserve native lipid asymmetry, biological composition and lateral diffusion makes this method a powerful tool for dissecting membrane fusion mechanisms and other membrane biological processes with unparalleled precision.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-06-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144216392","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Biomimetic membrane in a microfluidic chip for the electrical and optical monitoring of biological reactions. 用于生物反应电学和光学监测的微流控芯片中的仿生膜。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-06-03 DOI: 10.1038/s41596-025-01171-7

Delphine Mion, Louis Bunel, Sathish Ramakrishnan, Paul Heo, Frédéric Pincet

{"title":"Biomimetic membrane in a microfluidic chip for the electrical and optical monitoring of biological reactions.","authors":"Delphine Mion, Louis Bunel, Sathish Ramakrishnan, Paul Heo, Frédéric Pincet","doi":"10.1038/s41596-025-01171-7","DOIUrl":"https://doi.org/10.1038/s41596-025-01171-7","url":null,"abstract":"Biological membranes separate distinct inner and outer compartments through the organization of fluid lipids into two-dimensional bilayers. The specific lipid composition varies across different membrane types. Model membranes play a crucial role in replicating certain features of biological membranes. They provide invaluable insights to decipher reactions at biological membranes in physicochemical cues. In this Protocol, we present a comprehensive procedure for creating a biomimetic membrane that encompasses key characteristics of biological membranes. Each leaflet of this horizontal and large (~10,000 µm2) membrane is obtained from a separate set of liposomes, allowing control of the lipid distribution between the two bilayer leaflets. Suspended in a vertical conduit separating two controllable horizontal microfluidic channels, this membrane can be used for the reconstitution of chemical or molecular reactions in close proximity to the membrane on the desired leaflet. The microfluidic chip containing the two channels separated by the vertical conduit is made of poly(dimethylsiloxane) and is fabricated from resin molds. Initially, oil is trapped in the conduit. Liposome solutions are pushed in each channel and spread on the trapped oil-buffer interface, forming a separate leaflet facing each channel. As oil is absorbed by poly(dimethylsiloxane), the two leaflets assemble and form a bilayer. We outline four applications of this biomimetic membrane microfluidic setup, incorporating optical microscopy and/or electrical readouts (patch-clamp amplifiers): single-particle and global diffusion, membrane fusion and channel formation. The entire protocol, covering chip fabrication, membrane formation and various measurements, can be completed within 2-3 d.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-06-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144216391","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Generation and characterization of vestibular inner ear organoids from human pluripotent stem cells. 人多能干细胞制备前庭内耳类器官及其特性研究。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-06-02 DOI: 10.1038/s41596-025-01191-3

Wouter H van der Valk, Carl Nist-Lund, Jingyuan Zhang, Camila Perea, Jiahe Jin, Kelly Y Gim, Matthew R Steinhart, Jiyoon Lee, Karl R Koehler

{"title":"Generation and characterization of vestibular inner ear organoids from human pluripotent stem cells.","authors":"Wouter H van der Valk, Carl Nist-Lund, Jingyuan Zhang, Camila Perea, Jiahe Jin, Kelly Y Gim, Matthew R Steinhart, Jiyoon Lee, Karl R Koehler","doi":"10.1038/s41596-025-01191-3","DOIUrl":"https://doi.org/10.1038/s41596-025-01191-3","url":null,"abstract":"The inner ear has a pivotal role in auditory and vestibular perception. Despite the vast number of individuals worldwide affected by hearing loss and balance disorders, therapeutic options have been largely limited to technological aids. The recent advent of gene therapies for genetic hearing loss in human patients underscores the urgency of developing scalable platforms to investigate a broader spectrum of inner ear disorders. Although animal models are powerful for assessing auditory and vestibular dysfunction, in vitro human inner ear models have shown promise in disease modeling and as platforms for studying developmental biology. Several studies have demonstrated that stem cells can be guided to differentiate into otic progenitor cells by mimicking environmental cues present during normal fetal inner ear development. Here we present a step-by-step approach to creating inner ear organoids (IEOs), which is an extension of our previous method for skin organoid generation, with which it shares foundational methodology and reagents. We used these organoids to elucidate the subtle signaling cues that govern their developmental trajectories. Generating sensory hair cells takes about 40 d, and cultures can be maintained for up to 150 d to allow further development. Moreover, we outline methods for assessing late-stage organoids, including whole-mount imaging of cleared IEOs, vibratome sectioning of live and fixed IEOs and other endpoint analyses, to study inner ear biology. IEOs are ideal for investigating human inner ear development, studying the mechanisms of inner ear disorders and developing therapeutic strategies. This protocol requires proficiency in basic stem cell culture techniques.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-06-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144208930","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Integration of large genetic payloads using prime editing and site-specific integrases. 利用启动编辑和位点特异性整合酶整合大的遗传有效载荷。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-06-01 DOI: 10.1038/s41596-024-01094-9

Zongliang Gao, Rasmus O Bak

引用次数: 0

Visually guided in vivo single-cell electroporation for monitoring and manipulating mammalian hippocampal neurons. 视觉引导单细胞电穿孔监测和操纵哺乳动物海马神经元。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-06-01 Epub Date: 2025-02-10 DOI: 10.1038/s41596-024-01099-4

Kevin C Gonzalez, Asako Noguchi, George Zakka, Hyun Choong Yong, Satoshi Terada, Miklos Szoboszlay, Justin O'Hare, Adrian Negrean, Tristan Geiller, Franck Polleux, Attila Losonczy

{"title":"Visually guided in vivo single-cell electroporation for monitoring and manipulating mammalian hippocampal neurons.","authors":"Kevin C Gonzalez, Asako Noguchi, George Zakka, Hyun Choong Yong, Satoshi Terada, Miklos Szoboszlay, Justin O'Hare, Adrian Negrean, Tristan Geiller, Franck Polleux, Attila Losonczy","doi":"10.1038/s41596-024-01099-4","DOIUrl":"10.1038/s41596-024-01099-4","url":null,"abstract":"Sparse, single-cell labeling approaches enable high-resolution, high signal-to-noise recordings from subcellular compartments and intracellular organelles and allow precise manipulations of individual cells and local circuits while minimizing complex changes associated with global network manipulations. However, thus far, only a limited number of approaches have been developed to label single cells with unique combinations of genetically encoded indicators, target deep cortical structures or sustainably use the same chronic preparation for weeks. Here we developed a method to deliver plasmids selectively to single pyramidal neurons in the mouse dorsal hippocampus using two-photon visually guided in vivo single-cell electroporation to address these limitations. This method allows long-term plasmid expression in a controlled number of individual pyramidal neurons, facilitating subcellular resolution imaging, intracellular organelle tracking, monosynaptic input mapping, plasticity induction and targeted whole-cell patch-clamp recordings.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":"1468-1484"},"PeriodicalIF":13.1,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12151752/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143391369","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A multimodal imaging pipeline to decipher cell-specific metabolic functions and tissue microenvironment dynamics. 一个多模态成像管道破译细胞特异性代谢功能和组织微环境动力学。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-06-01 Epub Date: 2025-01-29 DOI: 10.1038/s41596-024-01118-4

Sharavan Vishaan Venkateswaran, Peter Kreuzaler, Catherine Maclachlan, Greg McMahon, Gina Greenidge, Lucy Collinson, Josephine Bunch, Mariia Yuneva

{"title":"A multimodal imaging pipeline to decipher cell-specific metabolic functions and tissue microenvironment dynamics.","authors":"Sharavan Vishaan Venkateswaran, Peter Kreuzaler, Catherine Maclachlan, Greg McMahon, Gina Greenidge, Lucy Collinson, Josephine Bunch, Mariia Yuneva","doi":"10.1038/s41596-024-01118-4","DOIUrl":"10.1038/s41596-024-01118-4","url":null,"abstract":"Tissue microenvironments are extremely complex and heterogeneous. It is challenging to study metabolic interaction between the different cell types in a tissue with the techniques that are currently available. Here we describe a multimodal imaging pipeline that allows cell type identification and nanoscale tracing of stable isotope-labeled compounds. This pipeline extends upon the principles of correlative light, electron and ion microscopy, by combining confocal microscopy reporter or probe-based fluorescence, electron microscopy, stable isotope labeling and nanoscale secondary ion mass spectrometry. We apply this method to murine models of hepatocellular and mammary gland carcinomas to study uptake of glucose derived carbon (13C) and glutamine derived nitrogen (15N) by tumor-associated immune cells. In vivo labeling with fluorescent-tagged antibodies (B220, CD3, CD8a, CD68) in tandem with confocal microscopy allows for the identification of specific cell types (B cells, T cells and macrophages) in the tumor microenvironment. Subsequent image correlation with electron microscopy offers the contrast and resolution to image membranes and organelles. Nanoscale secondary ion mass spectrometry tracks the enrichment of stable isotopes within these intracellular compartments. The whole protocol described here would take ~6 weeks to perform from start to finish. Our pipeline caters to a broad spectrum of applications as it can easily be adapted to trace the uptake and utilization of any stable isotope-labeled nutrient, drug or a probe by defined cellular populations in any tissue in situ.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":"1678-1699"},"PeriodicalIF":13.1,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7617660/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143066052","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

An accessible workflow for high-sensitivity proteomics using parallel accumulation-serial fragmentation (PASEF). 使用平行积累-序列片段（PASEF）的高灵敏度蛋白质组学可访问的工作流程。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-06-01 Epub Date: 2025-01-17 DOI: 10.1038/s41596-024-01104-w

Patricia Skowronek, Georg Wallmann, Maria Wahle, Sander Willems, Matthias Mann

{"title":"An accessible workflow for high-sensitivity proteomics using parallel accumulation-serial fragmentation (PASEF).","authors":"Patricia Skowronek, Georg Wallmann, Maria Wahle, Sander Willems, Matthias Mann","doi":"10.1038/s41596-024-01104-w","DOIUrl":"10.1038/s41596-024-01104-w","url":null,"abstract":"Deep and accurate proteome analysis is crucial for understanding cellular processes and disease mechanisms; however, it is challenging to implement in routine settings. In this protocol, we combine a robust chromatographic platform with a high-performance mass spectrometric setup to enable routine yet in-depth proteome coverage for a broad community. This entails tip-based sample preparation and pre-formed gradients (Evosep One) combined with a trapped ion mobility time-of-flight mass spectrometer (timsTOF, Bruker). The timsTOF enables parallel accumulation-serial fragmentation (PASEF), in which ions are accumulated and separated by their ion mobility, maximizing ion usage and simplifying spectra. Combined with data-independent acquisition (DIA), it offers high peak sampling rates and near-complete ion coverage. Here, we explain how to balance quantitative accuracy, specificity, proteome coverage and sensitivity by choosing the best PASEF and DIA method parameters. The protocol describes how to set up the liquid chromatography-mass spectrometry system and enables PASEF method generation and evaluation for varied samples by using the py_diAID tool to optimally position isolation windows in the mass-to-charge and ion mobility space. Biological projects (e.g., triplicate proteome analysis in two conditions) can be performed in 3 d with ~3 h of hands-on time and minimal marginal cost. This results in reproducible quantification of 7,000 proteins in a human cancer cell line in quadruplicate 21-min injections and 29,000 phosphosites for phospho-enriched quadruplicates. Synchro-PASEF, a highly efficient, specific and novel scan mode, can be analyzed by Spectronaut or AlphaDIA, resulting in superior quantitative reproducibility because of its high sampling efficiency.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":"1700-1729"},"PeriodicalIF":13.1,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008630","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0