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Current Protocols in Cell Biology最新文献

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In vitro Assays for Targeting and Insertion of Tail-Anchored Proteins Into the ER Membrane 尾锚定蛋白在内质网膜中的靶向和插入的体外实验
Current Protocols in Cell Biology Pub Date : 2018-09-25 DOI: 10.1002/cpcb.63
Hyunju Cho, Un Seng Chio, Shu-ou Shan
{"title":"In vitro Assays for Targeting and Insertion of Tail-Anchored Proteins Into the ER Membrane","authors":"Hyunju Cho,&nbsp;Un Seng Chio,&nbsp;Shu-ou Shan","doi":"10.1002/cpcb.63","DOIUrl":"10.1002/cpcb.63","url":null,"abstract":"<p>Membrane proteins mediate numerous essential cellular functions. Due to the aggregation propensity of hydrophobic transmembrane domains in aqueous environments, the targeting and insertion of membrane proteins pose major challenges to cells. In the Guided Entry of Tail-anchored protein (GET) pathway, an essential class of newly synthesized tail-anchored proteins (TAs) are chaperoned and guided by multiple targeting factors to the endoplasmic reticulum (ER) membrane. Deciphering the molecular mechanism of this cellular process has benefitted from successful <i>in vitro</i> reconstitution of individual molecular events in the GET pathway with purified components. Here we describe recently developed protocols for <i>in vitro</i> reconstitution of functional complexes of TA substrates with their targeting factors, for monitoring the transfer of TAs between targeting factors, and for the insertion of TA into the microsomal membrane. These procedures are generally applicable to the interrogation of other post-translational membrane protein targeting pathways. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"81 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.63","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36521411","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}
引用次数: 3
Monitoring Sphingolipid Trafficking in Cells using Fluorescence Microscopy 利用荧光显微镜监测细胞内鞘脂运输
Current Protocols in Cell Biology Pub Date : 2018-09-24 DOI: 10.1002/cpcb.67
Emma L. Sundberg, Yongqiang Deng, Christopher G. Burd
{"title":"Monitoring Sphingolipid Trafficking in Cells using Fluorescence Microscopy","authors":"Emma L. Sundberg,&nbsp;Yongqiang Deng,&nbsp;Christopher G. Burd","doi":"10.1002/cpcb.67","DOIUrl":"10.1002/cpcb.67","url":null,"abstract":"<p>Sphingolipids are structural components of organelle membranes that also participate in signal transduction pathways. Complex sphingolipids are trafficked from their site of synthesis in organelles of the early secretory pathway to the Golgi apparatus, the plasma membrane, and the endo-lysosomal system. We have developed fluorescence microscopy–based methods to monitor sphingolipid trafficking in coordination with secretory protein sorting. A sphingomyelin binding protein fused to a fluorescent protein, which we term “EQ-SM,” is implemented to monitor sphingomyelin trafficking from the Golgi apparatus to the plasma membrane via secretory vesicles. A protocol is provided to determine if a query protein of interest is secreted from the cell via vesicles enriched in EQ-SM, an indication that the vesicle membrane is enriched in sphingomyelin. A complementary protocol is described that implements a chemically modified form of sphingosine, a metabolic precursor to complex sphingolipids, to visualize ceramide and complex sphingolipids in fixed cells. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"82 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.67","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36516455","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}
引用次数: 6
An On-Chip Method for Long-Term Growth and Real-Time Imaging of Brain Organoids 一种用于脑类器官长期生长和实时成像的芯片方法
Current Protocols in Cell Biology Pub Date : 2018-09-21 DOI: 10.1002/cpcb.62
Eyal Karzbrun, Rami Yair Tshuva, Orly Reiner
{"title":"An On-Chip Method for Long-Term Growth and Real-Time Imaging of Brain Organoids","authors":"Eyal Karzbrun,&nbsp;Rami Yair Tshuva,&nbsp;Orly Reiner","doi":"10.1002/cpcb.62","DOIUrl":"10.1002/cpcb.62","url":null,"abstract":"<p>Brain organoids are an emerging technique for studying human neurodevelopment in vitro, with biomedical implications. However, three-dimensional tissue culture poses several challenges, including lack of nutrient exchange at the organoid core and limited imaging accessibility of whole organoids. Here we present a method for culturing organoids in a micro-fabricated device that enables in situ real-time imaging over weeks with efficient nutrient exchange by diffusion. Our on-chip approach offers a means for studying the dynamics of organoid development, cell differentiation, cell cycle, and motion. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"81 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-09-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.62","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36508098","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}
引用次数: 12
Genome Editing in Mice Using CRISPR/Cas9 Technology 利用CRISPR/Cas9技术编辑小鼠基因组
Current Protocols in Cell Biology Pub Date : 2018-09-04 DOI: 10.1002/cpcb.57
Bradford Hall, Andrew Cho, Advait Limaye, Kyoungin Cho, Jaspal Khillan, Ashok B. Kulkarni
{"title":"Genome Editing in Mice Using CRISPR/Cas9 Technology","authors":"Bradford Hall,&nbsp;Andrew Cho,&nbsp;Advait Limaye,&nbsp;Kyoungin Cho,&nbsp;Jaspal Khillan,&nbsp;Ashok B. Kulkarni","doi":"10.1002/cpcb.57","DOIUrl":"10.1002/cpcb.57","url":null,"abstract":"CRISPR/Cas9 technology has revolutionized genome editing in mice, allowing for simple and rapid development of knockouts and knockins. CRISPR relies on small guide RNAs that direct the RNA‐guided nuclease Cas9 to a designated genomic site using ∼20 bp of corresponding sequence. Cas9 then creates a double‐strand break in the targeted loci that is either patched in an error‐prone fashion to produce a frame‐shift mutation, a knockout, or is repaired by recombination with donor DNA containing homology arms, a knockin. This protocol covers the techniques needed to rapidly generate knockout and knockin mice with CRISPR via microinjection of Cas9, the guide RNA, and possible donor DNA into the mouse zygote. © 2018 by John Wiley & Sons, Inc.","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"81 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.57","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10752707","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}
引用次数: 17
Preparation and Culture of Human Liver Resident Immune Cells 人肝脏常驻免疫细胞的制备与培养
Current Protocols in Cell Biology Pub Date : 2018-08-22 DOI: 10.1002/cpcb.50
Hao Feng, Baochi Ou, Wei Dong, Wolfgang E. Thasler
{"title":"Preparation and Culture of Human Liver Resident Immune Cells","authors":"Hao Feng,&nbsp;Baochi Ou,&nbsp;Wei Dong,&nbsp;Wolfgang E. Thasler","doi":"10.1002/cpcb.50","DOIUrl":"10.1002/cpcb.50","url":null,"abstract":"<p>Co-cultivation of tumor cells and liver resident immune cells or other non-parenchymal cells (NPCs) from the same donor is important for the study of cancer metastasis. So far, little is known about the mechanism of tumor cell or pathogen clearance, leukocyte infiltration, and immune cell recruitment in the human liver. To investigate these processes <i>in vitro</i>, the use of primary human hepatocytes and non-parenchymal cell, especially immune cell, co-culture systems play essential roles in the establishment of cell–cell and cell–extracellular matrix communications similar to native liver tissues. Hepatic non-parenchymal cells mainly comprise liver sinusoid endothelial cells (LSECs), microvascular endothelial cells, hepatic stellate cells, Kupffer cells (KCs), natural killer T (iNKT) cells, and dendritic cells (DCs). Here we describe procedures for preparation, isolation, and culture of human liver resident immune cells and other non-parenchymal cells. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"80 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-08-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.50","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36417472","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}
引用次数: 0
Cell Adhesion Activity of Peptides Conjugated to Polysaccharides 多肽与多糖结合的细胞粘附活性
Current Protocols in Cell Biology Pub Date : 2018-08-20 DOI: 10.1002/cpcb.53
Kentaro Hozumi, Motoyoshi Nomizu
{"title":"Cell Adhesion Activity of Peptides Conjugated to Polysaccharides","authors":"Kentaro Hozumi,&nbsp;Motoyoshi Nomizu","doi":"10.1002/cpcb.53","DOIUrl":"10.1002/cpcb.53","url":null,"abstract":"<p>Many cell-adhesive peptides have been identified from extracellular matrix (ECM) proteins, such as collagen, fibronectin, laminin, and vitronectin. ECM proteins have various cell-adhesive sequences. Most peptides demonstrate cell-adhesive activity when simply coated on a tissue culture plate, but solubility, conformation, and coating efficiency of the peptides can significantly alter their biological function. Evaluation of peptide cell-adhesive activity using peptide-conjugated polysaccharide constructs is a useful strategy for overcoming peptide solubility and conformation problems. After a simple modification of the polysaccharides, various polysaccharides (chitosan, alginate, and hyaluronate) can fix the peptides on the tissue culture plate quantitatively. The peptide-polysaccharide strategy can be used to fix different active peptides to the polysaccharide at same time, thus, mimicking the biological functions of the ECM. This paper describes the modification of polysaccharides that are suitable for covalently coupling the peptides and evaluation of the cell-adhesive activity of peptide as a peptide-polysaccharide matrix. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"80 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.53","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36413111","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}
引用次数: 7
Triggered Cell-Cell Fusion Assay for Cytoplasmic and Organelle Intermixing Studies 触发细胞-细胞融合试验用于细胞质和细胞器混合研究
Current Protocols in Cell Biology Pub Date : 2018-08-13 DOI: 10.1002/cpcb.61
Daniel Feliciano, Jonathon Nixon-Abell, Jennifer Lippincott-Schwartz
{"title":"Triggered Cell-Cell Fusion Assay for Cytoplasmic and Organelle Intermixing Studies","authors":"Daniel Feliciano,&nbsp;Jonathon Nixon-Abell,&nbsp;Jennifer Lippincott-Schwartz","doi":"10.1002/cpcb.61","DOIUrl":"10.1002/cpcb.61","url":null,"abstract":"<p>Different multicellular organisms undergo cell-cell fusion to form functional syncytia that support specialized functions necessary for proper development and survival. For years, monitoring the structural consequences of this process using live-cell imaging has been challenging due to the unpredictable timing of cell fusion events in tissue systems. Here we present a triggered vesicular stomatitis virus G-protein (VSV-G)-mediated cell-cell fusion assay that can be used to synchronize fusion between cells. This allows the study of cellular changes that occur during cell fusion. The process is induced using a fast wash of low pH isotonic buffer, promoting the fusion of plasma membranes of two or more adjacent cells within seconds. This approach is suitable for studying mixing of small cytoplasmic molecules between fusing cells as well as changes in organelle distribution and dynamics. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"81 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.61","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36392415","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}
引用次数: 3
Imaging Membrane Repair in Single Cells Using Correlative Light and Electron Microscopy 单细胞膜修复的相关光镜和电镜成像
Current Protocols in Cell Biology Pub Date : 2018-08-07 DOI: 10.1002/cpcb.55
Coralie Croissant, Flora Bouvet, Sisareuth Tan, Anthony Bouter
{"title":"Imaging Membrane Repair in Single Cells Using Correlative Light and Electron Microscopy","authors":"Coralie Croissant,&nbsp;Flora Bouvet,&nbsp;Sisareuth Tan,&nbsp;Anthony Bouter","doi":"10.1002/cpcb.55","DOIUrl":"10.1002/cpcb.55","url":null,"abstract":"<p>Many cells possess the ability to repair plasma membrane disruption in physiological conditions. Growing evidence indicates a correlation between membrane repair and many human diseases. For example, a negative correlation is observed in muscle where failure to reseal sarcolemma may contribute to the development of muscular dystrophies. Instead, a positive correlation is observed in cancer cells where membrane repair may be exacerbated during metastasis. Here we describe a protocol that combines laser technology for membrane damage, immunostaining with gold nanoparticles and imaging by fluorescence microscopy and transmission electron microscopy (TEM), which allows the characterization of the molecular machinery involved in membrane repair. Fluorescence microscopy enables to determine the subcellular localization of candidate proteins in damaged cells while TEM offers high-resolution ultrastructural analysis of the µm²-disruption site, which enables to decipher the membrane repair mechanism. Here we focus on the study of human skeletal muscle cells, for obvious clinical interest, but this protocol is also suitable for other cell types. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"81 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.55","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36378219","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}
引用次数: 3
Quantifying the Proteolytic Cleavage of Plasma Membrane Proteins in Living Cells 活细胞中质膜蛋白水解裂解的定量研究
Current Protocols in Cell Biology Pub Date : 2018-08-07 DOI: 10.1002/cpcb.58
Martino Calamai, Francesco Saverio Pavone
{"title":"Quantifying the Proteolytic Cleavage of Plasma Membrane Proteins in Living Cells","authors":"Martino Calamai,&nbsp;Francesco Saverio Pavone","doi":"10.1002/cpcb.58","DOIUrl":"10.1002/cpcb.58","url":null,"abstract":"The standard approach to study the activity of proteases consists of lysing cells and measuring the changes in the fluorescence properties of a synthetic substrate after cleavage in vitro. Here, a general protocol that uses a bi‐fluorescent chimeric construct of a known substrate protein that follows the proteolytic processing in living cells is described. This approach is useful, in particular, to search for pharmacological conditions altering the cleavage rate of a certain protease, or to investigate the biological factors influencing a certain proteolytic mechanism. Three different methods (microscopy, flow cytometry, and spectroscopy) to detect fluorescence changes due to alteration in the processing are described. This approach was originally developed for studying conditions affecting the proteolytic activity of the β‐secretase Bace1 on the amyloid precursor protein APP, but can in principle be applied to investigate any membrane protein undergoing ectodomain shedding by proteolytic cleavage. © 2018 by John Wiley & Sons, Inc.","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"81 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.58","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36378617","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}
引用次数: 1
Expansion Microscopy: Protocols for Imaging Proteins and RNA in Cells and Tissues 扩增显微镜:细胞和组织中蛋白质和RNA成像的规程
Current Protocols in Cell Biology Pub Date : 2018-08-02 DOI: 10.1002/cpcb.56
Shoh M. Asano, Ruixuan Gao, Asmamaw T. Wassie, Paul W. Tillberg, Fei Chen, Edward S. Boyden
{"title":"Expansion Microscopy: Protocols for Imaging Proteins and RNA in Cells and Tissues","authors":"Shoh M. Asano,&nbsp;Ruixuan Gao,&nbsp;Asmamaw T. Wassie,&nbsp;Paul W. Tillberg,&nbsp;Fei Chen,&nbsp;Edward S. Boyden","doi":"10.1002/cpcb.56","DOIUrl":"10.1002/cpcb.56","url":null,"abstract":"<p>Expansion microscopy (ExM) is a recently developed technique that enables nanoscale-resolution imaging of preserved cells and tissues on conventional diffraction-limited microscopes via isotropic physical expansion of the specimens before imaging. In ExM, biomolecules and/or fluorescent labels in the specimen are linked to a dense, expandable polymer matrix synthesized evenly throughout the specimen, which undergoes 3-dimensional expansion by ∼4.5 fold linearly when immersed in water. Since our first report, versions of ExM optimized for visualization of proteins, RNA, and other biomolecules have emerged. Here we describe best-practice, step-by-step ExM protocols for performing analysis of proteins (protein retention ExM, or proExM) as well as RNAs (expansion fluorescence <i>in situ</i> hybridization, or ExFISH), using chemicals and hardware found in a typical biology lab. Furthermore, a detailed protocol for handling and mounting expanded samples and for imaging them with confocal and light-sheet microscopes is provided. © 2020 The Authors.</p>","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"80 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-08-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.56","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36365606","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 120
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