Current Protocols in Cell Biology最新文献_第2页

Production of Recombinant Transmembrane Proteins from Mammalian Cells for Biochemical and Structural Analyses 从哺乳动物细胞中生产重组跨膜蛋白用于生化和结构分析

Current Protocols in Cell Biology Pub Date : 2020-06-09 DOI: 10.1002/cpcb.106

Robbins Puthenveetil, Chul-Jin Lee, Anirban Banerjee

{"title":"Production of Recombinant Transmembrane Proteins from Mammalian Cells for Biochemical and Structural Analyses","authors":"Robbins Puthenveetil, Chul-Jin Lee, Anirban Banerjee","doi":"10.1002/cpcb.106","DOIUrl":"10.1002/cpcb.106","url":null,"abstract":"Eukaryotic integral membrane proteins are key components of various biological processes. Because they are implicated in multiple diseases, it is important to understand their mechanism of action by elucidating their structure and function. Complex technical challenges associated with the generation of recombinant membrane proteins severely impair our ability to understand them using structural and biochemical methods. Here, we provide a detailed procedure to address and mitigate difficulties involved in the large-scale heterologous overexpression and purification of eukaryotic membrane proteins using HEK293S GnTi− cells transduced with baculovirus. Two human proteins, hDHHC15 and hPORCN, are presented as examples, with step-by-step instructions for transient transfection and generation of baculoviruses, followed by overexpression and purification from HEK293S GnTi− cells. © 2020 Wiley Periodicals LLC.Basic Protocol 1: Small-scale protein expression in mammalian HEK293T cellsBasic Protocol 2: Generation of baculovirus from Sf9 (insect) cellsAlternate Protocol: Enumeration-free method for generating P2 viral stockSupport Protocol 1: Small-scale transduction of HEK293T cells with P2 baculovirusBasic Protocol 3: Large-scale viral transduction of HEK293S GnTi− cellsSupport Protocol 2: Large-scale membrane preparation from HEK293S GnTi− cellsBasic Protocol 4: Large-scale purification of membrane proteins from HEK293S GnTi− cells","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"87 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-06-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.106","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38024956","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

Detection of LC3-Associated Phagocytosis (LAP) lc3相关吞噬作用(LAP)检测

Current Protocols in Cell Biology Pub Date : 2020-05-21 DOI: 10.1002/cpcb.104

Jennifer Martinez

{"title":"Detection of LC3-Associated Phagocytosis (LAP)","authors":"Jennifer Martinez","doi":"10.1002/cpcb.104","DOIUrl":"10.1002/cpcb.104","url":null,"abstract":"Phagocytes, notably macrophages, are critical sentinels of their environment, patrolling for and eradicating unwanted components. The ability of cells to process extracellular cargo in an appropriate manner is important for both clearance of the cargo and eventual return to homeostasis. Although the evolutionarily conserved pathway of autophagy involves the degradation and recycling of unnecessary or dysfunctional cellular components during starvation, we now appreciate that the reach of autophagy extends beyond nutrient deprivation, notably including cellular quality control (e.g., mitophagy) and host defense against internalized pathogens (i.e., xenophagy). Despite being seemingly disparate, autophagic functions are unified as conserved mechanisms for containment and immunosuppression, suggesting an original immune function for autophagy. A recently described pathway called LC3-associated phagocytosis (LAP) marries the ancient concepts of phagocytosis and autophagy, revealing new ways in which the autophagy machinery, in a molecularly distinct pathway, contributes to the inflammatory response. In this article, protocols to detect LAP by electron microscopy, immunofluorescence, flow cytometry, and phagosome purification are described, allowing the user to detect multiple characteristics of LAP in both qualitative and quantitative manners. Published 2020. U.S. Government.Basic Protocol 1: Detection of LAP by electron microscopyBasic Protocol 2: Detection of LAP by confocal microscopy of LC3-GFP-expressing cellsAlternate Protocol 1: Detection of LAP by confocal microscopy using immunofluorescenceBasic Protocol 3: Detection of LAP using flow cytometry of LC3-GFP-expressing cellsAlternate Protocol 2: Detection of LAP using antibody staining and flow cytometryBasic Protocol 4: Detection of LAP by western blot of purified LAPosomes","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"87 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-05-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.104","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37961833","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: 11

Generation of 3D Tumor Spheroids with Encapsulating Basement Membranes for Invasion Studies 包封基底膜三维肿瘤球体的生成及其侵袭研究

Current Protocols in Cell Biology Pub Date : 2020-05-21 DOI: 10.1002/cpcb.105

Shayan S. Nazari

{"title":"Generation of 3D Tumor Spheroids with Encapsulating Basement Membranes for Invasion Studies","authors":"Shayan S. Nazari","doi":"10.1002/cpcb.105","DOIUrl":"10.1002/cpcb.105","url":null,"abstract":"In the past, in vitro studies of invasion and tumor progression were performed primarily using cancer cells cultured on a flat, two-dimensional (2D) surface in a monolayer. In recent years, however, many studies have demonstrated differences in cell signaling and cell migration between 2D and 3D cell cultures. Traditional 2D monolayer cancer cell invasion models do not fully recapitulate 3D cell-to-cell and cell−to−extracellular matrix interactions that in vivo models can provide. Moreover, although in vivo animal models are irreplaceable for studying tumor biology and metastasis, they are costly, time-consuming, and impractical for answering preliminary questions. Thus, emergent and evolving 3D spheroid cell culture models have changed the way we study tumors and their interactions with their surrounding extracellular matrix. In the case of breast cancer, metastasis of breast cancer tumors results in high mortality rates, and thus development of robust cell culture models that are reproducible and practical for studying breast cancer progression is important for ultimately developing preventatives for cancer metastasis. This article provides a set of protocols for generating uniform spheroids with a thin sheet of basement membrane for studying the initial invasion of mammary epithelial cells into a surrounding collagen-rich extracellular matrix. Details are provided for generating 3D spheroids with a basement membrane, polymerizing collagen I, embedding the spheroids in the 3D collagen gel, and immunostaining the spheroids for invasion studies. Published 2020. U.S. Government.Basic Protocol 1: Growth of uniformly sized tumor spheroids with an encapsulating basement membraneBasic Protocol 2: Polymerization and embedding of tumor spheroids in a 3D type I collagen gelAlternate Protocol: Embedding of tumor spheroids in collagen gels using a sandwich methodBasic Protocol 3: Fixing and immunostaining of tumor spheroids embedded in 3D collagen gels","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"87 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-05-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.105","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37959072","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: 14

Optogenetic Control of RhoA to Probe Subcellular Mechanochemical Circuitry 光遗传学控制RhoA探测亚细胞机械化学回路

Current Protocols in Cell Biology Pub Date : 2020-02-07 DOI: 10.1002/cpcb.102

Kate E. Cavanaugh, Patrick W. Oakes, Margaret L. Gardel

{"title":"Optogenetic Control of RhoA to Probe Subcellular Mechanochemical Circuitry","authors":"Kate E. Cavanaugh, Patrick W. Oakes, Margaret L. Gardel","doi":"10.1002/cpcb.102","DOIUrl":"10.1002/cpcb.102","url":null,"abstract":"Spatiotemporal localization of protein function is essential for physiological processes from subcellular to tissue scales. Genetic and pharmacological approaches have played instrumental roles in isolating molecular components necessary for subcellular machinery. However, these approaches have limited capabilities to reveal the nature of the spatiotemporal regulation of subcellular machineries like those of cytoskeletal organelles. With the recent advancement of optogenetic probes, the field now has a powerful tool to localize cytoskeletal stimuli in both space and time. Here, we detail the use of tunable light-controlled interacting protein tags (TULIPs) to manipulate RhoA signaling in vivo. This is an optogenetic dimerization system that rapidly, reversibly, and efficiently directs a cytoplasmic RhoGEF to the plasma membrane for activation of RhoA using light. We first compare this probe to other available optogenetic systems and outline the engineering logic for the chosen recruitable RhoGEFs. We also describe how to generate the cell line, spatially control illumination, confirm optogenetic control of RhoA, and mechanically induce cell-cell junction deformation in cultured tissues. Together, these protocols detail how to probe the mechanochemical circuitry downstream of RhoA signaling. © 2020 by John Wiley & Sons, Inc.Basic Protocol 1: Generation of a stable cell line expressing TULIP constructsBasic Protocol 2: Preparation of collagen substrate for imagingBasic Protocol 3: Transient transfection for visualization of downstream effectorsBasic Protocol 4: Calibration of spatial illuminationBasic Protocol 5: Optogenetic activation of a region of interest","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"86 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.102","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37620788","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: 8

Isolation of Highly Migratory and Invasive Cells in Three-Dimensional Gels 三维凝胶中高迁移性和侵袭性细胞的分离

Current Protocols in Cell Biology Pub Date : 2020-02-05 DOI: 10.1002/cpcb.103

Takahisa Takino, Takeshi Suzuki, Motoharu Seiki

{"title":"Isolation of Highly Migratory and Invasive Cells in Three-Dimensional Gels","authors":"Takahisa Takino, Takeshi Suzuki, Motoharu Seiki","doi":"10.1002/cpcb.103","DOIUrl":"10.1002/cpcb.103","url":null,"abstract":"We developed a modified invasion assay in three-dimensional (3D) gels that permits isolation of invading cells as living cells, termed an invading cell–trapping (iCT) assay. A small cell strainer consisting of nylon mesh with pores of 40-µm square is used in this assay. A layer of gel composed of extracellular-matrix components is formed on each side of the nylon mesh, which permits cell migration or invasion from one gel layer to the other. At the end of the assay, the two gel layers are removed from the apparatus and easily separated from each other. Invading cells from the primary gel are trapped in the secondary gel, which maintains the morphology and other properties of the invasive cells in a 3D matrix. The cells that have invaded are observed and counted with a standard light microscope without cell staining. There is no need for a specialized microscope, imaging analysis software, or advanced cell-biological technical knowledge in this assay. This assay can also reduce measurement of nonspecific cell invasion by monitoring the upward invasion of cells. The viability of both invading and non-invading cells trapped in the gels can be assessed by typical colorimetric assays, if desired. This assessment characterizes the total number of cells (invading and non-invading cells) and the ratio of invading cells to total cells. By repeating the iCT assay, further enrichment of invasive and noninvasive cells can be attained. Thus, this assay improves comparative analyses between invasive and noninvasive cells. © 2020 by John Wiley & Sons, Inc.Basic Protocol 1: Measuring upward cell invasion into collagen gelBasic Protocol 2: Measuring cell invasion from Matrigel into collagen gelBasic Protocol 3: Isolation and enrichment of highly invasive cells","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"86 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.103","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37613048","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

Machine Learning for Analysis of Microscopy Images: A Practical Guide 机器学习显微镜图像分析:实用指南

Current Protocols in Cell Biology Pub Date : 2020-01-06 DOI: 10.1002/cpcb.101

Vadim Zinchuk, Olga Grossenbacher-Zinchuk

引用次数: 17

High-Content Screening for Protein-Protein Interaction Modulators Using Proximity Ligation Assay in Primary Neurons 利用邻近结扎法在初级神经元中筛选高含量的蛋白质相互作用调节剂

Current Protocols in Cell Biology Pub Date : 2019-12-26 DOI: 10.1002/cpcb.100

Tiago Mendes, Adrien Herledan, Florence Leroux, Benoit Deprez, Jean-Charles Lambert, Devrim Kilinc

{"title":"High-Content Screening for Protein-Protein Interaction Modulators Using Proximity Ligation Assay in Primary Neurons","authors":"Tiago Mendes, Adrien Herledan, Florence Leroux, Benoit Deprez, Jean-Charles Lambert, Devrim Kilinc","doi":"10.1002/cpcb.100","DOIUrl":"10.1002/cpcb.100","url":null,"abstract":"The proximity ligation assay (PLA) allows the detection and subcellular localization of protein-protein interactions with high specificity. We recently developed a high-content screening model based on primary hippocampal neurons cultured in 384-well plates and screened a library of ∼1100 compounds using a PLA between tau and bridging integrator 1, a genetic risk factor for Alzheimer's disease. We developed image-segmentation and spot-detection algorithms to delineate PLA signals in the axonal network, but not in cell bodies, from confocal images acquired via a high-throughput microscope. To compare data generated from different plates and through different experiments, we developed a computational routine to optimize the image analysis parameters for each plate and devised a range of quality-control measures to ultimately identify compounds that consistently increase or decrease our read-out. We provide the following protocols. © 2019 by John Wiley & Sons, Inc.Basic Protocol 1: Routine culture of rat postnatal hippocampal neurons in 384-well platesBasic Protocol 2: Compound incubation using the high-content screening platformSupport Protocol 1: Preparation of intermediate plates for compound screeningSupport Protocol 2: Preparation of intermediate plates for hit validation (dose-response curves)Basic Protocol 3: Proximity ligation assay in 384-well platesBasic Protocol 4: Image acquisition and analysisSupport Protocol 3: Optimization of analysis parametersBasic Protocol 5: Identification of hitsBasic Protocol 6: Validation of hits based on dose-response curves","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"86 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-12-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.100","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37490491","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: 2

A Sensitive and Quantitative mKeima Assay for Mitophagy via FACS FACS法测定线粒体自噬的灵敏定量mKeima试验

Current Protocols in Cell Biology Pub Date : 2019-12-26 DOI: 10.1002/cpcb.99

Chunxin Wang

{"title":"A Sensitive and Quantitative mKeima Assay for Mitophagy via FACS","authors":"Chunxin Wang","doi":"10.1002/cpcb.99","DOIUrl":"10.1002/cpcb.99","url":null,"abstract":"Mitophagy is a selective autophagy process that specifically removes damaged mitochondria via general autophagy. The two major recessive Parkinson's disease genes PINK1 and Parkin play essential roles in mitophagy initiation, increasing the interest in mitophagy in both basic and translational research over the past 10 years. Initially, mitophagy was measured by the loss of mitochondria either through confocal imaging or immunoblot of mitochondrial proteins such as Tom20 or COXII. Confocal imaging of mitochondria DNA loss via anti-DNA staining is another option. All of these methods, which take considerable effort and labor, are not sensitive enough to detect early stages of mitophagy with unambiguous objectivity. The mKeima assay can be used for both confocal imaging and FACS analysis to provide a thorough picture of mitophagy with a wide dynamic range. The Keima fluorophore has bimodal excitation under neutral and acidic pH conditions. Thus, when Keima is targeted to mitochondria it can accurately reveal the formation of mitolysosomes. Here the author briefly describes the origin and history of mKeima and how it is adapted to measure mitophagy. The author presents detailed protocols for making stable cell lines for optimized mitophagy detection and discuss many parameters that might affect the assay. A troubleshooting section is also provided to discuss possible pitfalls to improve reproducibility and sensitivity of the assay. © 2019 The Authors.This article was corrected on 20 August 2022. See the end of the full text for details.Basic Protocol 1: Making stable lines expressing mito-mKeima and YFP-ParkinSupport Protocol: Retrovirus/lentivirus infectionBasic Protocol 2: FACS analysis of mitophagy","PeriodicalId":40051,"journal":{"name":"Current Protocols in Cell Biology","volume":"86 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-12-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcb.99","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37490142","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: 11

Issue Information TOC 发布信息TOC

Current Protocols in Cell Biology Pub Date : 2019-12-18 DOI: 10.1002/cpcb.75

引用次数: 0

In Situ Visualization of Telomere Length, Telomere Elongation, and TERT Expression in Single Cells 单细胞中端粒长度、端粒伸长和TERT表达的原位可视化

Current Protocols in Cell Biology Pub Date : 2019-12-01 DOI: 10.1002/cpcb.97

A. Ravindranathan, Morgan E. Diolaiti, B. Cimini, B. Stohr

引用次数: 1