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Current Protocols in Cell Biology

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https://read.qxmd.com/read/30768855/bioluminescence-resonance-energy-transfer-2-bret2-based-ras-biosensors-to-characterize-ras-inhibitors
#1
Nicolas Bery, Terence H Rabbitts
Protein-protein interactions (PPIs) are principle biological processes that control normal cell growth, differentiation, and homeostasis but are also crucial in diseases such as malignancy, neuropathy, and infection. Despite the importance of PPIs in biology, this target class has been very challenging to convert to therapeutics. In the last decade, much progress has been made in the inhibition of PPIs involved in diseases, but many remain difficult such as RAS-effector interactions in cancers. We describe here a protocol for using Bioluminescence Resonance Energy Transfer 2 (BRET2)-based RAS biosensors to detect and characterize RAS PPI inhibition by macromolecules and small molecules...
February 15, 2019: Current Protocols in Cell Biology
https://read.qxmd.com/read/30724481/visualization-of-trigeminal-ganglion-neuronal-activities-in-mice
#2
Minghan Hu
Visualization of dynamic cellular activity has greatly expanded our understanding of brain function. Recently, there has been an increasing number of studies imaging rodent brain activity in real time. However, traditional in vivo calcium imaging technology has been limited to superficial brain structures. Because the trigeminal ganglion (TG) is located deep within the cranial cavity of mice, few studies have been able to access to it. To circumvent this limitation, overlying brain tissue must be removed to expose the TG so that optical recording can access deep brain neural ensembles...
February 6, 2019: Current Protocols in Cell Biology
https://read.qxmd.com/read/30548444/generation-of-pancreatic-ductal-organoids-and-whole-mount-immunostaining-of-intact-organoids
#3
Habib Rezanejad, Jennifer Hollister Lock, Brooke A Sullivan, Susan Bonner-Weir
Traditionally, studies of cells and tissues have been performed on isolated primary cells or immortalized cell lines by culturing them in 2D culture dishes or flasks. However, a caveat regarding 2D culture is that the cells poorly recapitulate their in vivo counterparts, mainly due to a lack of 3D cell-cell and cell-extracellular matrix interactions. In recent years, the development of in vitro organoids as 3D culture has gained substantial attention as a model to study different tissues. In adults, pancreatic ductal cells are considered as a source of stem or progenitor cells, so developing new methods to study ductal cells would be useful...
December 12, 2018: Current Protocols in Cell Biology
https://read.qxmd.com/read/30489039/dynamic-organellar-maps-for-spatial-proteomics
#4
Daniel N Itzhak, Julia P Schessner, Georg H H Borner
Eukaryotic cells are highly compartmentalized and protein subcellular localization critically influences protein function. Identification of the subcellular localizations of proteins and their translocation events upon perturbation has mostly been confined to targeted studies or laborious microscopy-based methods. Here we describe a systematic mass spectrometry-based method for spatial proteomics. The approach uses simple fractionation profiling and has two applications: Firstly it can be used to infer subcellular protein localization on a proteome-wide scale, resulting in a protein map of the cell...
November 29, 2018: Current Protocols in Cell Biology
https://read.qxmd.com/read/30253068/in-vitro-assays-for-targeting-and-insertion-of-tail-anchored-proteins-into-the-er-membrane
#5
Hyunju Cho, Un Seng Chio, Shu-Ou Shan
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 in vitro reconstitution of individual molecular events in the GET pathway with purified components...
December 2018: Current Protocols in Cell Biology
https://read.qxmd.com/read/30431237/analysis-of-organelle-positioning-using-patterned-microdevices
#6
Anahi Capmany, Bruno Latgé, Kristine Schauer
The consequences of alterations in the distribution of intracellular organelles, observed in many diseases, are often not clear. Intracellular organelles alter their morphology and positioning to regulate cell homeostasis and function. We outline how organelle positioning can be studied employing a density-based analysis of 3D images applied to cells that show similar cellular geometries. Quantification is facilitated by the use of single cells seeded on micropatterned substrates that provide cues for controlled cell spreading...
November 15, 2018: Current Protocols in Cell Biology
https://read.qxmd.com/read/30414385/visualizing-secretory-cargo-transport-in-budding-yeast
#7
Jason C Casler, Benjamin S Glick
Budding yeast is an excellent model organism for studying the dynamics of the Golgi apparatus. To characterize Golgi function, it is important to visualize secretory cargo as it traverses the secretory pathway. We describe a recently developed approach that generates fluorescent protein aggregates in the lumen of the yeast endoplasmic reticulum and allows the fluorescent cargo to be solubilized for transport through the Golgi by addition of a small-molecule ligand. We further describe how to generate a yeast strain expressing the regulatable secretory cargo, and we provide protocols for visualizing the cargo by 4D confocal microscopy and immunoblotting...
November 10, 2018: Current Protocols in Cell Biology
https://read.qxmd.com/read/30394692/single-molecule-rna-fish-smfish-in-whole-mount-mouse-embryonic-organs
#8
Shaohe Wang
Single molecule RNA fluorescence in situ hybridization (smFISH) has become the standard tool for high spatial resolution analysis of gene expression in the context of tissue organization. This article describes protocols to perform smFISH on whole-mount mouse embryonic organs, where tissue organization can be compared to RNA expression by co-immunostaining of known protein markers. An enzymatic labeling strategy is also introduced to produce low-cost smFISH probes. Important considerations and practical guidelines for imaging smFISH samples using fluorescence confocal microscopy are described...
November 5, 2018: Current Protocols in Cell Biology
https://read.qxmd.com/read/30394683/generating-embryonic-salivary-gland-organoids
#9
Zeinab F Hosseini, Deirdre A Nelson, Nicholas Moskwa, Melinda Larsen
Organoids are important research tools for studying organ morphogenesis and differentiation because they recapitulate ex vivo the native 3D organization of cells that is essential for proper cell and organ function. The composition of organoids can be manipulated to incorporate specific cell types to facilitate molecular interrogation of cell-cell interactions during organoid formation. A method for generating organoids derived from both embryonic salivary gland epithelial progenitor cells and mesenchymal support cells is described...
November 5, 2018: Current Protocols in Cell Biology
https://read.qxmd.com/read/30375749/visualization-of-genomic-loci-in-living-cells-with-bifc-tale
#10
Huan Hu, Xiaojing Yang, Chao Tang
Tracking the dynamics of genomic loci is essential for understanding a variety of cellular processes. However, earlier methods have all suffered from a low signal-to-background ratio (SBR), mainly caused by the background fluorescence from diffuse full-length fluorescent proteins in the nucleus. We have developed a novel method (BiFC-TALE) for labeling and tracking genomic loci in live mammalian cells, combining bimolecular fluorescence complementation (BiFC) and transcription activator-like effector (TALE) technologies...
October 30, 2018: Current Protocols in Cell Biology
https://read.qxmd.com/read/30329225/studying-glycolytic-oscillations-in-individual-yeast-cells-by-combining-fluorescence-microscopy-with-microfluidics-and-optical-tweezers
#11
Anna-Karin Gustavsson, Amin A Banaeiyan, David D van Niekerk, Jacky L Snoep, Caroline B Adiels, Mattias Goksör
In this unit, we provide a clear exposition of the methodology employed to study dynamic responses in individual cells, using microfluidics for controlling and adjusting the cell environment, optical tweezers for precise cell positioning, and fluorescence microscopy for detecting intracellular responses. This unit focuses on the induction and study of glycolytic oscillations in single yeast cells, but the methodology can easily be adjusted to examine other biological questions and cell types. We present a step-by-step guide for fabrication of the microfluidic device, for alignment of the optical tweezers, for cell preparation, and for time-lapse imaging of glycolytic oscillations in single cells, including a discussion of common pitfalls...
October 17, 2018: Current Protocols in Cell Biology
https://read.qxmd.com/read/30277020/analyzing-integrin-dependent-adhesion
#12
A Paul Mould
In this unit, methods for the analysis of integrin-dependent adhesion are described. Two major types of assays are commonly used for this analysis. The first are cell adhesion assays. A key application of this type of assay is to identify which integrin(s) mediate cell-substrate interactions; a comprehensive list of antibodies suitable for this purpose is detailed. The second are solid-phase assays in which purified integrins and integrin ligands are used. These assays can be used, e.g., to measure apparent affinities of integrins for different ligands and IC50 values of pharmacological inhibitors...
October 1, 2018: Current Protocols in Cell Biology
https://read.qxmd.com/read/30265447/fluorescence-activated-cell-sorting-facs-in-genome-wide-genetic-screening-of-membrane-trafficking
#13
Bridget L Menasche, Lauren Crisman, Daniel R Gulbranson, Eric M Davis, Haijia Yu, Jingshi Shen
About one-third of cellular proteins in eukaryotic cells are localized to membrane-enclosed organelles in the endomembrane system. Trafficking of these membrane proteins (including soluble lumenal proteins) among the organelles is mediated by small sac-like vesicles. Vesicle-mediated membrane trafficking regulates a broad range of biological processes, many of which are still poorly understood at the molecular level. A powerful approach to dissect a vesicle-mediated membrane trafficking pathway is unbiased genome-wide genetic screening, which only recently became possible in mammalian cells with the isolation of haploid human cell lines and the development of CRISPR-Cas9 genome editing...
September 28, 2018: Current Protocols in Cell Biology
https://read.qxmd.com/read/30265443/initiation-expansion-and-cryopreservation-of-human-primary-tissue-derived-normal-and-diseased-organoids-in-embedded-three-dimensional-culture
#14
James Clinton, Penney McWilliams-Koeppen
Organoids are primary patient-derived micro tissues grown within a three-dimensional extracellular matrix that better represents in vivo physiology and genetic diversity than existing two-dimensional cell lines. Organoids rely on the self-renewal and differentiation of tissue-resident stem cells that expand in culture and self-organize into complex three-dimensional structures. Depending on the tissue, organoids typically lack stromal, vascular, neural, and immune cells but otherwise can contain cells from all the respective tissue-specific cell lineages found in vivo...
September 28, 2018: Current Protocols in Cell Biology
https://read.qxmd.com/read/30265440/isolation-culture-and-differentiation-of-mammary-epithelial-stem-progenitor-cells-from-fresh-or-ex-vivo-cultured-human-breast-tissue
#15
Guang Chen, Hakim Bouamar, Lu-Zhe Sun
In vivo transplantation is the gold standard method for characterization of stem/progenitor cell self-renewal, tissue regeneration, and tumorigenesis. The method requires an enriched population of stem cells that represent a small fraction of a given tissue. An enriched population of stem/progenitor cells increases the likelihood of engraftment and reduces the number of recipient animals needed for in vivo transplantation. Methods for mammosphere formation by mammary epithelial stem and progenitor cells have been widely adopted for enriching stem/progenitor cells, allowing researchers to study genetic and epigenetic properties, interaction with other cell types, and differentiation and oncogenic transformation...
September 28, 2018: Current Protocols in Cell Biology
https://read.qxmd.com/read/30265439/identification-localization-and-quantification-of-hiv-reservoirs-using-microscopy
#16
Lisa Prevedel, Nancy Ruel, Paul Castellano, Carla Smith, Shaily Malik, Courtney Villeux, Morgane Bomsel, Susan Morgello, Eliseo A Eugenin
The major barrier to eradicating human immunodeficiency virus-1 (HIV) infection is the generation and extended survival of HIV reservoirs. In order to eradicate HIV infection, it is essential to detect, quantify, and characterize circulating and tissue-associated viral reservoirs in infected individuals. Currently, PCR-based technologies and Quantitative Viral Outgrowth Assays (Q-VOA) are the gold standards to detect viral reservoirs. However, these methods are limited to detecting circulating viral reservoirs, and it has been shown that they misrepresent the size of the reservoirs, largely because they detect only one component of the HIV life cycle and are unable to detect viral reservoirs in tissues...
September 28, 2018: Current Protocols in Cell Biology
https://read.qxmd.com/read/30246944/monitoring-sphingolipid-trafficking-in-cells-using-fluorescence-microscopy
#17
Emma L Sundberg, Yongqiang Deng, Christopher G Burd
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...
September 24, 2018: Current Protocols in Cell Biology
https://read.qxmd.com/read/30239150/an-on-chip-method-for-long-term-growth-and-real-time-imaging-of-brain-organoids
#18
Eyal Karzbrun, Rami Yair Tshuva, Orly Reiner
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...
September 21, 2018: Current Protocols in Cell Biology
https://read.qxmd.com/read/30178917/genome-editing-in-mice-using-crispr-cas9-technology
#19
Bradford Hall, Andrew Cho, Advait Limaye, Kyoungin Cho, Jaspal Khillan, Ashok B Kulkarni
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...
September 4, 2018: Current Protocols in Cell Biology
https://read.qxmd.com/read/30133963/preparation-and-culture-of-human-liver-resident-immune-cells
#20
Hao Feng, Baochi Ou, Wei Dong, Wolfgang E Thasler
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 in vitro, 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...
September 2018: Current Protocols in Cell Biology
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