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Current Protocols in Cell Biology

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https://www.readbyqxmd.com/read/28627757/super-resolution-microscopy-and-single-protein-tracking-in-live-bacteria-using-a-genetically-encoded-photostable-fluoromodule
#1
Saumya Saurabh, Adam M Perez, Colin J Comerci, Lucy Shapiro, W E Moerner
Visualization of dynamic protein structures in live cells is crucial for understanding the mechanisms governing biological processes. Fluorescence microscopy is a sensitive tool for this purpose. In order to image proteins in live bacteria using fluorescence microscopy, one typically genetically fuses the protein of interest to a photostable fluorescent tag. Several labeling schemes are available to accomplish this. Particularly, hybrid tags that combine a fluorescent or fluorogenic dye with a genetically encoded protein (such as enzymatic labels) have been used successfully in multiple cell types...
June 19, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/28627756/microcontact-peeling-a-cell-micropatterning-technique-for-circumventing-direct-adsorption-of-proteins-to-hydrophobic-pdms
#2
Sho Yokoyama, Tsubasa S Matsui, Shinji Deguchi
Microcontact printing (μCPr) is one of the most popular techniques used for cell micropatterning. In conventional μCPr, a polydimethylsiloxane (PDMS) stamp with microfeatures is used to adsorb extracellular matrix (ECM) proteins onto the featured surface and transfer them onto particular areas of a cell culture substrate. However, some types of functional proteins other than ECM have been reported to denature upon direct adsorption to hydrophobic PDMS. Here we describe a detailed protocol of an alternative technique--microcontact peeling (μCPe)--that allows for cell micropatterning while circumventing the step of adsorbing proteins to bare PDMS...
June 19, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/28627755/labeling-dna-replication-foci-to-visualize-chromosome-territories-in-vivo
#3
Apolinar Maya-Mendoza, Dean A Jackson
While a detailed understanding of chromatin dynamics is needed to explain how higher-order chromatin organization influences nuclear function, the molecular principles that regulate chromatin mobility in mammalian nuclei remain largely unknown. Here we describe experimental tools to follow chromatin dynamics by labeling DNA during S phase. Using these methods, we have found that foci labeled during early and mid/late S phase have significantly different dynamic behavior. Spatially constrained heterochromatic foci restrict long-range transformations of the chromosome territory (CT) structure while providing a structural framework on which highly mobile euchromatic foci undergo positional oscillations that drive local changes in the chromosome shape...
June 19, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/28627754/labeling-and-magnetic-resonance-imaging-of-exosomes-isolated-from-adipose-stem-cells
#4
Alice Busato, Roberta Bonafede, Pietro Bontempi, Ilaria Scambi, Lorenzo Schiaffino, Donatella Benati, Manuela Malatesta, Andrea Sbarbati, Pasquina Marzola, Raffaella Mariotti
Adipose stem cells (ASC) represent a promising therapeutic approach for neurodegenerative diseases. Most biological effects of ASC are probably mediated by extracellular vesicles, such as exosomes, which influence the surrounding cells. Current development of exosome therapies requires efficient and noninvasive methods to localize, monitor, and track the exosomes. Among imaging methods used for this purpose, magnetic resonance imaging (MRI) has advantages: high spatial resolution, rapid in vivo acquisition, and radiation-free operation...
June 19, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/28627753/traction-force-microscopy-in-3-dimensional-extracellular-matrix-networks
#5
M Cóndor, J Steinwachs, C Mark, J M García-Aznar, B Fabry
Cell migration through a three-dimensional (3-D) matrix depends strongly on the ability of cells to generate traction forces. To overcome the steric hindrance of the matrix, cells need to generate sufficiently high traction forces but also need to distribute these forces spatially in a migration-promoting way. This unit describes a protocol to measure spatial maps of cell traction forces in 3-D biopolymer networks such as collagen, fibrin, or Matrigel. Traction forces are computed from the relationship between measured force-induced matrix deformations surrounding the cell and the known mechanical properties of the matrix...
June 19, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/28627752/patterning-on-topography-for-generation-of-cell-culture-substrates-with-independent-nanoscale-control-of-chemical-and-topographical-extracellular-matrix-cues
#6
Emily N Sevcik, John M Szymanski, Quentin Jallerat, Adam W Feinberg
The cell microenvironment plays an important role in many biological processes, including development and disease progression. Key to this is the extracellular matrix (ECM), a complex biopolymer network serving as the primary insoluble signaling network for physical, chemical, and mechanical cues. In vitro, the ability to engineer the ECM at the micro- and nanoscales is a critical tool to systematically interrogate the influence of ECM properties on cellular responses. Specifically, both topographical and chemical surface patterning has been shown to direct cell alignment and tissue architecture on biomaterial surfaces, however, it has proven challenging to independently control these surface properties...
June 19, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/28256723/immunoblotting-and-immunodetection
#7
Duojiao Ni, Peng Xu, Sean Gallagher
Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps, starting with solubilization of the protein samples, usually by means of SDS and reducing agents. Following solubilization, the material is separated by SDS-PAGE and the antigens are electrophoretically transferred to a membrane, a process that can be monitored by reversible staining with Ponceau S. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection reagents...
March 3, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/28256722/discovering-protein-protein-interactions-using-nucleic-acid-programmable-protein-arrays
#8
Yanyang Tang, Ji Qiu, Matthias Machner, Joshua LaBaer
We have developed a protocol enabling the study of protein-protein interactions (PPIs) at the proteome level using in vitro-synthesized proteins. Assay preparation requires molecular cloning of the query gene into a vector that supports in vitro transcription/translation (IVTT) and appends a HaloTag to the query protein of interest. In parallel, protein microarrays are prepared by printing plasmids encoding glutathione S-transferase (GST)-tagged target proteins onto a carrier matrix/glass slide coated with antibody directed against GST...
March 3, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/28256721/a-functional-microrna-screening-method-for-organ-morphogenesis
#9
Ivan T Rebustini
The increasing repertoire of microRNAs expressed during organ development and their role in regulating organ morphogenesis provide a compelling need to develop methods to assess microRNA function using various in vitro and in vivo experimental models. Methods to assess microRNA function during organ morphogenesis include transfection of microRNA inhibitors (antagomirs) and activators (mimics) into mouse embryonic explanted organs using liposomes, which can potentially result in low efficiency of transfection and off-target effects...
March 3, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/28256720/a-unique-high-throughput-assay-to-identify-novel-small-molecule-inhibitors-of-chemotaxis-and-migration
#10
Xin-Hua Liao, Alan R Kimmel
Chemotaxis and cell migration play pivotal roles in normal physiological processes such as embryogenesis, inflammation, and wound healing, as well as in pathological processes including chronic inflammatory disease and cancer metastasis. Novel chemotaxis/migration inhibitors are desirable for developing effective therapeutics and probing molecular mechanisms. We describe a fluorescence-based phenotypic assay in a 1536-well plate format for high-throughput screening of novel inhibitors of chemotaxis/migration within complex libraries of thousands of compounds...
March 3, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/28256719/rna-whole-mount-in-situ-hybridization-proximity-ligation-assay-rish-pla-an-assay-for-detecting-rna-protein-complexes-in-intact-cells
#11
Ioannis M Roussis, Fiona A Myers, Garry P Scarlett
Techniques for studying RNA-protein interactions have lagged behind those for DNA-protein interactions as a consequence of the complexities associated with working with RNA. This unit describes a method for the adaptation of the In Situ Hybridization-Proximity Ligation Assay (ISH-PLA) to the study of RNA regulation (rISH-PLA). The rISH-PLA assay allows the identification of a given RNA-protein complex at subcellular and single-cell resolution, thus avoiding the lack of spatial resolution and sensitivity associated with assaying heterogeneous cell populations from which conventional RNA-protein interaction detection techniques suffer...
March 3, 2017: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/27906451/proximity-dependent-biotinylation-for-identification-of-interacting-proteins
#12
Valerie Le Sage, Alessandro Cinti, Andrew J Mouland
Complex interaction networks orchestrate key cellular processes including but not limited to transcription, translation, metabolism, and cell signaling. Delineating these interactions will aid in deciphering the regulation and function of these pathways and potential for manipulation. Proximity-dependent biotin identification (BioID) is quickly gaining popularity as a powerful tool for identifying novel protein-protein and proximity-based interactions in live cells. This technique relies on a promiscuous biotin ligase, which is fused to a protein of interest and, upon expression in the desired cell, will biotinylate proximal endogenous proteins...
December 1, 2016: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/27906450/lovtrap-a-versatile-method-to-control-protein-function-with-light
#13
Hui Wang, Klaus M Hahn
We describe a detailed procedure for the use of LOVTRAP, an approach to reversibly sequester and release proteins from cellular membranes using light. In the application described here, proteins that act at the plasma membrane are held at mitochondria in the dark, and reversibly released by irradiation. The technique relies on binding of an engineered Zdk domain to a LOV2 domain, with affinity <30 nM in the dark and >500 nM upon irradiation between 400 and 500 nm. LOVTRAP can be applied to diverse proteins, as it requires attaching only one member of the Zdk/LOV2 pair to the target protein, and the other to the membrane where the target protein is to be sequestered...
December 1, 2016: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/27580706/isolation-of-lipid-droplets-from-cells-by-density-gradient-centrifugation
#14
Dawn L Brasaemle, Nathan E Wolins
Lipid droplets are organelles found in most mammalian cells, as well as in various plant tissues and yeast. They are composed of a core of neutral lipids surrounded by a membrane monolayer of phospholipids and cholesterol in which specific proteins are embedded. This unit provides protocols for isolating lipid droplets from mammalian cells by discontinuous density gradient centrifugation. © 2016 by John Wiley & Sons, Inc.
September 1, 2016: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/27580705/identification-of-protein-kinase-substrates-by-the-kinase-interacting-substrate-screening-kiss-approach
#15
Mutsuki Amano, Tomoki Nishioka, Yoshimitsu Yura, Kozo Kaibuchi
Identifying the substrates of protein kinases to understand their modes of action has been undertaken by various approaches and remains an ongoing challenge. Phosphoproteomic technologies have accelerated the accumulation of data concerning protein phosphorylation and have uncovered vast numbers of phosphorylation sites in vivo. In this unit, a novel in vitro screening approach for protein kinase substrates is presented, based on protein-protein interaction and mass spectrometry-based phosphoproteomic technology...
September 1, 2016: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/27580704/generation-of-3d-collagen-gels-with-controlled-diverse-architectures
#16
Andrew D Doyle
Rat tail collagen solutions have been used as polymerizable in vitro three dimensional (3D) extracellular matrix (ECM) gels for single and collective cell migration assays as well as spheroid formation. Factors such as ECM concentration, pH, ionic concentration, and temperature can alter collagen polymerization and ECM architecture. This unit describes how to generate 3D collagen gels that have distinct architectures ranging from a highly reticular meshwork of short thin fibrils with small pores to a loose matrix consisting of stiff, parallel-bundled long fibrils by changing collagen polymerization temperature...
September 1, 2016: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/27245427/fluorescent-detection-of-lipid-droplets-and-associated-proteins
#17
Laura L Listenberger, Andrea M Studer, Deborah A Brown, Nathan E Wolins
Excess lipid is stored in intracellular organelles known as lipid droplets. This unit discusses techniques for the visualization of lipid droplets and associated proteins in cultured mammalian cells. Protocols for the detection of lipid droplets in fixed or live cells with BODIPY 493/503 are included. The best method for combining visualization of intracellular lipid droplets with indirect immunofluorescent detection of lipid droplet-associated proteins is described. Techniques for sample fixation and permeabilization must be chosen carefully to avoid alterations to lipid droplet morphology...
June 1, 2016: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/27245426/use-of-in-situ-proximity-ligation-assays-for-systems-analysis-of-signaling-pathways
#18
Tzu-Chi Chen, Chi-Ying F Huang
Understanding signaling pathway networks via protein-protein interactions (PPIs) at the cellular level is a significant task that has not yet been completed. Here, a systems approach that computationally infers interlinked pathways from numerous PPIs is described. The endogenous PPIs can be empirically detected using an in situ proximity ligation assay (PLA), which detects and visualizes endogenous PPIs and post-translational modifications of proteins with a high sensitivity and specificity. This unit includes two parts: (1) conversion of gene lists into PPIs for investigation and (2) large-scale detection and analysis of endogenous PPIs for elucidating pathway networks...
June 1, 2016: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/27245425/preparation-of-extracellular-matrices-produced-by-cultured-and-primary-fibroblasts
#19
Janusz Franco-Barraza, Dorothy A Beacham, Michael D Amatangelo, Edna Cukierman
Fibroblasts secrete and organize extracellular matrix (ECM), which provides structural support for their adhesion, migration, and tissue organization, besides regulating cellular functions such as growth and survival. Cell-to-matrix interactions are vital for vertebrate development. Disorders in these processes have been associated with fibrosis, developmental malformations, cancer, and other diseases. This unit describes a method for preparing a three-dimensional matrix derived from fibroblastic cells; the matrix is three-dimensional, cell and debris free, and attached to a two-dimensional culture surface...
June 1, 2016: Current Protocols in Cell Biology
https://www.readbyqxmd.com/read/27245424/immunoprecipitation
#20
Juan S Bonifacino, David C Gershlick, Esteban C Dell'Angelica
Selective immunoprecipitation of proteins is a useful tool for characterizing proteins and protein-protein interactions. Clear step-by-step protocols are provided for preparing lysates of cells and yeast under a variety of conditions, for binding the antibody to a solid matrix, and for performing the actual immunoprecipitation. An additional method is provided for increasing the specificity of the technique by reprecipitating the antigen with the same or a different antibody. © 2016 by John Wiley & Sons, Inc...
June 1, 2016: Current Protocols in Cell Biology
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