Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] Journal Impact Factor & Information

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ISSN 1934-2616

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    ABSTRACT: Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This unit describes the basic skills required to maintain and preserve cell cultures: maintaining aseptic technique, preparing media with the appropriate characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells. © 2015 by John Wiley & Sons, Inc. Copyright © 2015 John Wiley & Sons, Inc.
    Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] 01/2015; 66:1.1.1-1.1.22. DOI:10.1002/0471143030.cb0101s66
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    ABSTRACT: Isolated spinal motoneurons are a powerful tool for studying basic mechanisms of neurite growth and survival. Since motoneurons are a minor population of developing spinal cord cells, they need to be purified and enriched to separate them from non-neuronal cells. Therefore, the particular feature of embryonic motoneurons to express the low affinity neurotrophin receptor p75(NTR) is used to separate the motoneurons from other contaminating cells. Two ways are described to isolate embryonic motoneurons: the basic protocol taking advantage of the ability of p75(NTR) to bind lectin, and an alternative method using an antibody against p75(NTR) for a panning procedure. These protocols comprise suggestions for the cultivation of the isolated motoneurons for experiments regarding neural outgrowth and survival as well as instruction for the preparation of proteins of the cells. © 2015 by John Wiley & Sons, Inc. Copyright © 2015 John Wiley & Sons, Inc.
    Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] 01/2015; 66:1.9.1-1.9.10. DOI:10.1002/0471143030.cb0109s66
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    ABSTRACT: The nucleus is generally found near the cell center; however its position can vary in response to extracellular or intracellular signals, leading to a polarized intracellular organization. Nuclear movement is mediated by the cytoskeleton and its associated motors. While the role of actin and microtubule cytoskeletons in nuclear positioning has been assessed in various systems, the contribution of intermediate filaments is less established due in part to the lack of tools to study intermediate filament functions. The methods described here use micropatterned substrates to impose reproducible cell shape and nucleus position. Intermediate filament organization can be perturbed using gene downregulation or upregulation; intermediate filaments can also be visualized using fluorescent intermediate filament proteins. This protocol is valuable for characterizing the role of intermediate filaments in a variety of live or fixed adherent cells. © 2015 by John Wiley & Sons, Inc. Copyright © 2015 John Wiley & Sons, Inc.
    Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] 01/2015; 66:13.7.1-13.7.19. DOI:10.1002/0471143030.cb1307s66
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    ABSTRACT: One concept in regenerative therapy is the replacement of a lost or damaged organ with a regenerated, fully functional organ. Three-dimensional cell manipulation techniques, designated "organ germ methods," enable the normal development of a bioengineered organ germ in several types of ectodermal organs, such as teeth, hair follicles, and secretory glands. This method is useful for both organ regeneration technology and developmental biology, including cell kinetic analysis and the elucidation of gene regulation during organogenesis. Here, we describe a protocol for salivary gland reconstitution using the organ germ method to transplant a bioengineered salivary gland germ. © 2015 by John Wiley & Sons, Inc. Copyright © 2015 John Wiley & Sons, Inc.
    Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] 01/2015; 66:19.17.1-19.17.13. DOI:10.1002/0471143030.cb1917s66
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    ABSTRACT: Transient gene expression in protoplasts, which has been used in several plant species, is an important and versatile tool for rapid functional gene analysis, protein subcellular localization, and biochemical manipulations. This unit describes transient gene expression by electroporation of DNA into protoplasts of Arabidopsis or tobacco suspension-cultured cells and by polyethylene glycol (PEG)-mediated DNA transformation into protoplasts derived from rice leaf sheaths. PEG-mediated DNA transformation for transient gene expression in rice protoplasts in suspension culture is also described as an alternative technique. Methods for collecting intracellular and secreted proteins are also provided. Curr. Protoc. Cell Biol. 63:2.8.1-2.8.17. © 2014 by John Wiley & Sons, Inc.
    Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] 01/2014; 63:2.8.1-2.8.17. DOI:10.1002/0471143030.cb0208s63
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    ABSTRACT: Atg8 modifier in yeast is conjugated to phosphatidylethanolamine via ubiquitylation-like reactions essential for autophagy. Mammalian Atg8 homologs (Atg8s) including LC3, GABARAP, and GATE-16, are also ubiquitin-like modifiers. The carboxyl termini of mammalian Atg8 homologs are cleaved by Atg4B, a cysteine protease, to expose carboxyl terminal Gly which is essential for this ubiquitylation-like reaction. Thereafter, the Atg8 homologs are activated by Atg7, an E1-like enzyme, to form unstable Atg7-Atg8 E1-substrate intermediates via a thioester bond. The activated Atg8 homologs are transferred to mammalian Atg3, an E2-like enzyme, to form unstable Atg3-Atg8 E2-substrate intermediates via a thioester bond. Finally, Atg8 homologs are conjugated to phospholipids, phosphatidylethanolamine, and phosphatidylserine. Here, we describe a protocol for the reconstituted conjugation systems for mammalian Atg8 homologs in vitro using purified recombinant Atg proteins and liposomes. Curr. Protoc. Cell Biol. 64:11.20.1-11.20.13. © 2014 by John Wiley & Sons, Inc.
    Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] 01/2014; 64:11.20.1-11.20.13. DOI:10.1002/0471143030.cb1120s64
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    ABSTRACT: Peroxisomes are the most recently discovered classical organelles, and only lately have their diverse functions been truly recognized. Peroxisomes are highly dynamic structures, changing both morphologically and in number in response to both extracellular and intracellular signals. This metabolic organelle came to prominence due to the many genetic disorders caused by defects in its biogenesis or enzymatic functions. There is now growing evidence that suggests peroxisomes are involved in lipid biosynthesis, innate immunity, redox homeostasis, and metabolite scavenging, among other functions. Therefore, it is important to have available suitable methods and techniques to visualize and quantify peroxisomes in response to various cellular signals. This unit includes a number of protocols that will enable researchers to image, qualify, and quantify peroxisome numbers and morphology-with both steady-state and time-lapse imaging using mammalian cells. The use of photoactivatable fluorescent proteins to detect and measure peroxisome biogenesis is also described. Altogether, the protocols described here will facilitate understanding of the dynamic changes that peroxisomes undergo in response to various cellular signals. Curr. Protoc. Cell Biol. 62:21.9.1-21.9.20. © 2014 by John Wiley & Sons, Inc.
    Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] 01/2014; 62:21.9.1-21.9.20. DOI:10.1002/0471143030.cb2109s62
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    ABSTRACT: In this unit, we describe a workflow that enables array comparative genomic hybridization (aCGH) of single cells. The unit first describes the isolation and preparation of single peripheral mononuclear cells from blood (PBMC) to prepare a suitable reference DNA for aCGH experiments. An alternative procedure is described for the preparation of single cells of GM14667 and GM05423 cell lines to use as reference DNA and for sex-mismatched control experiments. A guide is also provided for micromanipulation of single cells. Next, the unit describes whole-genome amplification using adapter-linker PCR (Ampli1 WGA Kit) and an alternative nonlinear WGA method (PicoPLEX WGA Kit) for single-cell amplification. A protocol is also included for reamplification of Ampli1 WGA products, which can be used for aCGH as well. Finally, the use of 4 × 180k oligonucleotide microarrays to perform aCGH with single-cell WGA products is described. Curr. Protoc. Cell Biol. 65:22.19.1-22.19.23. © 2014 by John Wiley & Sons, Inc. Copyright © 2014 John Wiley & Sons, Inc.
    Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] 01/2014; 65:22.19.1-22.19.23. DOI:10.1002/0471143030.cb2219s65
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    ABSTRACT: A method to directly measure the intracellular pressure of adherent, migrating cells is described in this unit. This approach is based on the servo-null method where a microelectrode is introduced into the cell to directly measure the physical pressure of the cytoplasm. We also describe the initial calibration of the microelectrode, as well as the application of the method to cells migrating inside three-dimensional (3-D) extracellular matrix (ECM). Curr. Protoc. Cell Biol. 63:12.9.1-12.9.9. © 2014 by John Wiley & Sons, Inc.
    Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] 01/2014; 63:12.9.1-9. DOI:10.1002/0471143030.cb1209s63
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    ABSTRACT: Understanding the regulatory principles ensuring complete DNA replication in each cell division is critical for deciphering the mechanisms that maintain genomic stability. Recent advances in genome sequencing technology facilitated complete mapping of DNA replication sites and helped move the field from observing replication patterns at a handful of single loci to analyzing replication patterns genome-wide. These advances address issues, such as the relationship between replication initiation events, transcription, and chromatin modifications, and identify potential replication origin consensus sequences. This unit summarizes the technological and fundamental aspects of replication profiling and briefly discusses novel insights emerging from mining large datasets, published in the last 3 years, and also describes DNA replication dynamics on a whole-genome scale. Curr. Protoc. Cell Biol. 64:22.18.1-22.18.13. © 2014 by John Wiley & Sons, Inc.
    Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] 01/2014; 64:22.18.1-22.18.13. DOI:10.1002/0471143030.cb2218s64
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    ABSTRACT: Biosensors are valuable tools used to monitor many different protein behaviors in vivo. Demand for new biosensors is high, but their development and characterization can be difficult. During biosensor design, it is necessary to evaluate the effects of different biosensor structures on specificity, brightness, and fluorescence responses. By co-expressing the biosensor with upstream proteins that either stimulate or inhibit the activity reported by the biosensor, one can determine the difference between the biosensor's maximally activated and inactivated state, and examine response to specific proteins. We describe here a method for biosensor validation in a 96-well plate format using an automated microscope. This protocol produces dose-response curves, enables efficient examination of many parameters, and unlike cell suspension assays, allows visual inspection (e.g., for cell health and biosensor or regulator localization). Optimization of single-chain and dual-chain Rho GTPase biosensors is addressed, but the assay is applicable to any biosensor that can be expressed or otherwise loaded in adherent cells. The assay can also be used for purposes other than biosensor validation, using a well-characterized biosensor as a readout for effects of upstream molecules. © 2014 by John Wiley & Sons, Inc. Copyright © 2014 John Wiley & Sons, Inc.
    Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] 01/2014; 65:14.15.1-14.15.31. DOI:10.1002/0471143030.cb1415s65
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    ABSTRACT: Cell adhesion, migration, and signaling in physiologically normal and pathological processes depend highly on the extracellular matrix that the cell interacts with. A variety of in vitro models of two-dimensional and three-dimensional extracellular matrices have been developed to study multiple aspects of cellular behavior. However, there is a profound need for in vitro models of extracellular matrices to closely mimic both biochemical and physical aspects of a three-dimensional in vivo cellular environment. This unit outlines the preparation of human-tissue-derived, cell-free, three-dimensional extracellular matrices for studying cellular behavior and cell-extracellular matrix interactions ex vivo. These protocols can be used to prepare cell-free matrices from a variety of normal and cancerous tissues. This unit also provides protocols for quality control of acellular matrix preparations, and for immunostaining of cells for specific cellular proteins as well as of extracellular matrices for their components. Curr. Protoc. Cell Biol. 62:19.16.1-19.16.20. © 2014 by John Wiley & Sons, Inc.
    Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] 01/2014; 62:19.16.1-19.16.20. DOI:10.1002/0471143030.cb1916s62
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    ABSTRACT: Recent advances in genome-sequencing technology have led to the complete mapping of DNA replication initiation sites in the human genome. This thorough origin mapping facilitates understanding of the relationship between replication initiation events, transcription, and chromatin modifications, and allows the characterization of consensus sequences of potential replication origins. This unit provides a detailed protocol for isolation and sequence analysis of nascent DNA strands. Two variations of the protocol based on non-overlapping assumptions are described below, addressing potential bias issues for whole-genome analyses. © 2014 by John Wiley & Sons, Inc. Copyright © 2014 John Wiley & Sons, Inc.
    Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] 01/2014; 65:22.20.1-22.20.17. DOI:10.1002/0471143030.cb2220s65
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    ABSTRACT: Integrins are cell surface receptors for cell adhesion. Integrin-mediated cell adhesion regulates various cellular processes, including cell survival, migration, proliferation, and differentiation. In vivo, ligands for integrins are immobilized within extracellular matrices, insoluble sheet-like or fibrous supramolecular complexes that associate with or surround cells. To better understand the molecular basis of integrin-mediated regulation of cellular behavior in vivo, it is of critical importance to collect information regarding the activities as well as spatial distributions of integrin ligands in situ. This unit describes a protocol for detecting the spatial distribution of the complement of integrin ligands in situ by overlaying soluble recombinant integrins. © 2014 by John Wiley & Sons, Inc. Copyright © 2014 John Wiley & Sons, Inc.
    Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] 01/2014; 65:10.19.1-10.19.17. DOI:10.1002/0471143030.cb1019s65