Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.]

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  • ISSN
    1934-2616

Publications in this journal

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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.
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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.
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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.
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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.
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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.
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    ABSTRACT: Genetically encoded actuators that allow control of protein-protein interactions using light, termed 'optical dimerizers', are emerging as new tools for experimental biology. In recent years, numerous new and versatile dimerizer systems have been developed. Here we discuss the design of optical dimerizer experiments, including choice of a dimerizer system, photoexcitation sources, and the coordinate use of imaging reporters. We provide detailed protocols for experiments using two dimerization systems we previously developed, CRY2/CIB and UVR8/UVR8, for use in controlling transcription, protein localization, and protein secretion using light. Additionally, we provide instructions and software for constructing a pulse-controlled LED device for use in experiments requiring extended light treatments. Curr. Protoc. Cell Biol. 64:17.16.1-17.16.20. © 2014 by John Wiley & Sons, Inc.
    Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] 01/2014; 64:17.16.1-17.16.20.
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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.
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    ABSTRACT: The auxin-inducible degron (AID) system allows the rapid and reversible proteolysis of proteins of interest, and enables the generation of conditional mutants of budding yeast. The construction of budding yeast AID mutants is simple, and the effect of depletion of essential proteins on proliferation can be confirmed by analyzing their phenotype. In this protocol, we describe a procedure to generate AID mutants of budding yeast via a simple transformation using PCR-amplified DNA. We also describe methods to confirm the depletion of proteins of interest that are required for proliferation by serial-dilution and liquid-culture assays. Curr. Protoc. Cell Biol. 64:20.9.1-20.9.16. © 2014 by John Wiley & Sons, Inc.
    Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] 01/2014; 64:20.9.1-20.9.16.
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    ABSTRACT: Freeze-fracture is a unique investigative tool for visualization of the en face topography of individual membrane leaflets of cell membranes at high resolution under the electron microscope. The development of a system of freeze-fracture cytochemical and immunocytochemical techniques has further advanced the utility of this methodological approach for high-resolution localization of specific membrane and intracellular macromolecules in tissues and cells. The unit focuses on description, in a step-by-step manner, of the experimental procedures for two specific freeze-fracture labeling techniques, namely fracture-label and label-fracture. Users are guided in a stepwise manner, starting from the preparation of tissue or cell samples to the final retrieval and mounting of fracture-label and label-fracture specimens for examination on the electron microscope. © 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; 65:4.28.1-4.28.15.
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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.
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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.
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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.
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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.
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    ABSTRACT: Cryopreservation is the use of low temperatures to preserve structurally intact living cells. The cells that survive the thermodynamic journey from the 37°C incubator to the -196°C liquid nitrogen storage tank are free from the influences of time. Thus, cryopreservation is a critical component of cell culture and cell manufacturing protocols. Successful cryopreservation of human cells requires that the cells be derived from patient samples that are collected in a standardized manner, and carefully handled from blood draw through cell isolation. Furthermore, proper equipment must be in place to ensure consistency, reproducibility, and sterility. In addition, the correct choice and amount of cryoprotectant agent must be added at the correct temperature, and a controlled rate of freezing (most commonly 1°C/min) must be applied prior to a standardized method of cryogenic storage. This appendix describes how human primary cells can be frozen for long-term storage and thawed for growth in a tissue culture vessel. Curr. Protoc. Cell Biol. 64:A.3I.1-A.3I.8. © 2014 by John Wiley & Sons, Inc.
    Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] 01/2014; 64:A.3I.1-A.3I.8.
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    ABSTRACT: Cells interact with their environment through receptor proteins expressed at their plasma membrane, and protein-protein interactions govern the transduction of signals across the membrane into the cell. Therefore, the ability to measure receptor densities and protein colocalization within the membrane of intact cells is of paramount importance. This unit describes a technique to extract these parameters from fluorescence microscopy images obtained using a commercial confocal laser scanning microscope (CLSM) and other similar types of microscopes. It is based on the analysis of spatial fluorescence intensity fluctuations in the images, which can then be related to particle density and aggregation state via calculation of a spatial autocorrelation function, or used to measure particle colocalization via calculation of a spatial cross-correlation function from dual-color images of proteins tagged with two different fluorophores and imaged in two detection channels. These parameters offer key insights on the interaction of the cell with its environment. Curr. Protoc. Cell Biol. 59:4.27.1-4.27.15. © 2013 by John Wiley & Sons, Inc.
    Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] 06/2013; Chapter 4:Unit4.27.
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    ABSTRACT: A simple, scalable, and fast procedure for the isolation of Chlamydomonas flagella is described. Chlamydomonas can be synchronously deflagellated by treatment with chemicals, pH shock, or mechanical shear. The Basic Protocol describes the procedure for flagellar isolation using dibucaine to induce flagellar abscission; we also describe the pH shock method as an Alternate Protocol when flagellar regeneration is desirable. Sub-fractionation of the isolated flagella into axonemes and the membrane + matrix fraction is described in a Support Protocol. Curr. Protoc. Cell Biol. 59:3.41.1-3.41.9. © 2013 by John Wiley & Sons, Inc.
    Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] 06/2013; Chapter 3:Unit3.41.
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    ABSTRACT: The cell's primary cilium is both a mechanical and chemical sensor involved in many signaling pathways. In order to ascertain protein enrichment in the primary cilium or study sub-ciliary localization of various proteins, it is advantageous to remove the primary cilium from the cell body. The protocol described here gives detailed instructions on purifying primary cilia by separating them from the cell body using shear force. This simple technique avoids using harsh purification conditions that may affect signaling proteins in the cilium or cause the ciliary membrane to disintegrate. In addition, as the cell body remains mostly intact, contamination of the isolated cilia by proteins from the cell body is minimized. This protocol is ideally suited for isolating cilia from renal cell lines, as primary cilia in these cells grow to greater lengths than in other cell types (up to 50-µm long in Xenopus A6 toad kidney cells as opposed to 1 to 5 µm in NIH3T3 fibroblast cells). Curr. Protoc. Cell Biol. 59:3.42.1-3.42.9. © 2013 by John Wiley & Sons, Inc.
    Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] 06/2013; Chapter 3:Unit3.42.