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: 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: Cells contain many important protein complexes involved in performing and regulating structural, metabolic, and signaling functions. Understanding physical and functional interactions between proteins in living systems is of vital importance in biology. The importance of protein-protein interactions (PPIs) has led to the development of several powerful methodologies and techniques to detect them. All of this information has enabled the creation of large protein-interaction networks. One important challenge in biology is to understand how protein complexes respond to genetic perturbations. Here we describe a systematic genetic assay termed "reverse PCA," which allows the identification of genes whose products are required for modulating the physical interaction between two given proteins. Our assay starts with a yeast strain in which the PPI of interest can be detected by resistance to the drug methotrexate, in the context of the protein-fragment complementation assay (PCA). By combining the synthetic genetic array (SGA) technology, we can systematically screen mutant libraries of the yeast Saccharomyces cerevisiae to identify trans-acting mutations that disrupt the physical interaction of interest. The identification of such mutants is valuable for unraveling important regulatory mechanisms, and for defining the response of the protein interactome to specific perturbations. Curr. Protoc. Cell Biol. 64:17.15.1-17.15.11. © 2014 by John Wiley & Sons, Inc.
    Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] 01/2014; 64:17.15.1-17.15.11.
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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: The spatial organization of the genome within the nucleus is now seen as a key contributor to genome function. Studying chromatin dynamics in living cells has been rendered possible by the development of fast microscopy coupled with fluorescent repressor operator systems (FROS). In these systems, arrays of protein-binding sites integrated at specific loci by homologous recombination are monitored through the fluorescence of tagged DNA-binding proteins. In the budding yeast, where homologous recombination is efficient, this technique, combined with targeting assay and genetic analysis, has been extremely powerful for studying the determinants and function of chromatin dynamics in living cells. However, issues have been recurrently raised in different species regarding the use of these systems. Here we discuss the different uses of gene tagging with FROS and their limitations, focusing in budding yeast as a model organism. Curr. Protoc. Cell Biol. 62:22.17.1-22.17.14. © 2014 by John Wiley & Sons, Inc.
    Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] 01/2014; 62:22.17.1-22.17.14.
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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: 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: 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: 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: 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: 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: 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: 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.
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    ABSTRACT: This unit focuses on the use of total internal reflection fluorescence (TIRF) microscopy and image analysis methods to study the dynamics of signal transduction mediated by class I phosphoinositide 3-kinases (PI3Ks) in mammalian cells. The first four protocols cover live-cell imaging experiments, image acquisition parameters, and basic image processing and segmentation. These methods are generally applicable to live-cell TIRF experiments. The remaining protocols outline more advanced image analysis methods, which were developed in our laboratory for the purpose of characterizing the spatiotemporal dynamics of PI3K signaling. These methods may be extended to analyze other cellular processes monitored using fluorescent biosensors. Curr. Protoc. Cell Biol. 61:14.14.1-14.14.24. © 2013 by John Wiley & Sons, Inc.
    Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] 01/2013; 61:14.14.1-14.14.24.
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    ABSTRACT: The knocksideways system inactivates proteins by using a small molecule to trap them onto mitochondria. It is typically ∼3 to 4 orders of magnitude faster than a knockdown. To get the best results out of a knocksideways, five parameters need to be optimized: the bait, the prey, the small molecule, the cell or organism, and the assay. Curr. Protoc. Cell Biol. 61:15.20.1-15.20.7. © 2013 by John Wiley & Sons, Inc.
    Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] 01/2013; 61:15.20.1-7.
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    ABSTRACT: Controlled proliferation of cardiomyocytes remains a major limitation in cell biology and one of the main underlying hurdles for true modern regenerative medicine. Here, a technique is described for robust expansion of early fetal-derived mouse ventricular cardiomyocytes on a platform usable for high-throughput molecular screening, tissue engineering and, potentially, in vivo translational experiments. This method provides a small-molecule approach to control proliferation or differentiation of early beating cardiomyocytes through modulation of the Wnt/β-catenin signaling pathway. Moreover, isolation and expansion of fetal cardiomyocytes takes less than 3 weeks, yields a relatively pure (∼70%) functional myogenic population, and is highly reproducible. Curr. Protoc. Cell Biol. 61:23.9.1-23.9.10. © 2013 by John Wiley & Sons, Inc.
    Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] 01/2013; 61:23.9.1-23.9.10.

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