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

Journal description

Current impact factor: 0.00

Impact Factor Rankings

Additional details

5-year impact 0.00
Cited half-life 0.00
Immediacy index 0.00
Eigenfactor 0.00
Article influence 0.00
ISSN 1934-2616

Publications in this journal

  • [Show abstract] [Hide abstract]
    ABSTRACT: G protein-coupled receptors (GPCRs) constitute the largest family of plasma membrane receptors, thus representing the more investigated drug targets in the design of new therapeutic strategies. The existence of receptor-receptor interactions has revolutionized the field, since GPCR oligomerization might confer new intervention opportunities in pharmacotherapy. However, demonstrating the existence of such receptor-receptor interactions in native tissue has been a bottleneck in GPCR pharmacology. Here, we discuss an experimental approach, the proximity ligation in situ assay (P-LISA), which provides enough sensitivity to evaluate a receptor's close proximity within a named GPCR oligomer. Indeed, we provide a detailed step-by-step protocol for P-LISA experiments visualizing receptor-receptor interactions in brain slices. Additionally, we provide instructions for slide observation, data acquisition and quantification. Finally, we also discuss these critical aspects determining the success of the technique, namely the fixation process and the validation of the primary antibodies used. Overall, the P-LISA is a powerful and straightforward technique to visualize receptor-receptor interactions when performed under optimal conditions. © 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; 67:17.17.1-17.17.16. DOI:10.1002/0471143030.cb1717s67
  • [Show abstract] [Hide abstract]
    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
  • [Show abstract] [Hide abstract]
    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
  • [Show abstract] [Hide abstract]
    ABSTRACT: In eukaryotes, damaged or unneeded proteins are typically degraded by the ubiquitin-proteasome system. In this system, the protein substrate is often first covalently modified with a chain of ubiquitin polypeptides. This chain serves as a signal for delivery to the 26S proteasome, a 2.5-MDa, ATP-dependent multisubunit protease complex. The proteasome consists of a barrel-shaped 20S core particle (CP) that is capped on one or both of its ends by a 19S regulatory particle (RP). The RP is responsible for recognizing the substrate, unfolding it, and translocating it into the CP for destruction. Here we describe simple, one-step purifications scheme for isolating the 26S proteasome and its 19S RP and 20S CP subcomplexes from the yeast Saccharomyces cerevisiae, as well as assays for measuring ubiquitin-dependent and ubiquitin-independent proteolytic activity in vitro. © 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; 67:3.43.1-3.43.20. DOI:10.1002/0471143030.cb0343s67
  • [Show abstract] [Hide abstract]
    ABSTRACT: The measurement of not only the location but also the organization of molecules in live cells is crucial to understanding diverse biological processes. Polarized light microscopy provides a nondestructive means to evaluate order within subcellular domains. When combined with fluorescence microscopy and GFP-tagged proteins, the approach can reveal organization within specific populations of molecules. This unit describes a protocol for measuring the architectural dynamics of cytoskeletal components using polarized fluorescence microscopy and OpenPolScope open-access software (http://www.openpolscope.org). The protocol describes installation of linear polarizers or a liquid crystal (LC) universal compensator, calibration of the system, polarized fluorescence imaging, and analysis. The use of OpenPolScope software and hardware allows for reliable, user-friendly image acquisition to measure and analyze polarized fluorescence. © 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; 67:4.29.1-4.29.13. DOI:10.1002/0471143030.cb0429s67
  • [Show abstract] [Hide abstract]
    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
  • [Show abstract] [Hide abstract]
    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
  • [Show abstract] [Hide abstract]
    ABSTRACT: The movement of mature VLDL particles from the TGN to the plasma membrane (PM) is a complex physiological process that plays a critical role in hepatic lipid homeostasis. However, the molecular mechanisms regulating these intracellular transport events had not been studied until recently because of the lack of appropriate molecular assays and techniques. This unit provides a detailed description of cell-free approaches and techniques to study the TGN-to-PM transport of the mature VLDL at the molecular level. A major emphasis is placed on the preparation and purification of sub-cellular organelles because the success of in vitro assays for the vesicle formation and fusion depends on the quality of the isolated TGN, hepatic PM and hepatic cytosol. A number of critical factors that control the formation of mature VLDL-containing vesicle, the PG-VTV, from the TGN and their subsequent targeting to and fusion with the hepatic PM have been discussed. © 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; 67:11.21.1-11.21.17. DOI:10.1002/0471143030.cb1121s67
  • [Show abstract] [Hide abstract]
    ABSTRACT: Tunneling nanotubes (TNTs) are thin membranous channels providing direct cytoplasmic connection between remote cells. They are commonly observed in different cell cultures and increasing evidence supports their role in intercellular communication and pathogen transfer. However, the study of TNTs presents several pitfalls (e.g., difficulty in preserving such delicate structures, possible confusion with other protrusions, structural and functional heterogeneity, etc.) and therefore requires thoroughly designed approaches. The methods described in this unit represent a guideline for the characterization of TNTs (or TNT-like structures) in cell culture. Specifically, optimized protocols to (1) identify TNTs and the cytoskeletal elements present inside them; (2) evaluate TNT frequency in cell culture; (3) unambiguously distinguish them from other cellular connections or protrusions; and (4) monitor their formation in living cells are provided. Finally, this unit describes how to assess TNT-mediated cell-to-cell transfer of cellular components, which is a fundamental criterion for identifying functional TNTs. © 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; 67:12.10.1-12.10.21. DOI:10.1002/0471143030.cb1210s67
  • [Show abstract] [Hide abstract]
    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.] 12/2014; 65:10.19.1-10.19.17. DOI:10.1002/0471143030.cb1019s65
  • [Show abstract] [Hide abstract]
    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
  • [Show abstract] [Hide abstract]
    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
  • [Show abstract] [Hide abstract]
    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
  • [Show abstract] [Hide abstract]
    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
  • [Show abstract] [Hide abstract]
    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