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Current Protocols in Cell Biology

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.
2016: Current Protocols in Cell Biology
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...
2016: Current Protocols in Cell Biology
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...
2016: Current Protocols in Cell Biology
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...
2016: Current Protocols in Cell Biology
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...
2016: Current Protocols in Cell Biology
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...
2016: Current Protocols in Cell Biology
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...
2016: Current Protocols in Cell Biology
Jiyun Kim, Kandice Tanner
This protocol describes a way to introduce topography to three-dimensional (3D) biomaterials. The self-assembling behavior of magnetic particles can be exploited to form nanoscale to microscale fibers, such that one can dissect the contribution of topography on cell behavior, which is independent of other physical properties of the biomaterial (e.g., stiffness). The magnetic particles are chemically cross-linked with several extracellular matrix (ECM) proteins and then using magnetic force-mediated assembly, one can program aligned nanofibers in a 3D hydrogel...
2016: Current Protocols in Cell Biology
Michael Timaner, Ofrat Beyar-Katz, Yuval Shaked
The tumor microenvironment consists of a variety of cell types. The contribution of each cell type to the tumor is an emerging subject in the field of cancer research. Here, we describe protocols for dissociating tumor tissues and Matrigel plugs into single cells for further analysis by flow cytometry. These protocols can be used for evaluating the cellular component of solid tumors from human or mouse origin or Matrigel plugs implanted in mice. The protocols describe the dissociation of tumor tissue with or without dissociation automatic devices...
2016: Current Protocols in Cell Biology
Vira V Artym
The stroma of invasive tumors becomes enriched in dense fibrillar collagen as a result of the desmoplastic reaction. This desmoplastic collagen exerts profound effects on tumor and normal cells. In view of these findings, it is important to develop novel in vitro cell systems that mimic this desmoplastic extracellular matrix in order to permit cell studies under in vivo-like conditions. This unit provides a protocol and troubleshooting guide for preparation of high-density fibrillar collagen (HDFC) matrices that closely model the desmoplastic collagenous matrix of malignant tumors...
2016: Current Protocols in Cell Biology
Julie G Donaldson
This unit provides a protocol for indirect immunofluorescence, which is a method that provides information about the locations of specific molecules and the structure of the cell. Antibody molecules for a specific target molecule are exposed to the cell or tissue being investigated. The binding of these molecules is detected by incubating the sample with a secondary antibody specific for immunoglobulin molecules and conjugated to a fluorophore. This provides both a visible signal and amplification of the signal and the results are observed with a fluorescence microscope...
December 1, 2015: Current Protocols in Cell Biology
John M Graham
This unit provides both a theoretical and a practical background to all the techniques associated with the application of differential and density gradient centrifugation for the analysis of subcellular membranes. The density gradient information focuses on the use of the modern gradient solute iodixanol, chosen for its ease of use, versatility, and compatibility with biological particles. Its use in both pre-formed discontinuous and continuous gradients and in self-generated gradients is discussed. Considerable emphasis is given to selection of the appropriate centrifuge rotors and tubes and their influence on the methods used for creation, fractionation, and analysis of density gradients...
December 1, 2015: Current Protocols in Cell Biology
Marvin Bentley, Gary Banker
Here we describe a method capable of identifying interactions between candidate trafficking proteins and a defined vesicle population in intact cells. The assay involves the expression of an FKBP12-rapamycin binding domain (FRB)-tagged candidate vesicle-binding protein that can be inducibly linked to an FKBP-tagged molecular motor. If the FRB-tagged candidate protein binds the labeled vesicles, then linking the FRB and FKBP domains recruits motors to the vesicles and causes a predictable, highly distinctive change in vesicle trafficking...
December 1, 2015: Current Protocols in Cell Biology
Keiko Mihara, Tomofumi Nakayama, Hisato Saitoh
Human myeloid HL-60 cells are usually cultured in suspension in medium containing 5% to 10% fetal bovine serum (FBS) and thus are often difficult to adhere to a coverslip. In this unit, we describe how removal of FBS from the culture medium facilitates adhesion of HL-60 cells to coverslips. Importantly, HL-60 cells that adhere to the coverslips immersed in FBS-free medium can be immobilized in situ by conventional chemical fixatives and thus permeabilized for probing cellular structures using specific dyes and/or reagents, followed by microscopic observation...
2015: Current Protocols in Cell Biology
Chad D Williamson, Daniel S Wong, Petros Bozidis, Aiping Zhang, Anamaris M Colberg-Poley
Increasingly mechanistic virology studies require dependable and sensitive methods for isolating purified organelles containing functional cellular sub-domains. The mitochondrial network is, in part, closely apposed to the endoplasmic reticulum (ER). The mitochondria-associated membrane (MAM) fraction provides direct physical contact between the ER and mitochondria. Characterization of the dual localization and trafficking of human cytomegalovirus (HCMV) UL37 proteins required establishing protocols in which the ER and mitochondria could be reliably separated...
2015: Current Protocols in Cell Biology
Travis J Crites, Michael Maddox, Kartika Padhan, James Muller, Calvin Eigsti, Rajat Varma
Glass-supported lipid bilayers presenting freely diffusing proteins have served as a powerful tool for studying cell-cell interfaces, in particular, T cell-antigen presenting cell (APC) interactions, using optical microscopy. Here we expand upon existing protocols and describe the preparation of liposomes by an extrusion method, and describe how this system can be used to study immune synapse formation by Jurkat cells. We also present a method for forming such lipid bilayers on silica beads for the study of signaling responses by population methods, such as western blotting, flow cytometry, and gene-expression analysis...
2015: Current Protocols in Cell Biology
Molly McQuilken, Shalin B Mehta, Amitabh Verma, Grant Harris, Rudolf Oldenbourg, Amy S Gladfelter
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...
2015: Current Protocols in Cell Biology
Yanjie Li, Robert J Tomko, Mark Hochstrasser
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...
2015: Current Protocols in Cell Biology
Sang-Soo Kim, Kathleen F Pirollo, Esther H Chang
In many human cancers including malignant glioblastoma multiforme (GBM), cancer stem cells (CSCs) are thought to be responsible for tumor initiation, metastasis and resistance to conventional anti-cancer therapies. Therefore, a CSC-targeted drug delivery strategy to eliminate CSCs is a desirable approach for developing a more effective therapeutic. Moreover, isolated CSCs will provide an invaluable tool for studying the underlying cellular mechanisms of tumor development and provide insight into therapeutic options for successful eradication of CSCs...
2015: Current Protocols in Cell Biology
Jaume Taura, Víctor Fernández-Dueñas, Francisco Ciruela
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...
2015: Current Protocols in Cell Biology
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