Current Protocols in Cell Biology最新文献

G-quadruplexes (G4s) are higher-order nucleic acid structures that fold from guanine (G)-rich DNA and RNA strands. This field of research gains traction as a major chemical biology area since it aims at uncovering many key cellular mechanisms in which quadruplexes are involved. The wealth of knowledge acquired over the past three decades strongly supports pivotal roles of G4 in the regulation of gene expression at both transcriptional (DNA quadruplexes) and translational levels (RNA quadruplexes). Recent biochemical discoveries uncovered myriad of additional G4 actions: from chromosomal stability to the firing of replication origins, from telomere homeostasis to functional dysregulations underlying genetic diseases (including cancers and neurodegeneration). Here, we listed a repertoire of protocols that we have developed over the past years to visualize quadruplexes in cells. These achievements were made possible thanks to the discovery of a novel family of versatile quadruplex-selective fluorophores, the twice-as-smart quadruplex ligands named TASQ (for template-assembled synthetic G-quartet). The versatility of this probe allows for multiple imaging techniques in both fixed and live cells, including the use of the multiphoton microscopy, confocal microscopy, and real-time fluorescent image collection. © 2017 by John Wiley & Sons, Inc.

Nuclear envelope transmembrane proteins (NETs) are synthesized on the endoplasmic reticulum and then transported from the outer nuclear membrane (ONM) to the inner nuclear membrane (INM) in eukaryotic cells. The abnormal distribution of NETs has been associated with many human diseases. However, quantitative determination of the spatial distribution and translocation dynamics of NETs on the ONM and INM is still very limited in currently existing approaches. Here we demonstrate a single-point single-molecule fluorescence recovery after photobleaching (FRAP) microscopy technique that enables quick determination of distribution and translocation rates for NETs in vivo. © 2017 by John Wiley & Sons, Inc.

Aberrant protein glycosylation is a hallmark of cancer, infectious diseases, and autoimmune or neurodegenerative disorders. Unlocking the potential of glycans as disease markers will require rapid and unbiased glycoproteomics methods for glycan biomarker discovery. The present method is a facile and rapid protocol for qualitative analysis of protein glycosylation in complex biological mixtures. While traditional lectin arrays only provide an average signal for the glycans in the mixture, which is usually dominated by the most abundant proteins, our method provides individual lectin binding profiles for all proteins separated in the gel electrophoresis step. Proteins do not have to be excised from the gel for subsequent analysis via the lectin array but are transferred by contact diffusion from the gel to a glass slide presenting multiple copies of printed lectin arrays. Fluorescently marked glycoproteins are trapped by the printed lectins via specific carbohydrate-lectin interactions and after a washing step their binding profile with up to 20 lectin probes is analyzed with a fluorescent scanner. The method produces the equivalent of 20 lectin blots in a single experiment, giving detailed insight into the binding epitopes present in the fractionated proteins. © 2017 by John Wiley & Sons, Inc.

Subcellular fractionation techniques are essential for cell biology and drug development studies. The emergence of organelle-targeted nanoparticle (NP) platforms necessitates the isolation of target organelles to study drug delivery and activity. Mitochondria-targeted NPs have attracted the attention of researchers around the globe, since mitochondrial dysfunctions can cause a wide range of diseases. Conventional mitochondria isolation methods involve high-speed centrifugation. The problem with high-speed centrifugation-based isolation of NP-loaded mitochondria is that NPs can pellet even if they are not bound to mitochondria. We report development of a mitochondria-targeted paramagnetic iron oxide nanoparticle, Mito-magneto, that enables isolation of mitochondria under the influence of a magnetic field. Isolation of mitochondria using Mito-magneto eliminates artifacts typically associated with centrifugation-based isolation of NP-loaded mitochondria, thus producing intact, pure, and respiration-active mitochondria. © 2017 by John Wiley & Sons, Inc.

Cell migration is essential for many biological processes including development, wound healing, and metastasis. However, studying cell migration often requires the time-consuming and labor-intensive task of manually tracking cells. To accelerate the task of obtaining coordinate positions of migrating cells, we have developed a graphical user interface (GUI) capable of automating the tracking of fluorescently labeled nuclei. This GUI provides an intuitive user interface that makes automated tracking accessible to researchers with no image-processing experience or familiarity with particle-tracking approaches. Using this GUI, users can interactively determine a minimum of four parameters to identify fluorescently labeled cells and automate acquisition of cell trajectories. Additional features allow for batch processing of numerous time-lapse images, curation of unwanted tracks, and subsequent statistical analysis of tracked cells. Statistical outputs allow users to evaluate migratory phenotypes, including cell speed, distance, displacement, and persistence, as well as measures of directional movement, such as forward migration index (FMI) and angular displacement. © 2017 by John Wiley & Sons, Inc.

Reconstitution of cellular organelles in vitro offers the possibility to perform quantitative and qualitative experiments in a controlled environment that cannot be done with the same accuracy in living cells. Following a previous report, the subsequent list of protocols describes how to reconstitute and quantify a tubular ER network in vitro based on purified microsomes from culture cells and cytosol from Xenopus laevis egg extracts. Biological material preparation and reconstitution assays require mostly basic laboratory instrumentation and chemicals, and can be executed without any specific training, making them appealing to a wide range of laboratories. Moreover, to promote conditions that are markedly more reflective of in vivo environments, this method describes for the first time in the literature, the purification of microsomes from HeLa cells in some detail. Basic Protocol 1 in this article describes the reconstitution process on different substrates including the associated fluorescence imaging process. Purification of ER microsomes and cytosol, both of which are needed for this approach, are described in detail in Support Protocols 1 and 2, respectively. Coating of surfaces with polyacrylamide gels is described in Support Protocol 3. Basic Protocol 2 outlines how to segment and skeletonize fluorescence images of ER networks, and how to quantify segment lengths between the network's branching points. The described quantitative evaluation provides a meaningful approach to analyze the topology and geometry of organelle structures. © 2017 by John Wiley & Sons, Inc.

Adipose stem cells (ASC) represent a promising therapeutic approach for neurodegenerative diseases. Most biological effects of ASC are probably mediated by extracellular vesicles, such as exosomes, which influence the surrounding cells. Current development of exosome therapies requires efficient and noninvasive methods to localize, monitor, and track the exosomes. Among imaging methods used for this purpose, magnetic resonance imaging (MRI) has advantages: high spatial resolution, rapid in vivo acquisition, and radiation-free operation. To be detectable with MRI, exosomes must be labeled with MR contrast agents, such as ultra-small superparamagnetic iron oxide nanoparticles (USPIO). Here, we set up an innovative approach for exosome labeling that preserves their morphology and physiological characteristics. We show that by labeling ASC with USPIO before extraction of nanovesicles, the isolated exosomes retain nanoparticles and can be visualized by MRI. The current work aims at validating this novel USPIO-based exosome labeling method by monitoring the efficiency of the labeling with MRI both in ASC and in exosomes. © 2017 by John Wiley & Sons, Inc.

While a detailed understanding of chromatin dynamics is needed to explain how higher-order chromatin organization influences nuclear function, the molecular principles that regulate chromatin mobility in mammalian nuclei remain largely unknown. Here we describe experimental tools to follow chromatin dynamics by labeling DNA during S phase. Using these methods, we have found that foci labeled during early and mid/late S phase have significantly different dynamic behavior. Spatially constrained heterochromatic foci restrict long-range transformations of the chromosome territory (CT) structure while providing a structural framework on which highly mobile euchromatic foci undergo positional oscillations that drive local changes in the chromosome shape. Despite often dramatic mobility, we have demonstrated a preservation of structural integrity which ensures that DNA from neighboring CTs is not able to mix freely within the same nuclear space. Finally, other potential applications of the presented protocols are discussed. © 2017 by John Wiley & Sons, Inc.

Microcontact printing (μCPr) is one of the most popular techniques used for cell micropatterning. In conventional μCPr, a polydimethylsiloxane (PDMS) stamp with microfeatures is used to adsorb extracellular matrix (ECM) proteins onto the featured surface and transfer them onto particular areas of a cell culture substrate. However, some types of functional proteins other than ECM have been reported to denature upon direct adsorption to hydrophobic PDMS. Here we describe a detailed protocol of an alternative technique––microcontact peeling (μCPe)––that allows for cell micropatterning while circumventing the step of adsorbing proteins to bare PDMS. This technique employs microfeatured materials with a relatively high surface energy such as copper, instead of using a microfeatured PDMS stamp, to peel off a cell-adhesive layer present on the surface of substrates. Consequently, cell-nonadhesive substrates are exposed at the specific surface that undergoes the physical contact with the microfeatured material. Thus, although μCPe and μCPr are apparently similar, the former does not comprise a process of transferring biomolecules through hydrophobic PDMS. © 2017 by John Wiley & Sons, Inc.

The cell microenvironment plays an important role in many biological processes, including development and disease progression. Key to this is the extracellular matrix (ECM), a complex biopolymer network serving as the primary insoluble signaling network for physical, chemical, and mechanical cues. In vitro, the ability to engineer the ECM at the micro- and nanoscales is a critical tool to systematically interrogate the influence of ECM properties on cellular responses. Specifically, both topographical and chemical surface patterning has been shown to direct cell alignment and tissue architecture on biomaterial surfaces, however, it has proven challenging to independently control these surface properties. This protocol describes a method termed Patterning on Topography (PoT) to engineer 2D nanopatterns of ECM proteins onto topographically complex substrates, which enables independent control of physical and chemical surface properties. Applications include interrogation of fundamental cell-surface interactions and engineering interfaces that can direct cell and/or tissue function. © 2017 by John Wiley & Sons, Inc.