Browsing by Subject "Staining and Labeling"

Now showing 1 - 5 of 5
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    A chemical method for labeling lysine methyltransferase substrates.
    (Chembiochem : a European journal of chemical biology, 2011-01) Binda, Olivier; Boyce, Michael; Rush, Jason S; Palaniappan, Krishnan K; Bertozzi, Carolyn R; Gozani, Or
    Several protein lysine methyltransferases (PKMTs) modify histones to regulate chromatin-dependent cellular processes, such as transcription, DNA replication and DNA damage repair. PKMTs are likely to have many additional substrates in addition to histones, but relatively few nonhistone substrates have been characterized, and the substrate specificity for many PKMTs has yet to be defined. Thus, new unbiased methods are needed to find PKMT substrates. Here, we describe a chemical biology approach for unbiased, proteome-wide identification of novel PKMT substrates. Our strategy makes use of an alkyne-bearing S-adenosylmethionine (SAM) analogue, which is accepted by the PKMT, SETDB1, as a cofactor, resulting in the enzymatic attachment of a terminal alkyne to its substrate. Such labeled proteins can then be treated with azide-functionalized probes to ligate affinity handles or fluorophores to the PKMT substrates. As a proof-of-concept, we have used SETDB1 to transfer the alkyne moiety from the SAM analogue onto a recombinant histone H3 substrate. We anticipate that this chemical method will find broad use in epigenetics to enable unbiased searches for new PKMT substrates by using recombinant enzymes and unnatural SAM cofactors to label and purify many substrates simultaneously from complex organelle or cell extracts.
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    Biomimetic nanoparticles with enhanced affinity towards activated endothelium as versatile tools for theranostic drug delivery.
    (Theranostics, 2018-01-05) Martinez, Jonathan O; Molinaro, Roberto; Hartman, Kelly A; Boada, Christian; Sukhovershin, Roman; De Rosa, Enrica; Kirui, Dickson; Zhang, Shanrong; Evangelopoulos, Michael; Carter, Angela M; Bibb, James A; Cooke, John P; Tasciotti, Ennio
    Activation of the vascular endothelium is characterized by increased expression of vascular adhesion molecules and chemokines. This activation occurs early in the progression of several diseases and triggers the recruitment of leukocytes. Inspired by the tropism of leukocytes, we investigated leukocyte-based biomimetic nanoparticles (i.e., leukosomes) as a novel theranostic platform for inflammatory diseases. Methods: Leukosomes were assembled by combining phospholipids and membrane proteins from leukocytes. For imaging applications, phospholipids modified with rhodamine and gadolinium were used. Leukosomes incubated with antibodies blocking lymphocyte function-associated antigen 1 (LFA-1) and CD45 were administered to explore their roles in targeting inflammation. In addition, relaxometric assessment of NPs was evaluated. Results: Liposomes and leukosomes were both spherical in shape with sizes ranging from 140-170 nm. Both NPs successfully integrated 8 and 13 µg of rhodamine and gadolinium, respectively, and demonstrated less than 4% variation in physicochemical features. Leukosomes demonstrated a 16-fold increase in breast tumor accumulation relative to liposomes. Furthermore, quantification of leukosomes in tumor vessels demonstrated a 4.5-fold increase in vessel lumens and a 14-fold increase in vessel walls. Investigating the targeting mechanism of action revealed that blockage of LFA-1 on leukosomes resulted in a 95% decrease in tumor accumulation. Whereas blockage of CD45 yielded a 60% decrease in targeting and significant increases in liver and spleen accumulation. In addition, when administered in mice with atherosclerotic plaques, leukosomes exhibited a 4-fold increase in the targeting of inflammatory vascular lesions. Lastly, relaxometric assessment of NPs demonstrated that the incorporation of membrane proteins into leukosomes did not impact the r1 and r2 relaxivities of the NPs, demonstrating 6 and 30 mM-1s-1, respectively. Conclusion: Our study demonstrates the ability of leukosomes to target activated vasculature and exhibit superior accumulation in tumors and vascular lesions. The versatility of the phospholipid backbone within leukosomes permits the incorporation of various contrast agents. Furthermore, leukosomes can potentially be loaded with therapeutics possessing diverse physical properties and thus warrant further investigation toward the development of powerful theranostic agents.
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    Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners.
    (Proceedings of the National Academy of Sciences of the United States of America, 2012-03-12) Yu, Seok-Ho; Boyce, Michael; Wands, Amberlyn M; Bond, Michelle R; Bertozzi, Carolyn R; Kohler, Jennifer J
    O-linked β-N-acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification found on hundreds of nuclear and cytoplasmic proteins in higher eukaryotes. Despite its ubiquity and essentiality in mammals, functional roles for the O-GlcNAc modification remain poorly defined. Here we develop a combined genetic and chemical approach that enables introduction of the diazirine photocrosslinker onto the O-GlcNAc modification in cells. We engineered mammalian cells to produce diazirine-modified O-GlcNAc by expressing a mutant form of UDP-GlcNAc pyrophosphorylase and subsequently culturing these cells with a cell-permeable, diazirine-modified form of GlcNAc-1-phosphate. Irradiation of cells with UV light activated the crosslinker, resulting in formation of covalent bonds between O-GlcNAc-modified proteins and neighboring molecules, which could be identified by mass spectrometry. We used this method to identify interaction partners for the O-GlcNAc-modified FG-repeat nucleoporins. We observed crosslinking between FG-repeat nucleoporins and nuclear transport factors, suggesting that O-GlcNAc residues are intimately associated with essential recognition events in nuclear transport. Further, we propose that the method reported here could find widespread use in investigating the functional consequences of O-GlcNAcylation.
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    Robust approaches to quantitative ratiometric FRET imaging of CFP/YFP fluorophores under confocal microscopy.
    (J Microsc, 2009-01) Tadross, MR; Park, SA; Veeramani, B; Yue, DT
    Ratiometric quantification of CFP/YFP FRET enables live-cell time-series detection of molecular interactions, without the need for acceptor photobleaching or specialized equipment for determining fluorescence lifetime. Although popular in widefield applications, its implementation on a confocal microscope, which would enable sub-cellular resolution, has met with limited success. Here, we characterize sources of optical variability (unique to the confocal context) that diminish the accuracy and reproducibility of ratiometric FRET determination and devise practical remedies. Remarkably, we find that the most popular configuration, which pairs an oil objective with a small pinhole aperture, results in intractable variability that could not be adequately corrected through any calibration procedure. By quantitatively comparing several imaging configurations and calibration procedures, we find that significant improvements can be achieved by combining a water objective and increased pinhole aperture with a uniform-dye calibration procedure. The combination of these methods permitted remarkably consistent quantification of sub-cellular FRET in live cells. Notably, this methodology can be readily implemented on a standard confocal instrument, and the dye calibration procedure yields a time savings over traditional live-cell calibration methods. In all, identification of key technical challenges and practical compensating solutions promise robust sub-cellular ratiometric FRET imaging under confocal microscopy.
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    Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling.
    (J Vis Exp, 2015-09-14) Arriaga, Gustavo; Macopson, Joshua J; Jarvis, Erich D
    Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This approach has been most extensively used in the brain by utilizing the swine pathogen pseudorabies virus (PRV)(1). PRV does not infect great apes, including humans, so it is most commonly used in studies on small mammals, especially rodents. The pseudorabies strain PRV152 expresses the enhanced green fluorescent protein (eGFP) reporter gene and only crosses functional synapses retrogradely through the hierarchical sequence of synaptic connections away from the infection site(2,3). Other PRV strains have distinct microbiological properties and may be transported in both directions (PRV-Becker and PRV-Kaplan)(4,5). This protocol will deal exclusively with PRV152. By delivering the virus at a peripheral site, such as muscle, it is possible to limit the entry of the virus into the brain through a specific set of neurons. The resulting pattern of eGFP signal throughout the brain then resolves the neurons that are connected to the initially infected cells. As the distributed nature of transsynaptic tracing with pseudorabies virus makes interpreting specific connections within an identified network difficult, we present a sensitive and reliable method employing biotinylated dextran amines (BDA) and cholera toxin subunit b (CTb) for confirming the connections between cells identified using PRV152. Immunochemical detection of BDA and CTb with peroxidase and DAB (3, 3'-diaminobenzidine) was chosen because they are effective at revealing cellular processes including distal dendrites(6-11).