Characterization of endoplasmic reticulum by co-localization of BiP and dicarbocyanine dyes

1992 ◽  
Vol 101 (2) ◽  
pp. 315-322 ◽  
Author(s):  
M. Terasaki ◽  
T.S. Reese
Keyword(s):  
Endoplasmic Reticulum ◽  
Cultured Cells ◽  
Protein A ◽  
Epithelial Cell Line ◽  
Cultured Fibroblasts ◽  
Whole Cells ◽  
Membrane Compartments ◽  
Immunoglobulin Binding Protein ◽  
Immunoglobulin Binding ◽  
Peripheral Regions

The original concept of endoplasmic reticulum derived from the observation of a reticular network in cultured fibroblasts by electron microscopy of whole cells. It was previously reported that the fluorescent dye, DiOC6(3), stains a similar network as well as mitochondria and other organelles in living cells. Here, we investigate the significance of the structures labeled by DiO6(3) in CV-1 cells, a monkey epithelial cell line. First, we show that the network stained in living CV-1 cells is preserved by glutaraldehyde fixation and then we co-label it with an antibody against BiP (immunoglobulin binding protein), a protein commonly accepted to be present in the endoplasmic reticulum. Anti-BiP labeled the same network as that labeled by DiOC6(3), so this network now is identified as being part of the endoplasmic reticulum. DiOC6(3) labels many other membrane compartments in addition to the endoplasmic reticulum. This, along with its lipophilic properties, suggests that DiOC6(3) stains all intracellular membranes. However, the extensive reticular network in the thin peripheral regions of cultured cells is easily distinguished from these other membranes. Thus, staining by DiOC6(3) is a useful method for localizing the endoplasmic reticulum, particularly in thin peripheral regions of cultured cells.

scholarly journals Small molecule control of neurotransmitter sulfonation

2020 ◽  
pp. jbc.RA120.015177
Author(s):  
Ian Cook ◽  
Mary Cacace ◽  
Ting Wang ◽  
Kristie Darrah ◽  
Alexander Deiters ◽  
...  
Keyword(s):  
Active Site ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Specific Binding ◽  
Protein A ◽  
Binding Pocket ◽  
Human Mammary Epithelial Cell ◽  
Primary Objective ◽  
Epithelial Cell Line

Controlling unmodified serotonin levels in brain synapses is a primary objective when treating major depressive disorder — a disease that afflicts ~20% of the world’s population. Roughly 60% of patients respond poorly to first-line treatments and thus new therapeutic strategies are sought. Toward this end, we have constructed isoform-specific inhibitors of the human cytosolic sulfotransferase 1A3 (SULT1A3) — the isoform responsible for sulfonating ~80% of the serotonin in extracellular brain fluid. The inhibitor design includes a core ring structure, which anchors the inhibitor into a SULT1A3-specific binding pocket located outside the active site, and a sidechain crafted to act as a latch to inhibit turnover by fastening down the SULT1A3 active-site cap. The inhibitors are allosteric, they bind with nanomolar affinity and are highly specific for the 1A3 isoform. The cap-stabilizing effects of the latch can be accurately calculated and are predicted to extend throughout the cap and into the surrounding protein. A free energy correlation demonstrates that the percent inhibition at saturating inhibitor varies linearly with cap stabilization — the correlation is linear because the rate-limiting step of the catalytic cycle, nucleotide release, scales linearly with the fraction of enzyme in the cap-open form. Inhibitor efficacy in cultured cells was studied using a human mammary epithelial cell line that expresses SULT1A3 at levels comparable to those found in neurons. The inhibitors perform similarly in ex vivo and in vitro studies; consequently, SULT1A3 turnover can now be potently suppressed in an isoform-specific manner in human cells.

scholarly journals Metabolism and intracellular localization of a fluorescently labeled intermediate in lipid biosynthesis within cultured fibroblasts.

1981 ◽  
Vol 91 (3) ◽  
pp. 872-877 ◽  
Author(s):  
R E Pagano ◽  
K J Longmuir ◽  
O C Martin ◽  
D K Struck
Keyword(s):  
Endoplasmic Reticulum ◽  
Phosphatidic Acid ◽  
Cultured Cells ◽  
Large Fraction ◽  
Intracellular Localization ◽  
Chinese Hamster ◽  
Cultured Fibroblasts ◽  
Acid Analogue ◽  
Unique Method

In this paper we report on the uptake and distribution of an exogenously supplied fluorescent phosphatidic acid analogue by Chinese hamster fibroblasts. Under appropriate in vitro incubation conditions, 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidic acid was rapidly and preferentially transferred from phospholipid vesicles to cells at 2 degrees C. However, unlike similar fluorescent derivatives of phosphatidylcholine and phosphatidylethanolamine that remain restricted to the plasma membrane under such incubation conditions (Struck, D. K., and R. E. Pagano. 1080. J. Biol. Chem. 255:5405--5410), most of the phosphatidic acid-derived fluorescence was localized at the nuclear membrane, endoplasmic reticulum, and mitochondria. This was shown by labeling cells with rhodamine-containing probes specific for mitochondria or endoplasmic reticulum, and comparing the patterns of intracellular NBD and rhodamine fluorescence. Extraction and analysis of the fluorescent lipids associated with the cells after treatment with vesicles at 2 degrees or 37 degrees C revealed that a large fraction of the fluorescent phosphatidic acid was converted to fluorescent diglyceride, phosphatidylcholine, and triglyceride. Our findings suggest that fluorescent phosphatidic acid may be useful in correlating biochemical studies of lipid metabolism in cultured cells and studies of the Intracellular localization of the metabolites by fluorescence microscopy. In addition, this compound provides a unique method for visualizing the endoplasmic reticulum in living cells.

scholarly journals Protein A Is Released into the Staphylococcus aureus Culture Supernatant with an Unprocessed Sorting Signal

2015 ◽  
Vol 83 (4) ◽  
pp. 1598-1609 ◽  
Author(s):  
Dara P. O'Halloran ◽  
Kieran Wynne ◽  
Joan A. Geoghegan
Keyword(s):  
Staphylococcus Aureus ◽  
Protein A ◽  
Covalent Attachment ◽  
Sortase A ◽  
Reporter Protein ◽  
Extracellular Medium ◽  
Content Type ◽  
Sorting Signal ◽  
Immunoglobulin Binding Protein ◽  
Immunoglobulin Binding

The immunoglobulin binding protein A (SpA) ofStaphylococcus aureusis synthesized as a precursor with a C-terminal sorting signal. The sortase A enzyme mediates covalent attachment to peptidoglycan so that SpA is displayed on the surface of the bacterium. Protein A is also found in the extracellular medium, but the processes involved in its release are not fully understood. Here, we show that a portion of SpA is released into the supernatant with an intact sorting signal, indicating that it has not been processed by sortase A. Release of SpA was reduced when the native sorting signal of SpA was replaced with the corresponding region of another sortase-anchored protein (SdrE). Similarly, a reporter protein fused to the sorting signal of SpA was released to a greater extent than the same polypeptide fused to the SdrE sorting signal. Released SpA protected bacteria from killing in human blood, indicating that it contributes to immune evasion.

scholarly journals Fractionation of Golgi, Endoplasmic Reticulum, and Plasma Membrane from Cultured Cells in a Preformed Continuous Iodixanol Gradient

2002 ◽  
Vol 2 ◽  
pp. 1435-1439 ◽  
Author(s):  
John Graham
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Cultured Cells ◽  
Density Range ◽  
Cultured Cell ◽  
Golgi Membranes ◽  
Cell Homogenate ◽  
Membrane Compartments

A continuous iodixanol gradient within the range 0-30% (w/v) iodixanol can resolve the major membrane compartments of the endoplasmic reticulum, Golgi membranes, and plasma membrane from a postnuclear supernatant prepared from a cultured cell homogenate. The precise density range of the gradient and the centrifugation conditions (100,000-200,000gfor 2-16 h) vary with the type of cell and the requirements of the separation. The strategy is widely used to study the processing of proteins within cells.

scholarly journals Induction of the FK506-binding protein, FKBP13, under conditions which misfold proteins in the endoplasmic reticulum

1994 ◽  
Vol 303 (3) ◽  
pp. 705-708 ◽  
Author(s):  
K T Bush ◽  
B A Hendrickson ◽  
S K Nigam
Keyword(s):  
Endoplasmic Reticulum ◽  
Mdck Cells ◽  
Binding Protein ◽  
Significant Similarity ◽  
Fk506 Binding Protein ◽  
Unfolded Protein ◽  
Immunoglobulin Binding Protein ◽  
The Unfolded Protein Response ◽  
Protein Response ◽  
Immunoglobulin Binding

In order to determine whether the endoplasmic reticulum (ER) luminal FK506-binding protein, FKBP13, shares properties of ER molecular chaperones, MDCK cells were treated with either tunicamycin or Ca2+ ionophores. By Northern-blot analysis, tunicamycin resulted in a 2-fold rise in FKBP13 mRNA, whereas ionophores (A23187 and ionomycin) caused a more impressive rise in FKBP13 mRNA (up to 5-fold with ionomycin). Actinomycin D chase experiments in ionomycin-treated cells revealed no change in the half-life of FKBP13 mRNA, indicating that the increase in FKBP13 mRNA observed was not due to greater message stability. Moreover, sequencing of the 5′ flanking region of the gene for murine FKBP13 revealed significant similarity to similar regions in human BiP (immunoglobulin-binding protein) and the human glucose-regulated protein grp94, including a 37 bp sequence in FKBP13 with approximately 50% identity with the unfolded protein response element of the BiP gene. Together, these data suggest a role for FKBP13 in ER protein folding.

scholarly journals Equilibrium and pre-equilibrium fluorescence spectroscopic studies of the binding of a single-immunoglobulin-binding domain derived from protein G to the Fc fragment from human IgG1

1995 ◽  
Vol 310 (1) ◽  
pp. 177-184 ◽  
Author(s):  
K N Walker ◽  
S P Bottomley ◽  
A G Popplewell ◽  
B J Sutton ◽  
M G Gore
Keyword(s):  
Protein A ◽  
Spectroscopic Studies ◽  
Binding Domain ◽  
Protein G ◽  
Binding Interaction ◽  
Fc Fragment ◽  
Igg Binding ◽  
Immunoglobulin Binding Protein ◽  
Human Igg1 ◽  
Immunoglobulin Binding

A single-immunoglobulin-binding protein based upon the C2 domain of Protein G from Streptococcus has been shown to bind tightly to the Fc fragment of IgG1. The binding interaction results in a decrease in the fluorescence intensity from the sole Trp residue (Trp-48) in this domain. This spectral change has been used to monitor the binding interactions between the two proteins using equilibrium and pre-equilibrium fluorescence spectroscopy. Comparison of the data from the two techniques suggests that a conformational change occurs after the initial formation of the complex. Mutagenesis studies have shown that the Trp residue is important for binding and that replacement by a Phe residue is important for binding and that replacement by a Phe residue leads to a 300-fold decrease in the affinity for Fc gamma 1. Determination of the rate constants kon and koff at different values of pH between 4.0 and 9.0 suggest that variations in Kd are mediated predominantly by changes in kon. Competition experiments between SpG1 and a single-IgG-binding domain from Protein A from Staphylococcus aureus have been used to determine the affinity of the latter for Fc gamma 1.

scholarly journals Changes in the Expression of Biofilm-Associated Surface Proteins inStaphylococcus aureusFood-Environmental Isolates Subjected to Sublethal Concentrations of Disinfectants

2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Lenka Cincarova ◽  
Ondrej Polansky ◽  
Vladimir Babak ◽  
Pavel Kulich ◽  
Petr Kralik
Keyword(s):  
Biofilm Formation ◽  
Protein A ◽  
Surface Proteins ◽  
Bacterial Cells ◽  
Abiotic Surfaces ◽  
Immunoglobulin Binding Protein ◽  
Chloramine T ◽  
Nucleic Acid Stain ◽  
Immunoglobulin Binding ◽  
Sublethal Concentrations

Sublethal concentrations (sub-MICs) of certain disinfectants are no longer effective in removing biofilms from abiotic surfaces and can even promote the formation of biofilms. Bacterial cells can probably adapt to these low concentrations of disinfectants and defend themselves by way of biofilm formation. In this paper, we report on threeStaphylococcus aureusbiofilm formers (strong B+++, moderate B++, and weak B+) that were cultivated with sub-MICs of commonly used disinfectants, ethanol or chloramine T, and quantified using Syto9 green fluorogenic nucleic acid stain. We demonstrate that 1.25–2.5% ethanol and 2500 μg/mL chloramine T significantly enhancedS. aureusbiofilm formation. To visualize differences in biofilm compactness betweenS. aureusbiofilms in control medium, 1.25% ethanol, or 2500 μg/mL chloramine T, scanning electron microscopy was used. To describe changes in abundance of surface-exposed proteins in ethanol- or chloramine T-treated biofilms, surface proteins were prepared using a novel trypsin shaving approach and quantified after dimethyl labeling by LC-LTQ/Orbitrap MS. Our data show that some proteins with adhesive functions and others with cell maintenance functions and virulence factor EsxA were significantly upregulated by both treatments. In contrast, immunoglobulin-binding protein A was significantly downregulated for both disinfectants. Significant differences were observed in the effect of the two disinfectants on the expression of surface proteins including some adhesins, foldase protein PrsA, and two virulence factors.

scholarly journals Intracellular Ca2+ stores of rat cerebellum: heterogeneity within and distinction from endoplasmic reticulum

1993 ◽  
Vol 291 (1) ◽  
pp. 199-204 ◽  
Author(s):  
A Nori ◽  
A Villa ◽  
P Podini ◽  
D R Witcher ◽  
P Volpe
Keyword(s):  
Endoplasmic Reticulum ◽  
Signal Sequence ◽  
Binding Protein ◽  
Ryanodine Receptors ◽  
High Capacity ◽  
Receptor Alpha ◽  
Rat Cerebellum ◽  
Immunoglobulin Binding Protein ◽  
Intracellular Organelles ◽  
Immunoglobulin Binding

Rat cerebellum microsomes were subfractionated on isopycnic linear sucrose (20-42%)-density gradients. The distribution of endoplasmic reticulum (ER) markers (RNA, signal-sequence receptor alpha, calnexin, calreticulin, the immunoglobulin-binding protein Bip) and markers of intracellular rapidly exchanging Ca2+ stores [Ca2+ channels sensitive to either Ins(1,4,5)P3 or ryanodine) was investigated biochemically and immunologically. The comparison indicates that: (a) vesicles bearing the InsP3 receptor were separated from those bearing the ryanodine receptor; (b) ER markers, i.e. Bip, calnexin, signal-sequence receptor alpha, RNA, did not sediment as either InsP3 or ryanodine receptors did; (c) calreticulin, an intralumenal low-affinity high-capacity Ca(2+)-binding protein, had a widespread distribution, similar to that of Bip and calnexin, and was present in Purkinje, granule, Golgi and stellate neurons, as indicated by immunofluorescent labelling of cerebellum cortex cryosections. The present results show that the ER is not a homogeneous entity, and that Ca2+ stores are heterogeneous insofar as InsP3 receptors and ryanodine receptors are segregated, either to discrete intracellular organelles or to specialized ER subcompartments.

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