CaBP1, a calcium binding protein of the thioredoxin family, is a resident KDEL protein of the ER and not of the intermediate compartment

1994 ◽  
Vol 107 (10) ◽  
pp. 2719-2727 ◽  
Author(s):  
J. Fullekrug ◽  
B. Sonnichsen ◽  
U. Wunsch ◽  
K. Arseven ◽  
P. Nguyen Van ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Laser Scanning ◽  
Vero Cells ◽  
Subcellular Fractionation ◽  
Laser Scanning Microscopy ◽  
Cos Cells ◽  
Marker Proteins ◽  
Intermediate Compartment ◽  
High Level ◽  
High Degree

A cDNA encoding rat CaBP1 has been isolated and sequenced. The deduced polypeptide chain consists of 440 amino acids including two internal thioredoxin-like domains and a C-terminal KDEL retention/retrieval signal. Regarding the high degree of identity to the hamster protein P5, CaBP1 is considered to be the homologous rat protein. Previous work has suggested that CaBP1 is a resident luminal protein of the intermediate compartment (Schweizer, A., Peter, F., Nguyen Van, P., Soling, H.D. and Hauri, H.P. (1993) Eur. J. Cell Biol. 60, 366–370). Our conclusion that CaBP1 is a resident protein of the endoplasmic reticulum and not of the intermediate compartment is based on three different approaches: subcellular fractionation, indirect immunofluorescence and overexpression of CaBP1. Subcellular fractionation of Vero cells in a velocity controlled step gradient led to copurification of CaBP1-containing vesicles and several marker proteins for the ER including calreticulin and alpha-SSRP. The intermediate compartment, as defined by a monoclonal antibody against the marker protein p53 (ERGIC-53), could be separated from these ER markers. Double immunofluorescence analysed by laser scanning microscopy showed no significant colocalization between CaBP1 and p53, but between CaBP1 and calreticulin. In addition experiments, Vero cells were infected with VSV tsO45. At 15 degrees C the VSV-G protein accumulated in punctuate structures representing the intermediate compartment, while CaBP1 maintained its original reticular localization. Even after high-level overexpression in COS cells, CaBP1 was not detected in the intermediate compartment, but was efficiently retained in the ER as judged by light microscopy.

Characterization of a novel 63 kDa membrane protein. Implications for the organization of the ER-to-Golgi pathway

1993 ◽  
Vol 104 (3) ◽  
pp. 671-683 ◽  
Author(s):  
A. Schweizer ◽  
M. Ericsson ◽  
T. Bachi ◽  
G. Griffiths ◽  
H.P. Hauri
Keyword(s):  
Membrane Protein ◽  
Laser Scanning ◽  
Protein Transport ◽  
Brefeldin A ◽  
Vero Cells ◽  
Subcellular Fractionation ◽  
Confocal Laser ◽  
Similar Degree ◽  
Fractionation Procedure ◽  
Intermediate Compartment

Owing to the lack of appropriate markers the structural organization of the ER-to-Golgi pathway and the dynamics of its membrane elements have been elusive. To elucidate this organization we have taken a monoclonal antibody (mAb) approach. A mAb against a novel 63 kDa membrane protein (p63) was produced that identifies a large tubular network of smooth membranes in the cytoplasm of primate cells. The distribution of p63 overlaps with the ER-Golgi intermediate compartment, defined by a previously described 53 kDa marker protein (here termed ERGIC-53), as visualized by confocal laser scanning immunofluorescence microscopy and immunoelectron microscopy. The p63 compartment mediates protein transport from the ER to Golgi apparatus, as indicated by partial colocalization of p63 and vesicular stomatitis virus G protein in Vero cells cultured at 15 degrees C. Low temperatures and brefeldin A had little effect on the cellular distribution of p63, suggesting that this novel marker is a stably anchored resident protein of these pre-Golgi membranes. p63 and ERGIC-53 were enriched to a similar degree by the same subcellular fractionation procedure. These findings demonstrate an unanticipated complexity of the ER-Golgi interface and suggest that the ER-Golgi intermediate compartment defined by ERGIC-53 may be part of a greater network of smooth membranes.

LTCC Based Multi-Electrode Arrays for In-Vitro Cell Culture

2015 ◽  
Vol 2015 (CICMT) ◽  
pp. 000269-000274
Author(s):  
Heike Bartsch ◽  
Dirk Stöpel ◽  
Marcel Himmerlich ◽  
Martin Baca ◽  
Philipp Stadie ◽  
...  
Keyword(s):  
Cell Culture ◽  
Laser Scanning ◽  
Neuronal Cell ◽  
Laser Scanning Microscopy ◽  
Gold Electrodes ◽  
Neural Cells ◽  
Electrode Arrays ◽  
Green Tape ◽  
High Level

Neurobiological concepts based on state-of-the art technology have so far lacked the complexity of actual high-level neurobiological systems. Two key advances are needed to improve our understanding of such systems: in vitro 3D-neuronal cell culture and 3D MEA systems for measuring such 3D-cultures. These requirements call for smart multilayer and packaging technology. The material Green Tape TM from DuPont Nemours is chosen for the presented works, because its compatibility and those of available metallisation with cell cultures is already proven. An LTCC multilayer circuit with gold electrodes is the base of the 3D MEA. The layout of the 3D MEA is designed to fit the MEA2100-System for in vitro recording from Multi Channel Systems and enable thus a comparable data processing to established 2D MEAs Slots. The surface topography of the thick film electrodes and the surface state is investigated with laser scanning microscopy, SEM, XPS and measurements of the wetting angle of contact. The impedance of the screen printed electrodes is discussed taking these data into account. Their impedance amounts to 24 kΩ and are falls thus below the impedance of commercially available electroplated gold electrodes of 30 kΩ. First promising results have been achieved using 3D MEAs for 2D culture of human pluripotent stem cell derived neural cells.

scholarly journals Assessment of the In Vitro Antischistosomal Activities of the Extracts and Compounds from Solidago Microglossa DC (Asteraceae) and Aristolochia Cymbifera Mart. & Zucc. (Aristolochiaceae)

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Poliana da Silva Costa ◽  
Ohana Oliveira Zuza da Silva ◽  
Danilo de Souza Costa ◽  
Lara Aparecida de Oliveira Silva ◽  
Priscila de Faria Pinto ◽  
...  
Keyword(s):  
Methyl Ester ◽  
Motor Activity ◽  
Laser Scanning ◽  
Acid Methyl Ester ◽  
Vero Cells ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Acid Methyl ◽  
Antischistosomal Activity

Schistosomiasis, caused by helminth flatworms of the genus Schistosoma, is a neglected tropical disease that afflicts over 230 million people worldwide. Currently, treatment is achieved with only one drug, praziquantel (PZQ). In this regard, the roots of Solidago microglossa (Asteraceae) and Aristolochia cymbifera (Aristolochiaceae) are popularly used as anthelmintic. Despite their medicinal use against helminthiasis, such as schistosomiasis, A. cymbifera, and S. microglossa have not been evaluated against S. mansoni. Then, in this work, the in vitro antischistosomal activity of the crude extracts of A. cymbifera (Ac) and S. microglossa (Sm) and their isolated compounds were investigated against S. mansoni adult worms. Sm (200 μg/mL) and Ac (100–200 μg/mL) were lethal to all male and female worms at the 24 h incubation. In addition, Sm (10–50 μg/mL) and Ac (10 μg/mL) caused significant reduction in the parasite’s movements, showing no significant cytotoxicity to Vero cells at the same range of schistosomicidal concentrations. Confocal laser scanning microscopy revealed that Sm and Ac caused tegumental damages and reduced the numbers of tubercles of male schistosomes. Chromatographic fractionation of Sm leads to isolation of bauerenol, α-amirin, and spinasterol, while populifolic acid, cubebin, 2-oxopopulifolic acid methyl ester, and 2-oxopopulifolic acid were isolated from Ac. At concentrations of 25–100 μM, bauerenol, α-amirin, spinasterol, populifolic acid, and cubebin showed significant impact on motor activity of S. mansoni. 2-oxopopulifolic acid methyl ester and 2-oxopopulifolic acid caused 100% mortality and decreased the motor activity of adult schistosomes at 100 μM. This study has reported, for the first time, the in vitro antischistosomal effects of S. microglossa and A. cymbifera extracts, also showing promising compounds against adult schistosomes.

scholarly journals Acidic stress induces protective autophagy in SGC7901 cells

2018 ◽  
Vol 46 (8) ◽  
pp. 3285-3295 ◽  
Author(s):  
Jie Sheng ◽  
Li-Bin Sun ◽  
Shu-Fen Zhao ◽  
Wei-Wei Qi ◽  
Jing Lv ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Proliferation ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Acidic Environment ◽  
Bafilomycin A1 ◽  
Autophagosome Formation ◽  
Acidic Conditions ◽  
High Level

Objective To investigate the effect of acidity on gastric cancer SGC7901 cells in terms of autophagy and provide a new strategy for therapeutically targeting gastric cancer autophagy in an acidic environment. Methods Transmission electron microscopy (TEM) and confocal laser scanning microscopy were used to examine the effect of an acidic environment on autophagosome formation. Light chain 3 (LC3) and p62 levels in SGC7901 cells exposed to acidic conditions were measured using Western blot analysis. To explore changes in autophagy flux, the cells were treated with an inhibitor of autophagy bafilomycin A1. The CCK-8 assay was performed to determine if inhibiting acid-induced autophagy affected cell proliferation. Results Increased autophagosome formation was observed by TEM. Punctate LC3 structures were observed in cells cultured under acidic conditions, whereas untreated cells exhibited diffuse and weak staining for punctate LC3 structures. Cytoplasmic LC3-I translocated to the autophagic membrane (LC3-II) levels increased under acidic conditions, whereas p62 levels decreased. The bafilomycin A1-induced inhibition of autophagy caused by the acidic environment inhibited cell proliferation. Conclusion The acidic environment upregulates autophagy in SGC7901 cells. In long-term culture, a stable and high level of autophagy is maintained in an acidic environment, which has a protective effect on cells.

Rapid Mobilization of Intracellularly Stored RANTES in Response to Interferon-γ in Human Eosinophils

Blood ◽  
1999 ◽  
Vol 94 (1) ◽  
pp. 23-32 ◽  
Author(s):  
Paige Lacy ◽  
Salahaddin Mahmudi-Azer ◽  
Ben Bablitz ◽  
Stacey C. Hagen ◽  
Juan R. Velazquez ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Human Peripheral Blood ◽  
Subcellular Fractionation ◽  
Secretory Vesicle ◽  
Interferon Γ ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Enzyme Linked Immunosorbent Assay ◽  
Gm Csf ◽  
Ifn Γ

The CC chemokine RANTES is synthesized, stored, and upregulated in response to interferon-γ (IFN-γ) in human peripheral blood eosinophils. In this report, we propose that RANTES is rapidly mobilized from eosinophil crystalloid granules during agonist-induced degranulation. We stimulated purified eosinophils (>99%) from atopic asthmatics with 500 U/mL IFN-γ to analyze the kinetics of mobilization and release of RANTES (0 to 240 minutes). We used subcellular fractionation, immunogold analysis, two-color confocal laser scanning microscopy (CLSM), and enzyme-linked immunosorbent assay (ELISA) to trace the movement of eosinophil-derived RANTES from intracellular stores to release. RANTES was rapidly mobilized (10 minutes) and released after 120 minutes of stimulation (80 ± 15 pg/mL per 2 × 106 cells). RANTES appeared to be stored in at least two intracellular compartments: the matrix of crystalloid granules, detected by major basic protein and eosinophil peroxidase activities, and a specialized small secretory vesicle present in light membrane fractions. The extragranular RANTES was mobilized more rapidly than that of crystalloid granules during IFN-γ stimulation. This effect was not observed in eosinophils treated with IFN-, interleukin-3 (IL-3), IL-5, granulocyte-macrophage colony-stimulating factor (GM-CSF), or genistein followed by IFN-γ. Our findings suggest that RANTES may be mobilized and released by piecemeal degranulation upon stimulation, involving transport through a putative pool of small secretory vesicles.

scholarly journals Transport of an external Lys-Asp-Glu-Leu (KDEL) protein from the plasma membrane to the endoplasmic reticulum: studies with cholera toxin in Vero cells.

1996 ◽  
Vol 133 (4) ◽  
pp. 777-789 ◽  
Author(s):  
I V Majoul ◽  
P I Bastiaens ◽  
H D Söling
Keyword(s):  
Endoplasmic Reticulum ◽  
Cholera Toxin ◽  
Retrograde Transport ◽  
Vero Cells ◽  
Subcellular Fractionation ◽  
Pulse Treatment ◽  
Resistant Form ◽  
Immunofluorescence Studies ◽  
Intermediate Compartment ◽  
Kdel Sequence

The A2 chain of cholera toxin (CTX) contains a COOH-terminal Lys-Asp-Glu-Leu (KDEL) sequence. We have, therefore, analyzed by immunofluorescence and by subcellular fractionation in Vero cells whether CTX can used to demonstrate a retrograde transport of KDEL proteins from the Golgi to the ER. Immunofluorescence studies reveal that after a pulse treatment with CTX, the CTX-A and B subunits (CTX-A and CTX-B) reach Golgi-like structures after 15-20 min (maximum after 30 min). Between 30 and 90 min, CTX-A (but not CTX-B) appear in the intermediate compartment and in the ER, whereas the CTX-B are translocated to the lysosomes. Subcellular fractionation studies confirm these results: after CTX uptake for 15 min, CTX-A is associated only with endosomal and Golgi compartments. After 30 min, a small amount of CTX-A appears in the ER in a trypsin-resistant form, and after 60 min, a significant amount appears. CTX-A seems to be transported mainly in its oxidized form (CTX-A1-S-S-CTX-A2) from the Golgi to the ER, where it becomes slowly reduced to form free CTX A1 and CTX-A2, as indicated by experiments in which cells were homogenized 30 and 90 min after the onset of CTX uptake in the presence of N-ethylmaleimide. Nocodazol applied after accumulation of CTX in Golgi inhibits the appearance of CTX-A in the ER and delays the increase of 3',5'cAMP, indicating the participation of microtubules in the retrograde Golgi-ER transport.

scholarly journals Determination of nonlinear absorption and scattering in a single plasmonic nanostructure using X-scan technique

2019 ◽  
Author(s):  
Tushar C Jagadale ◽  
Dhanya Murali ◽  
Shi-Wei Chu
Keyword(s):  
Laser Scanning ◽  
Detection System ◽  
Optical Nonlinearity ◽  
Excitation Intensity ◽  
Laser Scanning Microscopy ◽  
Thin Sample ◽  
Channel Detection ◽  
Laser Focus ◽  
Novel Method ◽  
High Level

Nonlinear nano-plasmonics opens up many exciting opportunities, such as nano-laser, nano-antenna, nano-modulator, etc. A highly desirable tool in the field of nonlinear nano-plasmonics is to characterize nonlinearity of optical absorption and scattering in single nanostructures. Currently, the most widely used method to quantify optical nonlinearity is z-scan, which can derive real and imaginary parts of permittivity through translating a thin sample across a laser focus. However, z-scan typically works with thin films, and thus acquires nonlinear responses from ensemble of nanostructures, not a single one. In this work, we present an X-scan technique, which is based on laser scanning microscopy equipped with forward and backward detectors. The two-channel detection allows simultaneous quantification of nonlinear behaviours of scattering, absorption, as well as total attenuation, from a single nanostructure. At low excitation intensity, both scattering and absorption responses are linear, thus confirming the linearity of detection system. At high excitation intensity, we found that the nonlinear response can be derived directly from the point spread function of X-scan images. Surprisingly high level of nonlinearities in both scattering and absorption are unravelled simultaneously for the first time. Our study not only provides a novel method for characterizing single-nanostructure nonlinearity, but also reports exceptionally large plasmonic nonlinearities.

scholarly journals Aminopeptidase I Is Targeted to the Vacuole by a Nonclassical Vesicular Mechanism

1997 ◽  
Vol 138 (1) ◽  
pp. 37-44 ◽  
Author(s):  
Sidney V. Scott ◽  
Misuzu Baba ◽  
Yoshinori Ohsumi ◽  
Daniel J. Klionsky
Keyword(s):  
Mutant Strain ◽  
Subcellular Fractionation ◽  
Marker Proteins ◽  
In Vivo Analysis ◽  
Autophagy Pathway ◽  
Intermediate Compartment ◽  
Vacuolar Protein ◽  
Molecular Components

The yeast vacuolar protein aminopeptidase I (API) is synthesized as a cytosolic precursor that is transported to the vacuole by a nonclassical targeting mechanism. Recent genetic studies indicate that the biosynthetic pathway that transports API uses many of the same molecular components as the degradative autophagy pathway. This overlap coupled with both in vitro and in vivo analysis of API import suggested that, like autophagy, API transport is vesicular. Subcellular fractionation experiments demonstrate that API precursor (prAPI) initially enters a nonvacuolar cytosolic compartment. In addition, subvacuolar vesicles containing prAPI were purified from a mutant strain defective in breakdown of autophagosomes, further indicating that prAPI enters the vacuole inside a vesicle. The purified subvacuolar vesicles do not appear to contain vacuolar marker proteins. Immunogold EM confirms that prAPI is localized in cytosolic and in subvacuolar vesicles in a mutant strain defective in autophagic body degradation. These data suggest that cytosolic vesicles containing prAPI fuse with the vacuole to release a membrane-bounded intermediate compartment that is subsequently broken down, allowing API maturation.

scholarly journals Pro-Apoptotic Apoptosis Protease–Activating Factor 1 (Apaf-1) Has a Cytoplasmic Localization Distinct from Bcl-2 or Bcl-XL

2000 ◽  
Vol 149 (3) ◽  
pp. 623-634 ◽  
Author(s):  
George Hausmann ◽  
Lorraine A. O'Reilly ◽  
Rosemary van Driel ◽  
Jennifer G. Beaumont ◽  
Andreas Strasser ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cytochrome C ◽  
Laser Scanning ◽  
Subcellular Fractionation ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Immunogold Electron Microscopy ◽  
Cytoplasmic Localization ◽  
In Vitro Activation

How Bcl-2 and its pro-survival relatives prevent activation of the caspases that mediate apoptosis is unknown, but they appear to act through the caspase activator apoptosis protease–activating factor 1 (Apaf-1). According to the apoptosome model, the Bcl-2–like proteins preclude Apaf-1 activity by sequestering the protein. To explore Apaf-1 function and to test this model, we generated monoclonal antibodies to Apaf-1 and used them to determine its localization within diverse cells by subcellular fractionation and confocal laser scanning microscopy. Whereas Bcl-2 and Bcl-xL were prominent on organelle membranes, endogenous Apaf-1 was cytosolic and did not colocalize with them, even when these pro-survival proteins were overexpressed or after apoptosis was induced. Immunogold electron microscopy confirmed that Apaf-1 was dispersed in the cytoplasm and not on mitochondria or other organelles. After the death stimuli, Bcl-2 and Bcl-xL precluded the release of the Apaf-1 cofactor cytochrome c from mitochondria and the formation of larger Apaf-1 complexes, which are steps that presage apoptosis. However, neither Bcl-2 nor Bcl-xL could prevent the in vitro activation of Apaf-1 induced by the addition of exogenous cytochrome c. Hence, rather than sequestering Apaf-1 as proposed by the apoptosome model, Bcl-2–like proteins probably regulate Apaf-1 indirectly by controlling upstream events critical for its activation.

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