scholarly journals Molecular and Functional Characterization of Caveolae in Mixed Cultures of Human NT-2 Neurons and Astrocytes

Neuroglia ◽  
2021 ◽  
Vol 2 (1) ◽  
pp. 68-88
Author(s):  
Jagdeep K. Sandhu ◽  
Maria Ribecco-Lutkiewicz ◽  
Abedelnasser Abulrob
Keyword(s):  
Plasma Membrane ◽  
Functional Characterization ◽  
Mixed Cultures ◽  
Cholera Toxin B Subunit ◽  
Mrna Levels ◽  
Caveolin 1 ◽  
B Subunit ◽  
A Cells ◽  
Human Neurons ◽  
Health And Disease

Caveolae are plasma membrane invaginations that are enriched in cholesterol-binding proteins called caveolins. The presence of caveolae and caveolins in mixed cultures of human neurons and glia has not been investigated. Here, we sought to determine the presence of caveolae and caveolins in human NTera-2 (NT2/D1) cells, differentiated with retinoic acid into neuron-like (NT2/N) and astrocyte-like (NT2/A) cells. We found that while caveolin-3 mRNA levels remained relatively constant, caveolin-1 and -2 levels were upregulated in NT2/A and downregulated in NT2/N. No caveolin-1 immunoreactivity was detected in NT2/N. Electron microscopy revealed numerous flask-shaped invaginations (~86–102 nm in diameter) in the plasma membrane of NT2/A and NT2/N cells, while only few were detected in NT2/D1 cells. Immunoelectron microscopy localized caveolin-1 gold particles in the flask-shaped structures on plasmalemma and cytoplasmic vesicles of NT2/A cells. Furthermore, NT2/A endocytosed Alexa 488 conjugated-cholera toxin B subunit (CTX-B) through a caveolae- and clathrin-dependent pathway, whereas NT2/N endocytosed CTX-B through a caveolae-independent pathway. We have established that while NT2/A expressed functional caveolae, the molecular identity of the plasma membrane invaginations in NT2/N is unknown. The expression of caveolin proteins was differentially regulated in these cells. Taken together, our findings support the usefulness of the human NT2 model system to study the role of caveolins in neuron–glia communication, and their involvement in brain health and disease.

8 FLOW CYTOMETRIC MONITORING OF CHOLERA TOXIN B SUBUNIT BINDING TO BOVINE SPERMATOZOA

2008 ◽  
Vol 20 (1) ◽  
pp. 84
Author(s):  
M. Boilard ◽  
M. Beaulieu ◽  
P. Blondin
Keyword(s):  
Flow Cytometry ◽  
Plasma Membrane ◽  
Lipid Rafts ◽  
Molecular Probes ◽  
Cholera Toxin B Subunit ◽  
Enhanced Fluorescence ◽  
Toxin B ◽  
B Subunit ◽  
Bovine Spermatozoa ◽  
High Fluorescence

In order to become able to fertilize, mammalian spermatozoa must undergo a series of biochemical modifications. This process called capacitation involves several changes of the content and the ultrastucture of the plasma membrane. Among these changes, loss of cholesterol from the plasma membrane is required. Lipid rafts are detergent-insoluble plasma membrane domains rich in cholesterol and sphingolipids. Some proteins are confined to lipid rafts while others are excluded. It has been hypothesized in the past that the loss of cholesterol could destabilize and relocate lipid rafts and would thus affect protein interactions in the plasma membrane, thereby leading to downstream events involved in the capacitation process. Thus, quantification of lipid rafts within the membrane of spermatozoa would become useful to monitor sperm functions and maturation level. The present study aimed to quantify lipid rafts in bovine spermatozoa using the Vibrant Lipid Raft detection kit from Molecular Probes (Invitrogen Canada, Inc., Burlingame, Ontario, Canada) and flow cytometry. The Vibrant kit uses the cholera toxin B subunit (CT-B) and claims to detect ganglioside Gm1 that sublocalizes within lipid rafts. Briefly, freshly ejaculated and frozen/thawed spermatozoa were washed once by centrifugation at 250g for five min in sp-Talp and were then re-suspended in sp-Talp containing 1 �g mL–1 CT-B. Then, cells were incubated at 4�C for 10 min, washed in chilled sp-Talp, incubated for 15 min in the presence of an anti-CT-B antibody coupled to the Alexa Fluor� 488 dye (Molecular Probes), and washed again to remove excess antibody. Spermatozoa were then analyzed with a BD LSR II flow cytometer (BD Biosciences, San Jose, CA, USA). Two populations showing different fluorescence levels were observed in all samples. Greater proportions of spermatozoa displayed the high fluorescence pattern in cryopreserved samples (37.9%) when compared to freshly ejaculated spermatozoa (8.2%) (P < 0.01). Also, when compared to freshly ejaculated spermatozoa, increased proportions of high fluorescence was detected following a 6-h incubation in sp-Talp containing bicarbonate and BSA. These results suggest that capacitation and cryopreservation both promote exposure of CT-B binding molecules in bovine spermatozoa. Microscopic observation of labeled cryopreserved spermatozoa did not yield the expected raft labeling patterns, but rather 5 different patterns of labeling. In the past, some of these patterns were recognized to be associated with capacitation and acrosome reaction. At this point, more work is needed to confirm which of the fluorescent patterns observed in microscopy corresponds to the enhanced fluorescence sperm population observed by flow cytometry and to directly associate this enhanced fluorescence to capacitation or the acrosome reaction. In conclusion, it appears that the Vibrant kit from Molecular Probes cannot be used to quantify lipid rafts by flow cytometry. Nevertheless, it might be an interesting tool to use in flow cytometry to monitor membrane changes associated with capacitation or cryo-damage.

scholarly journals Immunization with the Recombinant Cholera Toxin B Fused to Fimbria 2 Protein Protects againstBordetella pertussisInfection

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Noelia Olivera ◽  
Celina E. Castuma ◽  
Daniela Hozbor ◽  
María E. Gaillard ◽  
Martín Rumbo ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Cholera Toxin ◽  
Bordetella Pertussis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cholera Toxin B Subunit ◽  
Mrna Levels ◽  
Toxin B ◽  
Coding Sequence ◽  
Cholera Toxin B ◽  
B Subunit

This study examined the immunogenic properties of the fusion protein fimbria 2 ofBordetella pertussis(Fim2)—cholera toxin B subunit (CTB) in the intranasal murine model of infection. To this endB. pertussisFim2 coding sequence was cloned downstream of the cholera toxin B subunit coding sequence. The expression and assembly of the fusion protein into pentameric structures (CTB-Fim2) were evaluated by SDS-PAGE and monosialotetrahexosylgaglioside (GM1-ganglioside) enzyme-linked immunosorbent assay (ELISA). To evaluate the protective capacity of CTB-Fim2, an intraperitoneal or intranasal mouse immunization schedule was performed with 50 μg of CTB-Fim2. Recombinant (rFim2) or purified (BpFim2) Fim2, CTB, and phosphate-buffered saline (PBS) were used as controls. The results showed that mice immunized with BpFim2 or CTB-Fim2 intraperitoneally or intranasally presented a significant reduction in bacterial lung counts compared to control groups (P<0.01orP<0.001,resp.). Moreover, intranasal immunization with CTB-Fim2 induced significant levels of Fim2-specific IgG in serum and bronchoalveolar lavage (BAL) and Fim2-specific IgA in BAL. Analysis of IgG isotypes and cytokines mRNA levels showed that CTB-Fim2 results in a mixed Th1/Th2 (T-helper) response. The data presented here provide support for CTB-Fim2 as a promising recombinant antigen againstBordetella pertussisinfection.

scholarly journals High-Resolution FRET Microscopy of Cholera Toxin B-Subunit and GPI-anchored Proteins in Cell Plasma Membranes

2000 ◽  
Vol 11 (5) ◽  
pp. 1645-1655 ◽  
Author(s):  
Anne K. Kenworthy ◽  
Nadezda Petranova ◽  
Michael Edidin
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Cholera Toxin ◽  
Lipid Rafts ◽  
Plasma Membranes ◽  
Membrane Lipid ◽  
Cholera Toxin B Subunit ◽  
Toxin B ◽  
Cholera Toxin B ◽  
B Subunit

“Lipid rafts” enriched in glycosphingolipids (GSL), GPI-anchored proteins, and cholesterol have been proposed as functional microdomains in cell membranes. However, evidence supporting their existence has been indirect and controversial. In the past year, two studies used fluorescence resonance energy transfer (FRET) microscopy to probe for the presence of lipid rafts; rafts here would be defined as membrane domains containing clustered GPI-anchored proteins at the cell surface. The results of these studies, each based on a single protein, gave conflicting views of rafts. To address the source of this discrepancy, we have now used FRET to study three different GPI-anchored proteins and a GSL endogenous to several different cell types. FRET was detected between molecules of the GSL GM1 labeled with cholera toxin B-subunit and between antibody-labeled GPI-anchored proteins, showing these raft markers are in submicrometer proximity in the plasma membrane. However, in most cases FRET correlated with the surface density of the lipid raft marker, a result inconsistent with significant clustering in microdomains. We conclude that in the plasma membrane, lipid rafts either exist only as transiently stabilized structures or, if stable, comprise at most a minor fraction of the cell surface.

Na+/H+ exchanger isoform 6 (NHE6/SLC9A6) is involved in clathrin-dependent endocytosis of transferrin

2011 ◽  
Vol 301 (6) ◽  
pp. C1431-C1444 ◽  
Author(s):  
Lou Xinhan ◽  
Masafumi Matsushita ◽  
Manami Numaza ◽  
Akira Taguchi ◽  
Keiji Mitsui ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Ph Regulation ◽  
Microscopic Analysis ◽  
Cholera Toxin B Subunit ◽  
Wild Type ◽  
B Subunit ◽  
Epidermal Growth ◽  
Transferrin Uptake ◽  
In Cells

In mammalian cells, nine conserved isoforms of the Na+/H+ exchanger (NHE) are known to be important for pH regulation of the cytoplasm and organellar lumens. NHE1–5 are localized to the plasma membrane, whereas NHE6–9 are localized to distinct organelles. NHE6 is localized predominantly in endosomal compartments but is also found in the plasma membrane. To investigate the role of NHE6 in endocytosis, we established NHE6-knockdown HeLa cells and analyzed the effect of this knockdown on endocytotic events. The expression level of NHE6 in knockdown cells was decreased to ∼15% of the level seen in control cells. Uptake of transferrin was also decreased. No effect was found on the endocytosis of epidermal growth factor or on the cholera toxin B subunit. Moreover, in the NHE6-knockdown cells, transferrin uptake was found to be affected in the early stages of endocytosis. Microscopic analysis revealed that, at 2 min after the onset of endocytosis, colocalization of NHE6, clathrin, and transferrin was observed, which suggests that NHE6 was localized to endocytotic, clathrin-coated vesicles. In addition, in knockdown cells, transferrin-positive endosomes were acidified, but no effect was found on cytoplasmic pH. In cells overexpressing wild-type NHE6, increased transferrin uptake was observed, but no such increase was seen in cells overexpressing mutant NHE6 deficient in ion transport. The luminal pH in transferrin-positive endosomes was alkalized in cells overexpressing wild-type NHE6 but normal in cells overexpressing mutant NHE6. These observations suggest that NHE6 regulates clathrin-dependent endocytosis of transferrin via pH regulation.

scholarly journals Ceramide structure dictates glycosphingolipid nanodomain assembly and function

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Senthil Arumugam ◽  
Stefanie Schmieder ◽  
Weria Pezeshkian ◽  
Ulrike Becken ◽  
Christian Wunder ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Acyl Chain ◽  
Cholera Toxin B Subunit ◽  
Cellular Functions ◽  
Toxin B ◽  
Cellular Processes ◽  
B Subunit ◽  
Outer Leaflet ◽  
And Function ◽  
Nanodomain Structure

AbstractGangliosides in the outer leaflet of the plasma membrane of eukaryotic cells are essential for many cellular functions and pathogenic interactions. How gangliosides are dynamically organized and how they respond to ligand binding is poorly understood. Using fluorescence anisotropy imaging of synthetic, fluorescently labeled GM1 gangliosides incorporated into the plasma membrane of living cells, we found that GM1 with a fully saturated C16:0 acyl chain, but not with unsaturated C16:1 acyl chain, is actively clustered into nanodomains, which depends on membrane cholesterol, phosphatidylserine and actin. The binding of cholera toxin B-subunit (CTxB) leads to enlarged membrane domains for both C16:0 and C16:1, owing to binding of multiple GM1 under a toxin, and clustering of CTxB. The structure of the ceramide acyl chain still affects these domains, as co-clustering with the glycosylphosphatidylinositol (GPI)-anchored protein CD59 occurs only when GM1 contains the fully saturated C16:0 acyl chain, and not C16:1. Thus, different ceramide species of GM1 gangliosides dictate their assembly into nanodomains and affect nanodomain structure and function, which likely underlies many endogenous cellular processes.

scholarly journals Analysis of NIS Plasma Membrane Interactors Discloses Key Regulation by a SRC/RAC1/PAK1/PIP5K/EZRIN Pathway with Potential Implications for Radioiodine Re-Sensitization Therapy in Thyroid Cancer

Cancers ◽  
2021 ◽  
Vol 13 (21) ◽  
pp. 5460
Author(s):  
Márcia Faria ◽  
Rita Domingues ◽  
Maria João Bugalho ◽  
Ana Luísa Silva ◽  
Paulo Matos
Keyword(s):  
Plasma Membrane ◽  
Thyroid Cancer ◽  
Actin Polymerization ◽  
Functional Characterization ◽  
Thyroid Tissue ◽  
Small Gtpase ◽  
Mrna Levels ◽  
Iodide Uptake ◽  
Thyroid Cells ◽  
And Function

The functional expression of the sodium–iodide symporter (NIS) at the membrane of differentiated thyroid cancer (DTC) cells is the cornerstone for the use of radioiodine (RAI) therapy in these malignancies. However, NIS gene expression is frequently downregulated in malignant thyroid tissue, and 30% to 50% of metastatic DTCs become refractory to RAI treatment, which dramatically decreases patient survival. Several strategies have been attempted to increase the NIS mRNA levels in refractory DTC cells, so as to re-sensitize refractory tumors to RAI. However, there are many RAI-refractory DTCs in which the NIS mRNA and protein levels are relatively abundant but only reduced levels of iodide uptake are detected, suggesting a posttranslational failure in the delivery of NIS to the plasma membrane (PM), or an impaired residency at the PM. Because little is known about the molecules and pathways regulating NIS delivery to, and residency at, the PM of thyroid cells, we here employed an intact-cell labeling/immunoprecipitation methodology to selectively purify NIS-containing macromolecular complexes from the PM. Using mass spectrometry, we characterized and compared the composition of NIS PM complexes to that of NIS complexes isolated from whole cell (WC) lysates. Applying gene ontology analysis to the obtained MS data, we found that while both the PM-NIS and WC-NIS datasets had in common a considerable number of proteins involved in vesicle transport and protein trafficking, the NIS PM complexes were particularly enriched in proteins associated with the regulation of the actin cytoskeleton. Through a systematic validation of the detected interactions by co-immunoprecipitation and Western blot, followed by the biochemical and functional characterization of the contribution of each interactor to NIS PM residency and iodide uptake, we were able to identify a pathway by which the PM localization and function of NIS depends on its binding to SRC kinase, which leads to the recruitment and activation of the small GTPase RAC1. RAC1 signals through PAK1 and PIP5K to promote ARP2/3-mediated actin polymerization, and the recruitment and binding of the actin anchoring protein EZRIN to NIS, promoting its residency and function at the PM of normal and TC cells. Besides providing novel insights into the regulation of NIS localization and function at the PM of TC cells, our results open new venues for therapeutic intervention in TC, namely the possibility of modulating abnormal SRC signaling in refractory TC from a proliferative/invasive effect to the re-sensitization of these tumors to RAI therapy by inducing NIS retention at the PM.

scholarly journals Selective Caveolin-1–dependent Endocytosis of Glycosphingolipids

2003 ◽  
Vol 14 (8) ◽  
pp. 3254-3265 ◽  
Author(s):  
Raman Deep Singh ◽  
Vishwajeet Puri ◽  
Jacob T. Valiyaveettil ◽  
David L. Marks ◽  
Robert Bittman ◽  
...  
Keyword(s):  
Cell Types ◽  
Cholera Toxin B Subunit ◽  
Cell Type ◽  
Toxin B ◽  
Caveolin 1 ◽  
B Subunit ◽  
Internalization Mechanism ◽  
Rat Fibroblasts ◽  
Low Levels ◽  
In Cells

We studied the endocytosis of fluorescent glycosphingolipid (GSL) analogs in various cell types using pathway-specific inhibitors and colocalization studies with endocytic markers and DsRed caveolin-1 (cav-1). Based on inhibitor studies, all GSLs tested were internalized predominantly (>80%) by a clathrin-independent, caveolar-related mechanism, regardless of cell type. In addition, fluorescent lactosylceramide (LacCer) colocalized with DsRed-cav-1 in vesicular structures upon endocytosis in rat fibroblasts. The internalization mechanism for GSLs was unaffected by varying the carbohydrate headgroup or sphingosine backbone chain length; however, a fluorescent phosphatidylcholine analog was not internalized via caveolae, suggesting that the GSL ceramide core may be important for caveolar uptake. Internalization of fluorescent LacCer was reduced 80–90% in cell types with low cav-1, but was dramatically stimulated by cav-1 overexpression. However, even in cells with low levels of cav-1, residual LacCer internalization was clathrin independent. In contrast, cholera toxin B subunit (CtxB), which binds endogenous GM1, was internalized via clathrin-independent endocytosis in cells with high cav-1 expression, whereas significant clathrin-dependent uptake occurred in cells with low cav-1. Fluorescent GM1, normally internalized by clathrin-independent endocytosis in HeLa cells with low cav-1, was induced to partially internalize via the clathrin pathway in the presence of CtxB. These results suggest that GSL analogs are selectively internalized via a caveolar-related mechanism in most cell types, whereas CtxB may undergo “pathway switching” when cav-1 levels are low.

scholarly journals Spray-Dried Formulation of Epicertin, a Recombinant Cholera Toxin B Subunit Variant That Induces Mucosal Healing

Pharmaceutics ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 576
Author(s):  
Micaela A. Reeves ◽  
Joshua M. Royal ◽  
David A. Morris ◽  
Jessica M. Jurkiewicz ◽  
Nobuyuki Matoba ◽  
...  
Keyword(s):  
Gastric Acid ◽  
Cholera Toxin ◽  
Oral Formulation ◽  
Size Exclusion ◽  
Cholera Toxin B Subunit ◽  
Toxin B ◽  
Cholera Toxin B ◽  
B Subunit ◽  
Enteric Coated ◽  
Spray Dried

Epicertin (EPT) is a recombinant variant of the cholera toxin B subunit, modified with a C-terminal KDEL endoplasmic reticulum retention motif. EPT has therapeutic potential for ulcerative colitis treatment. Previously, orally administered EPT demonstrated colon epithelial repair activity in dextran sodium sulfate (DSS)-induced acute and chronic colitis in mice. However, the oral dosing requires cumbersome pretreatment with sodium bicarbonate to conserve the acid-labile drug substance while transit through the stomach, hampering its facile application in chronic disease treatment. Here, we developed a solid oral formulation of EPT that circumvents degradation in gastric acid. EPT was spray-dried and packed into enteric-coated capsules to allow for pH-dependent release in the colon. A GM1-capture KDEL-detection ELISA and size-exclusion HPLC indicated that EPT powder maintains activity and structural stability for up to 9 months. Capsule disintegration tests showed that EPT remained encapsulated at pH 1 but was released over 180 min at pH 6.8, the approximate pH of the proximal colon. An acute DSS colitis study confirmed the therapeutic efficacy of encapsulated EPT in C57BL/6 mice upon oral administration without gastric acid neutralization pretreatment compared to vehicle-treated mice (p < 0.05). These results provide a foundation for an enteric-coated oral formulation of spray-dried EPT.

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