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(a) Total internal reflection fluorescence microscopy (TIRFM) to reveal vesicle trafficking. Left top: the schematics of evanescence illumination of the thin subplasmalemmal region (o200 nm). Right: A typical TIRFM image of the footprints of secretory vesicles in PC12 cells. The vesicles (bright dots, highlighted in circles) are enlightened by overexpression of NPY-EGFP. The cell contour is outlined by the dashed line. The scale bar = 5 mm. Left bottom: A typical trajectory of vesicle lateral movement. The rectangle (dotted line) that just encases all the footprints of the vesicle gives the first estimation of the coverage area of vesicle lateral motion. The scale bars = 100 nm. (b)–(e) The statistics of the average vesicle number in the subplasmalemmal region (b), the vesicles arrived from the cytosol (c), the diffusion constant (d), and the motion coverage area (e) for cells grown on PLL(open columns) or CTB-coated (dark columns) coverslips without (w.o. MbCD) or with MbCD treatment (w. MbCD). Each data is analyzed from the time-lapse images with a duration of 2 min and an interval of 0.5 s, from 11 cells with 618 vesicles (PLL, w.o. MbCD), 11 cells with 960 vesicles (CTB, w.o. MbCD), 10 cells with 329 vesicles (PLL, w. MbCD) and 10 cells with 300 vesicles (CTB, w. MbCD). The error bars indicate SE. *** p o 0.001 in Student's t-test.
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The subunit B of cholera toxin (CTB), which specifically binds with ganglioside GM1 enriched in membrane lipid rafts, is known to interfere with multiple cell functions. However, the specific, stable and spatially defined membrane signaling induced by CTB binding is often difficult to investigate by applying CTB molecules in bulk solution due to qu...
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... and priming to become readily releasable, and final fusion with plasma membrane upon triggering. The recently emerged total internal reflection fluorescence microscopy (TIRFM), which evanescently and selectively illuminate the thin subplasmalemmal region (o200 nm thick) just above the interface between the coverslip and the cell membrane ( Fig. 4a, left top), is instrumental in revealing the vesicle dynamics near the cell membrane. 19,20 The large dense core vesicles (LDCV) inside PC12 cells were enlightened by overexpressing EGFP tagged neuropeptide Y (NPY-EGFP) which is a specific LDCV marker. 21 The fluor- escently labeled secretory vesicles in the subplasmalemmal region can ...
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... in revealing the vesicle dynamics near the cell membrane. 19,20 The large dense core vesicles (LDCV) inside PC12 cells were enlightened by overexpressing EGFP tagged neuropeptide Y (NPY-EGFP) which is a specific LDCV marker. 21 The fluor- escently labeled secretory vesicles in the subplasmalemmal region can be individually resolved under TIRFM. Fig. 4a (right) presents a typical TIRFM image of a PC12 cell grown on the CTB functionalized coverslip. As revealed by TIRFM 14,19,20 secretory vesicles undertake constant lateral movement (parallel to the cell membrane) and vertical movement (transition between inner cytosol and subplasma- lemmal region). The trafficking dynamics of the ...
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... lemmal region). The trafficking dynamics of the secretory vesicles are highly relevant to the vesicle fusion competence and exocytotic kinetics. 20,22,23 The LDCV vesicles in PC12 cells were individually tracked at 2 Hz for 2 min to obtain the trajectory of their lateral movement, diffusion constant, motion area, and the vertical transport. Fig. 4a (left bottom) depicts a typical trajectory of a vesicle's lateral movement, whose coverage area can be approximated by the rectangular region that just encases all the vesicle ...
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... the subplasmalemmal vesicles stays nearly constant as the arrival and retrieval rates are balanced. It was found that the mean total number of the subplasmalemmal vesicles in the cells grown on CTB coated coverslips was significantly greater than that in the cells on PLL coated coverslips, implying that CTB interaction facilitates vesicle docking (Fig. 4b). In addition, CTB coating enhanced the vertical trafficking as evidenced by the increase in vesicle arrival from the inner cytosol ( Fig. 4c) and enhanced the lateral motion as evidenced by the increased average diffusion constant (Fig. 4d) and motion coverage area (Fig. ...
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... the subplasmalemmal vesicles in the cells grown on CTB coated coverslips was significantly greater than that in the cells on PLL coated coverslips, implying that CTB interaction facilitates vesicle docking (Fig. 4b). In addition, CTB coating enhanced the vertical trafficking as evidenced by the increase in vesicle arrival from the inner cytosol ( Fig. 4c) and enhanced the lateral motion as evidenced by the increased average diffusion constant (Fig. 4d) and motion coverage area (Fig. ...
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... than that in the cells on PLL coated coverslips, implying that CTB interaction facilitates vesicle docking (Fig. 4b). In addition, CTB coating enhanced the vertical trafficking as evidenced by the increase in vesicle arrival from the inner cytosol ( Fig. 4c) and enhanced the lateral motion as evidenced by the increased average diffusion constant (Fig. 4d) and motion coverage area (Fig. ...
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... coverslips, implying that CTB interaction facilitates vesicle docking (Fig. 4b). In addition, CTB coating enhanced the vertical trafficking as evidenced by the increase in vesicle arrival from the inner cytosol ( Fig. 4c) and enhanced the lateral motion as evidenced by the increased average diffusion constant (Fig. 4d) and motion coverage area (Fig. ...
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... cyclodextrin (MbCD), a cholesterol extracting agent which disrupts lipid rafts, diminishes the clustering of GM1 on the cell membrane 24 and impairs CTB binding to the lipid rafts. 25 Here, we found that MbCD treatment (incubation with 5 mM MbCD for 30 min at 37 1C) 14 completely abolished the CTB effects on vesicle docking and trafficking (Fig. 4), indicating that the observed CTB effects are cholesterol dependent. Membrane cholesterol was removed by 37.04 AE 2.57% (3 independent experiments) after MbCD treatment (measured by Amplex Red Cholesterol Assay Kit from ...
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... facilitating roles of CTB on vesicle docking and trafficking were corroborated by the TIRFM experiments on cells grown on dual micropatterns of CTB and PLL. As shown in Fig. 5a and b, more vesicles were localized on the CTB patterns rather than on the PLL patterns. Consistent with the results demonstrated in Fig. 4, vesicles on the CTB patterns exhibited enhanced vertical and lateral trafficking, and cholesterol depletion by MbCD completely eliminated the facilitating effects of the CTB patterns (Fig. 5). Deprivation of membrane cholesterol by culturing the cells in the lipo- protein deficient serum (LPDS, Sigma) for 5 days gave similar results ...
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... to test this hypothesis. As demonstrated in Fig. 6a and b, the extent of exocytosis was significantly augmented when the cells grew on CTB substrates as compared to those grown on PLL sub- strates. Such increase in the frequency of exocytotic response is likely attributable, at least in part, to the enhanced vesicle docking and trafficking (Fig. 4 and 5). The total charge (quantal size) of each amperometric spike, which reflects the total number of released molecules, was also signifi- cantly increased by CTB coating ( Fig. 6c and d). The CTB- enhanced quantal vesicle release was completely abolished by deprivation of membrane cholesterol using MbCD. Interestingly, although ...
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The AB5 toxins cholera toxin (CT) from Vibrio cholerae and heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli are notorious for their roles in diarrheal disease, but their effect on other intestinal bacteria remains unexplored. Another foodborne pathogen, Campylobacter jejuni, can mimic the GM1 ganglioside receptor of CT and LT. Her...
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Antibodies to the ganglioside GM1 are associated with various forms of acute and chronic immune-mediated neuropathy, including Guillain-Barré syndrome (GBS) and multifocal motor neuropathy. In diagnostics and research, these antibodies are usually detected by GM1 preparations derived from bovine brain tissue, which are non-covalently attached to so...
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... However, there is still no consensus on whether cell exposure to CTxB elicits the secretion of pro-inflammatory or anti-inflammatory cytokines [12][13][14][15][16]. As both signaling pathways and lipid rafts play a crucial role in endocytosis [17], we hypothesized that treating cells with CTxB could potentially modulate the endocytosis and trafficking pathways of silica NPs. As a positive control, we treated cells with lipopolysaccharide (LPS), which is known to induce a proinflammatory response and promote the NP uptake in J774A.1 cells via binding to Toll-like receptor 4 (TLR4) [18]. ...
The gastrointestinal tract is the main target of orally ingested nanoparticles (NPs) and at the same time is exposed to noxious substances, such as bacterial components. We investigated the interaction of 59 nm silica (SiO2) NPs with differentiated Caco-2 intestinal epithelial cells in the presence of cholera toxin subunit B (CTxB) and compared the effects to J774A.1 macrophages. CTxB can affect cellular functions and modulate endocytosis via binding to the monosialoganglioside (GM1) receptor, expressed on both cell lines. After stimulating macrophages with CTxB, we observed notable changes in the membrane structure but not in Caco-2 cells, and no secretion of the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) was detected. Cells were then exposed to 59 nm SiO2 NPs and CtxB sequentially and simultaneously, resulting in a high NP uptake in J774A.1 cells, but no uptake in Caco-2 cells was detected. Flow cytometry analysis revealed that the exposure of J774A.1 cells to CTxB resulted in a significant reduction in the uptake of SiO2 NPs. In contrast, the uptake of NPs by highly selective Caco-2 cells remained unaffected following CTxB exposure. Based on colocalization studies, CTxB and NPs might enter cells via shared endocytic pathways, followed by their sorting into different intracellular compartments. Our findings provide new insights into CTxB’s function of modulating SiO2 NP uptake in phagocytic but not in differentiated intestine cells.
... (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) associated with the CTA subunit (Soo et al., 2010). It has been shown that CTB acts as a strong adjuvant to uncoupled antigens by administration through the nasal route but less so when administered orally (Fukuyama et al., 2015). ...
Tetanus is an infective disease caused by the Gram-negative bacilli, the Clostridium tetani which releases its toxin causing muscular spasm which may terminate with death. Current tetanus vaccine, in the time it is effective, it is laborious and costly to produce and needs alum as an adjuvant. The aim of this work was to produce non-toxic part of tetanus toxin genetically joined to the potent adjuvant cholera toxin subunit B (CTB) to be used as a vaccine. The CTB was genetically linked to heavy chain of tetanus toxin (Hc) gene to replace alum. Computational analysis of the proposed CTB-Hc gene cassette (insert) to test restriction sites, primary, secondary and tertiary structure of proposed protein, open reading frames, protein folding and interaction and testing of antigenicity were done. Design of final synthetic insert containing restriction sites in both ends was done and submitted to GenBank. The insert was cloned into pUC57 cloning plasmid and then subcloned into pQE-30 expression plasmid. M15 bacteria were transformed with CTB-Hc-pQE30 recombinant vector and recombinant CTB-Hc protein was expressed and purified. Identity of purified protein was tested using SDS-PAGE. The gene construct submitted to GenBank was given the accession No: KX022510. Analysis of expected fusion protein showed that the proposed protein is immunogenic and in right 3D structure with no interference of both proteins on antigenic determinant of each other. The insert in both pUC57 and pQE-30 was found to be at right orientation and open reading frames. Analysis of recombinant CTB-Hc fusion protein showed that the protein at right expected molecular weight. In conclusion, construction of DNA segment encoding for the non-toxic Hc gene genetically linked to the highly immunogenic non-toxic CTB gene in one cassette was possible. This lead to the production of recombinant tetanus-cholera double protein in one expression system with a save and economic feedback. Further studies to test doses and prolongation of the vaccine are needed.
Few studies regarding the anatomical distribution of motor neurons innervating muscles of the arm have been demonstrated in avian brains. The purpose of this study was to finely determine the localization of cerebral neurons innervating the biceps brachii muscle in the pigeon. The cholera toxin B subunit (CTB) was employed as a retrograde tracer to determine the location of neurons controlling the biceps brachii muscle in the telencephalon following intramuscular injection in male pigeons (n = 7), which were killed 14 days after intramuscular injection with CTB. We found that CTB-labelled neurons were located contralaterally in the hyperpallium apicale of the rostral telencephalon and that most of the CTB-labelled neurons were pyramidal in shape. This study shows that CTB is easily taken up by nerve terminals which innervate the biceps brachii muscle of the pigeon and that cerebral motor neurons controlling the biceps brachii muscle are located in the hyperpallium apicale.
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Patterning substrates with versatile chemical functionalities from micro- to nanometer scale is a long-standing and interesting topic. This review provides an overview of a range of techniques commonly used for surface patterning. The first section briefly introduces conventional micropatterning tools, such as photolithography and microcontact printing. The second section focuses on the currently used nanolithographic techniques, for example, scanning probe lithography (SPL), and their applications in surface patterning. Their advantages and disadvantages are also demonstrated. In the last section, dip-pen nanolithography (DPN) is emphatically illustrated, with a particular stress on the patterning and applications of biomolecules.
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Surface modification with functional polymers or molecules offers great promise for the development of smart materials and applications. Here, we describe a versatile and easy-to-use method of site-selective surface modification based on the ease of microcontact printing and the exquisite selectivity of enzymatic degradation. A micropatterned poly-L-lysine (PLL) layer on solid substrates was prepared by enzymatic degradation using trypsin enzyme immobilized on a prestructured poly(dimethlylsiloxane) (PDMS) stamp. After the enzymatic degradation of PLL and the removal of the degradation products, very well defined patterning was revealed over a large scale by fluorescence microscopy and atomic force microscopy (AFM). We investigate the advantage of our method by comparison with traditional microcontact printing and found that lateral diffusion was reduced, yielding a more accurate reproduction of the master. We also demonstrate that the stamp can be reused without reinking. The patterned surface was used for site-selective modification. The strategy was applied to two applications: the first is dedicated to the creation of amino-silane patterned surfaces, and the second illustrates the possibility of patterning polyelectrolyte multilayered thin films.
