Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Klein, Ofir; Roded, Amit; Hirschberg, Koret; Fukuda, Mitsunori; Galli, Stephen J.; Sagi‐Eisenberg, Ronit

doi:10.3791/57936

Cited by 5 publications

(5 citation statements)

References 77 publications

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“…The lysosome-mediated pathway was also studied. Cu could activate lysosome exocytosis that was usually associated with lysosome alkalization. − Therefore, cells were preincubated with FITC-dextran for evaluating the exocytosis process. , After being exposed with Au@CuO NPs, lysosome alkalization was observed in all cells (Figure S3). In addition, the inhibitor vacuolin-1 was used as it could block the Ca 2+ -dependent lysosome exocytosis.…”

Section: Resultsmentioning

confidence: 99%

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Cell-Type-Dependent Dissolution of CuO Nanoparticles and Efflux of Cu Ions following Cellular Internalization

Wang

2022

Environ. Sci. Technol.

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CuO nanoparticles (NPs) show promising applications in biosensors, waste treatment, and energy materials, but the growing manufacture of CuO NPs also leads to the concerns for their potential environmental and health risks. However, the cellular fates of CuO NPs such as Cu ion dissolution, transformation, and efflux remain largely speculative. In the present study, we for the first time combined the gold-core labeling and Cu ion bioimaging technologies to reveal the intracellular fates of CuO NPs in different cells following cellular internalization of NPs. We demonstrated that the dissolution rate of CuO NPs depended on the cell type. Following CuO dissolution, limited transformation of Cu(II) to Cu(I) occurred within the cellular microenvironment. Instead, Cu(II) was rapidly eliminated from the cells, and such rapid efflux in different cells was highly dependent on the GSH-mediated pathway and lysosome exocytosis. The labile Cu(I) level in the two cancerous cell lines was immediately regulated upon Cu exposure, which explained their tolerance to Au@CuO NPs. Overall, our study demonstrated a very rapid turnover of Cu in the cells following CuO internalization, which subsequently determined the cellular toxicity of CuO. The results will have important implications for assessing the health risk of CuO NPs.

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Section: Resultsmentioning

confidence: 99%

“…65−67 Therefore, cells were preincubated with FITC-dextran for evaluating the exocytosis process. 68,69 After being exposed with Au@CuO NPs, lysosome alkalization was observed in all cells (Figure S3). In addition, the inhibitor vacuolin-1 was used as it could block the Ca 2+ -dependent lysosome exocytosis.…”

Section: Toxicity Mechanisms Of Cuo Nps In Cellsmentioning

confidence: 99%

Cell-Type-Dependent Dissolution of CuO Nanoparticles and Efflux of Cu Ions following Cellular Internalization

Wang

2022

Environ. Sci. Technol.

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“…The observed increase of barrier function in endothelial cells (ECs) cultured with pericytes (PCs) on the opposing side of a transwell insert is in agreement with several previous studies [ 11 , 12 , 21 , 30 ]. The fact that the permeability to higher molecular weight dextrans (40 kDa) is decreased in presence of PCs indicates that trans-cellular transport pathways are affected in our co-culture model as such large molecules most probably travers the endothelium via non-specific transcytosis [ 31 ]. This observation is in agreement with a previous study showing increased trans-cellular pathways in pericyte-deficient mice [ 1 , 2 ].…”

Section: Discussionmentioning

confidence: 99%

Transcryptomic Analysis of Human Brain-Microvascular Endothelial Response to -Pericytes: Cell Orientation Defines Barrier Function

Kurmann

Okoniewski

Ogunshola

et al. 2021

Cells

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Pericytes facilitate blood–brain barrier (BBB) integrity; however, the mechanisms involved remain unclear. Hence, using co-cultures of human cerebral microvascular endothelial cells (ECs) and vascular pericytes (PCs) in different spatial arrangements, as well as PC conditioned media, we investigated the impact of PC-EC orientation and PC-derived soluble factors on EC barrier function. We provide the first evidence that barrier-inducing properties of PCs require basolateral contact with ECs. Gene expression analysis (GEA) in ECs co-cultured with PCs versus ECs alone showed significant upregulation of 38 genes and downregulation of 122 genes. Pathway enrichment analysis of modulated genes showed significant regulation of several pathways, including transforming growth factor-β and interleukin-1 regulated extracellular matrix, interferon and interleukin signaling, immune system signaling, receptor of advanced glycation end products (RAGE), and cytokine–cytokine receptor interaction. Transcriptomic analysis showed a reduction in molecules such as pro-inflammatory cytokines and chemokines, which are known to be induced during BBB disruption. Moreover, cytokine proteome array confirmed the downregulation of key pro-inflammatory cytokines and chemokines on the protein level. Other molecules which influence BBB and were favorably modulated upon EC-PC co-culture include IL-18 binding protein, kallikrein-3, CSF2 CSF3, CXCL10, CXCL11 (downregulated) and IL-1-R4; HGF, PDGF-AB/BB, PECAM, SERPIN E1 (upregulated). In conclusion, we provide the first evidence that (1) basolateral contact between ECs and PCs is essential for EC barrier function and integrity; (2) in ECs co-cultured with PCs, the profile of BBB disrupting pro-inflammatory molecules and cytokines/chemokines is downregulated; (3) PCs significantly modulate EC mechanisms known to improve barrier function, including TGF-β regulated ECM pathway, anti-inflammatory cytokines, growth factors and matrix proteins. This human PC-EC co-culture may serve as a viable in vitro model for investigating BBB function and drug transport.

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“…In this approach, the dye or transfected reporter is quenched when inside the acidic SG. However, once a fusion pore is formed and the SG's lumen alkalinizes due to its exposure to the external milieu, the dye/reporter regains their fluorescence, thus emitting a fluorescent signal concomitantly to the formation of the fusion pore [66, 82]. Based on this principle, FITC-dextran and β -hexosaminidase-pHluorin have been recently used for monitoring exocytosis in MCs [54, 63–65].…”

Section: Methods For Monitoring Compound Exocytosismentioning

confidence: 99%

“…Recording the robustness and duration of fluorescent signals also allows to distinguish between the different modes of exocytosis. For example, compound exocytosis can be distinguished from other exocytic modes simply through recording only long-lasting secretory events by setting long time intervals between acquisitions [82]. These modern live cell imaging techniques mark a new era, where exocytic events can be monitored in real time alongside protein trafficking and signaling events that occur in the cell.…”

Section: Methods For Monitoring Compound Exocytosismentioning

confidence: 99%

Anaphylactic Degranulation of Mast Cells: Focus on Compound Exocytosis

Klein

Sagi‐Eisenberg

2019

Journal of Immunology Research

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Anaphylaxis is a notorious type 2 immune response which may result in a systemic response and lead to death. A precondition for the unfolding of the anaphylactic shock is the secretion of inflammatory mediators from mast cells in response to an allergen, mostly through activation of the cells via the IgE-dependent pathway. While mast cells are specialized secretory cells that can secrete through a variety of exocytic modes, the most predominant mode exerted by the mast cell during anaphylaxis is compound exocytosis—a specialized form of regulated exocytosis where secretory granules fuse to one another. Here, we review the modes of regulated exocytosis in the mast cell and focus on compound exocytosis. We review historical landmarks in the research of compound exocytosis in mast cells and the methods available for investigating compound exocytosis. We also review the molecular mechanisms reported to underlie compound exocytosis in mast cells and expand further with reviewing key findings from other cell types. Finally, we discuss the possible reasons for the mast cell to utilize compound exocytosis during anaphylaxis, the conflicting evidence in different mast cell models, and the open questions in the field which remain to be answered.

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Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Cited by 5 publications

References 77 publications

Cell-Type-Dependent Dissolution of CuO Nanoparticles and Efflux of Cu Ions following Cellular Internalization

Cell-Type-Dependent Dissolution of CuO Nanoparticles and Efflux of Cu Ions following Cellular Internalization

Transcryptomic Analysis of Human Brain-Microvascular Endothelial Response to -Pericytes: Cell Orientation Defines Barrier Function

Anaphylactic Degranulation of Mast Cells: Focus on Compound Exocytosis

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