The Experts below are selected from a list of 1299 Experts worldwide ranked by ideXlab platform
Elizabeth M Fini - One of the best experts on this subject based on the ideXlab platform.
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Dynasore protects ocular surface mucosal epithelia subjected to oxidative stress by maintaining upr and calcium homeostasis
Free Radical Biology and Medicine, 2020Co-Authors: Rafael Martinezcarrasco, Pablo Argueso, Elizabeth M FiniAbstract:Abstract The mucosal epithelia of the ocular surface protect against external threats to the eye. Using a model of human stratified corneal epithelial cells with mucosal differentiation, we previously demonstrated that a small molecule inhibitor of dynamin GTPases, Dynasore, prevents damage to cells and their transcellular barriers when subjected to oxidative stress. Investigating mechanisms, we now report the novel finding that Dynasore acts by maintaining Ca+2 homeostasis, thereby inhibiting the PERK branch of the unfolded protein response (UPR) that promotes cell death. Dynasore was found to protect mitochondria by preventing mitochondrial permeability transition pore opening (mPTP), but, unlike reports using other systems, this was not mediated by dynamin family member DRP1. Necrostatin-1, an inhibitor of RIPK1 and lytic forms of programmed cell death, also inhibited mPTP opening and further protected the plasma membrane barrier. Significantly, necrostatin-1 did not protect the mucosal barrier. Oxidative stress increased mRNA for sXBP1, a marker of the IRE1 branch of the UPR, and CHOP, a marker of the PERK branch. It also stimulated phosphorylation of eIF2α, the upstream regulator of CHOP, as well as an increase in intracellular Ca2+. Dynasore selectively inhibited the increase in PERK branch markers, and also prevented the increase intracellular Ca2+ in response to oxidative stress. The increase in PERK branch markers were also inhibited when cells were treated with the cell permeable Ca2+ chelator, BAPTA-AM. To our knowledge, this is the first time that Dynasore has been shown to have an effect on the UPR and suggests therapeutic applications.
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Dynasore protects the ocular surface against damaging oxidative stress
PLOS ONE, 2018Co-Authors: Andrew Webster, Pablo Argueso, Shravan K Chintala, Michelle Ngan, Tatsuo Itakura, Noorjahan Panjwani, Joseph T Barr, Shinwu Jeong, Elizabeth M FiniAbstract:“Vital” dyes such as fluorescein and rose bengal are used clinically to evaluate ocular surface health; however, staining mechanisms remain poorly understood. Recent evidence suggests that sublethal cell damage stimulates fluorescein dye uptake. Since damage can also stimulate reparative plasma membrane remodeling, we hypothesized that dye uptake occurs via endocytic vesicles. Using an oxidative stress model, we show that damage to relatively undifferentiated monolayer cultures of human corneal epithelial cells stimulates uptake of fluorescein and rose bengal dyes and also stimulates endocytosis. Importantly, dye uptake was blocked by co-treatment with three different endocytosis inhibitors. Damage to stratified and differentiated corneal epithelial cell cultures, which are a better model of the ocular surface, also stimulated dye uptake; however, endocytosis was not stimulated in this case, and two of the inhibitors did not block dye uptake. The exception was the inhibitor Dynasore and its more potent analogue Dyngo-4a, small molecules that target dynamin family GTPases, but also have off-target effects on the plasma membrane. Significantly, while Dynasore blocked stress-stimulated dye uptake at the ocular surface of ex vivo mouse eyes when treatment was performed at the same time as eyes were stressed, it had no effect when used after stress was applied and the ocular surface was already damaged. Thus, Dynasore could not be working by inhibiting endocytosis. Employing cytotoxicity and western blotting assays, we demonstrate an alternative mechanism, showing that Dynasore is remarkably protective of cells and their surface glycocalyx, preventing damage due to oxidative stress, and thus precluding dye entry. These unexpected and novel findings provide greater insight into mechanisms of vital dye uptake and emphasize the importance of using a differentiated cell culture model for such studies. They also suggest that Dynasore and analogues might be used therapeutically to protect the ocular surface and to treat ocular surface disease.
Shinichi Ohno - One of the best experts on this subject based on the ideXlab platform.
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Function of Dynamin-2 in the Formation of Discoid Vesicles in Urinary Bladder Umbrella Cells
In Vivo Cryotechnique in Biomedical Research and Application for Bioimaging of Living Animal Organs, 2020Co-Authors: Nobuo Terada, Yurika Saitoh, Nobuhiko Ohno, Yasuhisa Fujii, Shinichi OhnoAbstract:Dynamin (Dyn) is a GTPase that promotes endocytic processes through scission of cell membranes. In this chapter, we present the contribution of Dyn2 to the structural integrity of discoid vesicles (DVs) and to the endocytic activity of urinary bladder umbrella cells (UCs) which have capacity to control bladder volume. Dyn2 was abundantly expressed in UCs, especially near the apical cytoplasmic regions. By immunoelectron microscopy, Dyn2 was found on and around DV membranes in UCs. Ultrastructural analysis with a quick-freezing and deep-etching method revealed the existence of distinct Dyn2-bound microfilaments in close association with DV membranes. Treatment of bladders with a Dyn-GTPase inhibitor, Dynasore, markedly reduced the number of DVs in UCs. In infected UCs, Escherichia coli was encased in compartments enriched in Dyn2, and treatment of bladders with Dynasore inhibits E. coli invasion of UCs.
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Involvement of dynamin-2 in formation of discoid vesicles in urinary bladder umbrella cells
Cell and Tissue Research, 2009Co-Authors: Nobuo Terada, Yurika Saitoh, Nobuhiko Ohno, Yasuhisa Fujii, Sei Saitoh, Tetsuo Kondo, Ryohei Katoh, Cheryl Chan, Soman N. Abraham, Shinichi OhnoAbstract:Umbrella cells (UCs) of the epithelium of the urinary bladder have the capacity to control bladder volume by regulating exocytosis/endocytosis of their intracellular discoid vesicles (DVs). Dynamin (Dyn) is a GTPase that promotes endocytic processes through scission of cell membranes. We have examined whether Dyn2, the most abundant Dyn form, is expressed in UCs and contributes to their endocytic actions. A specific antibody against Dyn2 was used to localize Dyn2 in human and rodent UCs by immunohistochemistry. To clarify the functional roles of Dyn2, mouse bladders were treated with a Dyn-GTPase inhibitor, Dynasore, and its effects on their UC structure were assessed. Since uropathogenic Escherichia coli can be encased into UCs during infection, we used immunohistochemistry to determine whether bacteria-encasing compartments in the infected UCs were also enriched with Dyn2. Light microscopy showed that Dyn2 was abundantly expressed in UCs, especially near the apical cytoplasmic regions. By immunoelectron microscopy, Dyn2 was found on and around DV membranes in UCs. Ultrastructural analysis with a quick-freezing and deep-etching method confirmed these findings and revealed the existence of distinct Dyn2-bound microfilaments in close association with DV membranes. Dynasore treatment of bladders markedly reduced the number of DVs in UCs. In infected UCs, E. coli was encased in compartments enriched in Dyn2. Therefore, Dyn2 is highly enriched in UCs and mostly associated with membranes of DVs and microfilaments in the UCs. Pretreatment of bladders with Dynasore inhibits E. coli invasion of UCs. Dyn2 thus contributes to the structural integrity of DVs and to the endocytic activity of UCs.
Magdalena Cañete - One of the best experts on this subject based on the ideXlab platform.
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Vehiculization determines the endocytic internalization mechanism of Zn(II)-phthalocyanine
Histochemistry and Cell Biology, 2012Co-Authors: Jorge Soriano, Juan C. Stockert, Angeles Villanueva, Magdalena CañeteAbstract:It is generally accepted that compounds of nanomolecular size penetrate into cells by different endocytic processes. The vehiculization strategy of a compound is a factor that could determine its uptake mechanism. Understanding the influence of the vehicle in the precise mechanism of drug penetration into cells makes possible to improve or modify the therapeutic effects. In this study, using human A-549 cells, we have characterized the possible internalization mechanism of the photosensitizer Zn(II)-phthalocyanine (ZnPc), either dissolved in dimethylformamide (ZnPc–DMF) or included in liposomes of dipalmitoyl-phosphatidyl-choline. Specific inhibitors involved in the main endocytic pathways were used. Co-incubation of cells with ZnPc–liposomes and Dynasore (dinamin-mediated endocytosis inhibitor) resulted in a significant decrease of photodamage, whereas other inhibitors did not alter the photodynamic effect of ZnPc. On the contrary, cells treated with ZnPc–DMF in the presence of Dynasore, genistein (caveolin-mediated endocytosis inhibitor) or cytochalasin D (macropinocytosis and caveolin-mediated endocytosis inhibitor) showed a significant decrease in ZnPc uptake and photodynamic damage. These results suggest that ZnPc–DMF penetrates into cells mainly by caveolin-mediated endocytosis, whereas ZnPc–liposomes are internalized into cells preferentially by clathrin-mediated endocytosis. We conclude that using different drug vehiculization systems, it is possible to modify the internalization mechanism of a therapeutic compound, which could be of great interest in clinical research.
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Cell uptake of Zn(II)-phthalocyanine-containing liposomes by clathrin-mediated endocytosis
Histochemistry and Cell Biology, 2010Co-Authors: Jorge Soriano, Juan C. Stockert, Angeles Villanueva, Magdalena CañeteAbstract:The study of uptake mechanisms of therapeutic drugs has a growing interest in biomedical research. In this work the cell uptake and phototoxicity of the photosensitizer Zn(II)-phthalocyanine (ZnPc) in dipalmitoyl-phosphatidyl-choline liposomes have been studied in the presence or absence of inhibitors of macropinocytosis (cytochalasin D), and clathrin-mediated endocytosis (Dynasore). No differences in the uptake or photodynamic damage were observed in A-549 cells subjected to incubation with either ZnPc alone or in combination with cytochalasin D. On the contrary, co-incubation of A-549 cells with ZnPc and Dynasore resulted in a significant decrease of photodamage as well as negligible uptake of the photosensitizer. These results indicate that ZnPc is internalized into cells preferentially by a mechanism of clathrin-mediated endocytosis.
Nobuo Terada - One of the best experts on this subject based on the ideXlab platform.
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Function of Dynamin-2 in the Formation of Discoid Vesicles in Urinary Bladder Umbrella Cells
In Vivo Cryotechnique in Biomedical Research and Application for Bioimaging of Living Animal Organs, 2020Co-Authors: Nobuo Terada, Yurika Saitoh, Nobuhiko Ohno, Yasuhisa Fujii, Shinichi OhnoAbstract:Dynamin (Dyn) is a GTPase that promotes endocytic processes through scission of cell membranes. In this chapter, we present the contribution of Dyn2 to the structural integrity of discoid vesicles (DVs) and to the endocytic activity of urinary bladder umbrella cells (UCs) which have capacity to control bladder volume. Dyn2 was abundantly expressed in UCs, especially near the apical cytoplasmic regions. By immunoelectron microscopy, Dyn2 was found on and around DV membranes in UCs. Ultrastructural analysis with a quick-freezing and deep-etching method revealed the existence of distinct Dyn2-bound microfilaments in close association with DV membranes. Treatment of bladders with a Dyn-GTPase inhibitor, Dynasore, markedly reduced the number of DVs in UCs. In infected UCs, Escherichia coli was encased in compartments enriched in Dyn2, and treatment of bladders with Dynasore inhibits E. coli invasion of UCs.
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Involvement of dynamin-2 in formation of discoid vesicles in urinary bladder umbrella cells
Cell and Tissue Research, 2009Co-Authors: Nobuo Terada, Yurika Saitoh, Nobuhiko Ohno, Yasuhisa Fujii, Sei Saitoh, Tetsuo Kondo, Ryohei Katoh, Cheryl Chan, Soman N. Abraham, Shinichi OhnoAbstract:Umbrella cells (UCs) of the epithelium of the urinary bladder have the capacity to control bladder volume by regulating exocytosis/endocytosis of their intracellular discoid vesicles (DVs). Dynamin (Dyn) is a GTPase that promotes endocytic processes through scission of cell membranes. We have examined whether Dyn2, the most abundant Dyn form, is expressed in UCs and contributes to their endocytic actions. A specific antibody against Dyn2 was used to localize Dyn2 in human and rodent UCs by immunohistochemistry. To clarify the functional roles of Dyn2, mouse bladders were treated with a Dyn-GTPase inhibitor, Dynasore, and its effects on their UC structure were assessed. Since uropathogenic Escherichia coli can be encased into UCs during infection, we used immunohistochemistry to determine whether bacteria-encasing compartments in the infected UCs were also enriched with Dyn2. Light microscopy showed that Dyn2 was abundantly expressed in UCs, especially near the apical cytoplasmic regions. By immunoelectron microscopy, Dyn2 was found on and around DV membranes in UCs. Ultrastructural analysis with a quick-freezing and deep-etching method confirmed these findings and revealed the existence of distinct Dyn2-bound microfilaments in close association with DV membranes. Dynasore treatment of bladders markedly reduced the number of DVs in UCs. In infected UCs, E. coli was encased in compartments enriched in Dyn2. Therefore, Dyn2 is highly enriched in UCs and mostly associated with membranes of DVs and microfilaments in the UCs. Pretreatment of bladders with Dynasore inhibits E. coli invasion of UCs. Dyn2 thus contributes to the structural integrity of DVs and to the endocytic activity of UCs.
Pablo Argueso - One of the best experts on this subject based on the ideXlab platform.
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Dynasore protects ocular surface mucosal epithelia subjected to oxidative stress by maintaining upr and calcium homeostasis
Free Radical Biology and Medicine, 2020Co-Authors: Rafael Martinezcarrasco, Pablo Argueso, Elizabeth M FiniAbstract:Abstract The mucosal epithelia of the ocular surface protect against external threats to the eye. Using a model of human stratified corneal epithelial cells with mucosal differentiation, we previously demonstrated that a small molecule inhibitor of dynamin GTPases, Dynasore, prevents damage to cells and their transcellular barriers when subjected to oxidative stress. Investigating mechanisms, we now report the novel finding that Dynasore acts by maintaining Ca+2 homeostasis, thereby inhibiting the PERK branch of the unfolded protein response (UPR) that promotes cell death. Dynasore was found to protect mitochondria by preventing mitochondrial permeability transition pore opening (mPTP), but, unlike reports using other systems, this was not mediated by dynamin family member DRP1. Necrostatin-1, an inhibitor of RIPK1 and lytic forms of programmed cell death, also inhibited mPTP opening and further protected the plasma membrane barrier. Significantly, necrostatin-1 did not protect the mucosal barrier. Oxidative stress increased mRNA for sXBP1, a marker of the IRE1 branch of the UPR, and CHOP, a marker of the PERK branch. It also stimulated phosphorylation of eIF2α, the upstream regulator of CHOP, as well as an increase in intracellular Ca2+. Dynasore selectively inhibited the increase in PERK branch markers, and also prevented the increase intracellular Ca2+ in response to oxidative stress. The increase in PERK branch markers were also inhibited when cells were treated with the cell permeable Ca2+ chelator, BAPTA-AM. To our knowledge, this is the first time that Dynasore has been shown to have an effect on the UPR and suggests therapeutic applications.
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Dynasore Protects Ocular Surface Mucosal Epithelia Subjected to Oxidative Stress by Maintaining UPR and Calcium Homeostasis
Free Radical Biology and Medicine, 2020Co-Authors: Rafael Martinez-carrasco, Pablo Argueso, M. Elizabeth FiniAbstract:Abstract The mucosal epithelia of the ocular surface protect against external threats to the eye. Using a model of human stratified corneal epithelial cells with mucosal differentiation, we previously demonstrated that a small molecule inhibitor of dynamin GTPases, Dynasore, prevents damage to cells and their transcellular barriers when subjected to oxidative stress. Investigating mechanisms, we now report the novel finding that Dynasore acts by maintaining Ca+2 homeostasis, thereby inhibiting the PERK branch of the unfolded protein response (UPR) that promotes cell death. Dynasore was found to protect mitochondria by preventing mitochondrial permeability transition pore opening (mPTP), but, unlike reports using other systems, this was not mediated by dynamin family member DRP1. Necrostatin-1, an inhibitor of RIPK1 and lytic forms of programmed cell death, also inhibited mPTP opening and further protected the plasma membrane barrier. Significantly, necrostatin-1 did not the protect the mucosal barrier. Oxidative stress increased mRNA for sXBP1, a marker of the IRE1 branch of the UPR, and CHOP, a marker of the PERK branch. It also stimulated phosphorylation of eIF2α, the upstream regulator of CHOP, as well as an increase in intracellular Ca2+. Dynasore selectively inhibited the increase in PERK branch markers, and also prevented the increase intracellular Ca2+ in response to oxidative stress. The increase in PERK branch markers were also inhibited when cells were treated with the cell permeable Ca2+ chelator, BAPTA-AM. To our knowledge, this is the first time that Dynasore has been shown to have an effect on the UPR and suggests therapeutic applications.
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Dynasore protects the ocular surface against damaging oxidative stress
PLOS ONE, 2018Co-Authors: Andrew Webster, Pablo Argueso, Shravan K Chintala, Michelle Ngan, Tatsuo Itakura, Noorjahan Panjwani, Joseph T Barr, Shinwu Jeong, Elizabeth M FiniAbstract:“Vital” dyes such as fluorescein and rose bengal are used clinically to evaluate ocular surface health; however, staining mechanisms remain poorly understood. Recent evidence suggests that sublethal cell damage stimulates fluorescein dye uptake. Since damage can also stimulate reparative plasma membrane remodeling, we hypothesized that dye uptake occurs via endocytic vesicles. Using an oxidative stress model, we show that damage to relatively undifferentiated monolayer cultures of human corneal epithelial cells stimulates uptake of fluorescein and rose bengal dyes and also stimulates endocytosis. Importantly, dye uptake was blocked by co-treatment with three different endocytosis inhibitors. Damage to stratified and differentiated corneal epithelial cell cultures, which are a better model of the ocular surface, also stimulated dye uptake; however, endocytosis was not stimulated in this case, and two of the inhibitors did not block dye uptake. The exception was the inhibitor Dynasore and its more potent analogue Dyngo-4a, small molecules that target dynamin family GTPases, but also have off-target effects on the plasma membrane. Significantly, while Dynasore blocked stress-stimulated dye uptake at the ocular surface of ex vivo mouse eyes when treatment was performed at the same time as eyes were stressed, it had no effect when used after stress was applied and the ocular surface was already damaged. Thus, Dynasore could not be working by inhibiting endocytosis. Employing cytotoxicity and western blotting assays, we demonstrate an alternative mechanism, showing that Dynasore is remarkably protective of cells and their surface glycocalyx, preventing damage due to oxidative stress, and thus precluding dye entry. These unexpected and novel findings provide greater insight into mechanisms of vital dye uptake and emphasize the importance of using a differentiated cell culture model for such studies. They also suggest that Dynasore and analogues might be used therapeutically to protect the ocular surface and to treat ocular surface disease.