Biotinylated θ-toxin derivative as a probe to examine intracellular cholesterol-rich domains in normal and Niemann-Pick type C1 cells

BCθ is a proteolytically nicked and biotinylated derivative of a cholesterol binding protein perfringolysin O (θ-toxin), and has been used to detect cholesterol-rich domains at the plasma membrane (PM). Here we show that by modifying the cell fixation condition, BCθ can also be used to detect choles...
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Autor*in:	Shigeki Sugii [verfasserIn] Patrick C. Reid [verfasserIn] Nobutaka Ohgami [verfasserIn] Yukiko Shimada [verfasserIn] Robert A. Maue [verfasserIn] Haruaki Ninomiya [verfasserIn] Yoshiko Ohno-Iwashita [verfasserIn] Ta-Yuan Chang [verfasserIn]

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2003

Schlagwörter:	BC θ perfringolysin O toxin cholesterol metabolism cholesterol stain intracellular cholesterol trafficking cholesterol mutants

Übergeordnetes Werk:	In: Journal of Lipid Research - Elsevier, 2021, 44(2003), 5, Seite 1033-1041
Übergeordnetes Werk:	volume:44 ; year:2003 ; number:5 ; pages:1033-1041

Links:	Link aufrufen Link aufrufen Link aufrufen Journal toc

DOI / URN:	10.1194/jlr.D200036-JLR200

Katalog-ID:	DOAJ061596841

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spelling	10.1194/jlr.D200036-JLR200 doi (DE-627)DOAJ061596841 (DE-599)DOAJ126374b3ddf84e7dac98a60a4e3ae71d DE-627 ger DE-627 rakwb eng QD415-436 Shigeki Sugii verfasserin aut Biotinylated θ-toxin derivative as a probe to examine intracellular cholesterol-rich domains in normal and Niemann-Pick type C1 cells 2003 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier BCθ is a proteolytically nicked and biotinylated derivative of a cholesterol binding protein perfringolysin O (θ-toxin), and has been used to detect cholesterol-rich domains at the plasma membrane (PM). Here we show that by modifying the cell fixation condition, BCθ can also be used to detect cholesterol-rich domains intracellularly. When cells were processed for PM cholesterol staining, the difference in BCθ signals between the CT43 (CT) cell, a mutant Chinese hamster ovary cell line lacking the Niemann-Pick type C1 (NPC1) protein, and its parental cell 25RA (RA) was minimal. However, when cells were fixed with 4% paraformaldehyde, they became permeable to BCθ. Under this condition, BCθ mainly stained cholesterol-rich domains inside the cells, with the signal being much stronger in CT cells than in RA cells. The sensitivity of BCθ staining was superior to that of filipin staining. The staining of cholesterol-rich domain(s) inside RA cells was sensitive to β-cyclodextrin treatment, while most of the staining inside CT cells was relatively resistant to cyclodextrin treatment. Clear differences in intracellular BCθ staining were also seen between the normal and mutant NPC1 fibroblasts of human or mouse origin.Thus, BCθ is a powerful tool for visually monitoring cholesterol-rich domains inside normal and NPC cells. BC θ perfringolysin O toxin cholesterol metabolism cholesterol stain intracellular cholesterol trafficking cholesterol mutants Biochemistry Patrick C. Reid verfasserin aut Nobutaka Ohgami verfasserin aut Yukiko Shimada verfasserin aut Robert A. Maue verfasserin aut Haruaki Ninomiya verfasserin aut Yoshiko Ohno-Iwashita verfasserin aut Ta-Yuan Chang verfasserin aut In Journal of Lipid Research Elsevier, 2021 44(2003), 5, Seite 1033-1041 (DE-627)26601593X (DE-600)1466675-3 15397262 nnns volume:44 year:2003 number:5 pages:1033-1041 https://doi.org/10.1194/jlr.D200036-JLR200 kostenfrei https://doaj.org/article/126374b3ddf84e7dac98a60a4e3ae71d kostenfrei http://www.sciencedirect.com/science/article/pii/S0022227520311494 kostenfrei https://doaj.org/toc/0022-2275 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_252 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2006 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 44 2003 5 1033-1041
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allfieldsGer	10.1194/jlr.D200036-JLR200 doi (DE-627)DOAJ061596841 (DE-599)DOAJ126374b3ddf84e7dac98a60a4e3ae71d DE-627 ger DE-627 rakwb eng QD415-436 Shigeki Sugii verfasserin aut Biotinylated θ-toxin derivative as a probe to examine intracellular cholesterol-rich domains in normal and Niemann-Pick type C1 cells 2003 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier BCθ is a proteolytically nicked and biotinylated derivative of a cholesterol binding protein perfringolysin O (θ-toxin), and has been used to detect cholesterol-rich domains at the plasma membrane (PM). Here we show that by modifying the cell fixation condition, BCθ can also be used to detect cholesterol-rich domains intracellularly. When cells were processed for PM cholesterol staining, the difference in BCθ signals between the CT43 (CT) cell, a mutant Chinese hamster ovary cell line lacking the Niemann-Pick type C1 (NPC1) protein, and its parental cell 25RA (RA) was minimal. However, when cells were fixed with 4% paraformaldehyde, they became permeable to BCθ. Under this condition, BCθ mainly stained cholesterol-rich domains inside the cells, with the signal being much stronger in CT cells than in RA cells. The sensitivity of BCθ staining was superior to that of filipin staining. The staining of cholesterol-rich domain(s) inside RA cells was sensitive to β-cyclodextrin treatment, while most of the staining inside CT cells was relatively resistant to cyclodextrin treatment. Clear differences in intracellular BCθ staining were also seen between the normal and mutant NPC1 fibroblasts of human or mouse origin.Thus, BCθ is a powerful tool for visually monitoring cholesterol-rich domains inside normal and NPC cells. BC θ perfringolysin O toxin cholesterol metabolism cholesterol stain intracellular cholesterol trafficking cholesterol mutants Biochemistry Patrick C. Reid verfasserin aut Nobutaka Ohgami verfasserin aut Yukiko Shimada verfasserin aut Robert A. Maue verfasserin aut Haruaki Ninomiya verfasserin aut Yoshiko Ohno-Iwashita verfasserin aut Ta-Yuan Chang verfasserin aut In Journal of Lipid Research Elsevier, 2021 44(2003), 5, Seite 1033-1041 (DE-627)26601593X (DE-600)1466675-3 15397262 nnns volume:44 year:2003 number:5 pages:1033-1041 https://doi.org/10.1194/jlr.D200036-JLR200 kostenfrei https://doaj.org/article/126374b3ddf84e7dac98a60a4e3ae71d kostenfrei http://www.sciencedirect.com/science/article/pii/S0022227520311494 kostenfrei https://doaj.org/toc/0022-2275 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_252 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2006 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 44 2003 5 1033-1041
allfieldsSound	10.1194/jlr.D200036-JLR200 doi (DE-627)DOAJ061596841 (DE-599)DOAJ126374b3ddf84e7dac98a60a4e3ae71d DE-627 ger DE-627 rakwb eng QD415-436 Shigeki Sugii verfasserin aut Biotinylated θ-toxin derivative as a probe to examine intracellular cholesterol-rich domains in normal and Niemann-Pick type C1 cells 2003 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier BCθ is a proteolytically nicked and biotinylated derivative of a cholesterol binding protein perfringolysin O (θ-toxin), and has been used to detect cholesterol-rich domains at the plasma membrane (PM). Here we show that by modifying the cell fixation condition, BCθ can also be used to detect cholesterol-rich domains intracellularly. When cells were processed for PM cholesterol staining, the difference in BCθ signals between the CT43 (CT) cell, a mutant Chinese hamster ovary cell line lacking the Niemann-Pick type C1 (NPC1) protein, and its parental cell 25RA (RA) was minimal. However, when cells were fixed with 4% paraformaldehyde, they became permeable to BCθ. Under this condition, BCθ mainly stained cholesterol-rich domains inside the cells, with the signal being much stronger in CT cells than in RA cells. The sensitivity of BCθ staining was superior to that of filipin staining. The staining of cholesterol-rich domain(s) inside RA cells was sensitive to β-cyclodextrin treatment, while most of the staining inside CT cells was relatively resistant to cyclodextrin treatment. Clear differences in intracellular BCθ staining were also seen between the normal and mutant NPC1 fibroblasts of human or mouse origin.Thus, BCθ is a powerful tool for visually monitoring cholesterol-rich domains inside normal and NPC cells. BC θ perfringolysin O toxin cholesterol metabolism cholesterol stain intracellular cholesterol trafficking cholesterol mutants Biochemistry Patrick C. Reid verfasserin aut Nobutaka Ohgami verfasserin aut Yukiko Shimada verfasserin aut Robert A. Maue verfasserin aut Haruaki Ninomiya verfasserin aut Yoshiko Ohno-Iwashita verfasserin aut Ta-Yuan Chang verfasserin aut In Journal of Lipid Research Elsevier, 2021 44(2003), 5, Seite 1033-1041 (DE-627)26601593X (DE-600)1466675-3 15397262 nnns volume:44 year:2003 number:5 pages:1033-1041 https://doi.org/10.1194/jlr.D200036-JLR200 kostenfrei https://doaj.org/article/126374b3ddf84e7dac98a60a4e3ae71d kostenfrei http://www.sciencedirect.com/science/article/pii/S0022227520311494 kostenfrei https://doaj.org/toc/0022-2275 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_252 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2006 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 44 2003 5 1033-1041
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callnumber-first	Q - Science
author	Shigeki Sugii
spellingShingle	Shigeki Sugii misc QD415-436 misc BC θ misc perfringolysin O toxin misc cholesterol metabolism misc cholesterol stain misc intracellular cholesterol trafficking misc cholesterol mutants misc Biochemistry Biotinylated θ-toxin derivative as a probe to examine intracellular cholesterol-rich domains in normal and Niemann-Pick type C1 cells
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topic_title	QD415-436 Biotinylated θ-toxin derivative as a probe to examine intracellular cholesterol-rich domains in normal and Niemann-Pick type C1 cells BC θ perfringolysin O toxin cholesterol metabolism cholesterol stain intracellular cholesterol trafficking cholesterol mutants
topic	misc QD415-436 misc BC θ misc perfringolysin O toxin misc cholesterol metabolism misc cholesterol stain misc intracellular cholesterol trafficking misc cholesterol mutants misc Biochemistry
topic_unstemmed	misc QD415-436 misc BC θ misc perfringolysin O toxin misc cholesterol metabolism misc cholesterol stain misc intracellular cholesterol trafficking misc cholesterol mutants misc Biochemistry
topic_browse	misc QD415-436 misc BC θ misc perfringolysin O toxin misc cholesterol metabolism misc cholesterol stain misc intracellular cholesterol trafficking misc cholesterol mutants misc Biochemistry
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title	Biotinylated θ-toxin derivative as a probe to examine intracellular cholesterol-rich domains in normal and Niemann-Pick type C1 cells
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title_full	Biotinylated θ-toxin derivative as a probe to examine intracellular cholesterol-rich domains in normal and Niemann-Pick type C1 cells
author_sort	Shigeki Sugii
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author_browse	Shigeki Sugii Patrick C. Reid Nobutaka Ohgami Yukiko Shimada Robert A. Maue Haruaki Ninomiya Yoshiko Ohno-Iwashita Ta-Yuan Chang
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title_sort	biotinylated θ-toxin derivative as a probe to examine intracellular cholesterol-rich domains in normal and niemann-pick type c1 cells
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title_auth	Biotinylated θ-toxin derivative as a probe to examine intracellular cholesterol-rich domains in normal and Niemann-Pick type C1 cells
abstract	BCθ is a proteolytically nicked and biotinylated derivative of a cholesterol binding protein perfringolysin O (θ-toxin), and has been used to detect cholesterol-rich domains at the plasma membrane (PM). Here we show that by modifying the cell fixation condition, BCθ can also be used to detect cholesterol-rich domains intracellularly. When cells were processed for PM cholesterol staining, the difference in BCθ signals between the CT43 (CT) cell, a mutant Chinese hamster ovary cell line lacking the Niemann-Pick type C1 (NPC1) protein, and its parental cell 25RA (RA) was minimal. However, when cells were fixed with 4% paraformaldehyde, they became permeable to BCθ. Under this condition, BCθ mainly stained cholesterol-rich domains inside the cells, with the signal being much stronger in CT cells than in RA cells. The sensitivity of BCθ staining was superior to that of filipin staining. The staining of cholesterol-rich domain(s) inside RA cells was sensitive to β-cyclodextrin treatment, while most of the staining inside CT cells was relatively resistant to cyclodextrin treatment. Clear differences in intracellular BCθ staining were also seen between the normal and mutant NPC1 fibroblasts of human or mouse origin.Thus, BCθ is a powerful tool for visually monitoring cholesterol-rich domains inside normal and NPC cells.
abstractGer	BCθ is a proteolytically nicked and biotinylated derivative of a cholesterol binding protein perfringolysin O (θ-toxin), and has been used to detect cholesterol-rich domains at the plasma membrane (PM). Here we show that by modifying the cell fixation condition, BCθ can also be used to detect cholesterol-rich domains intracellularly. When cells were processed for PM cholesterol staining, the difference in BCθ signals between the CT43 (CT) cell, a mutant Chinese hamster ovary cell line lacking the Niemann-Pick type C1 (NPC1) protein, and its parental cell 25RA (RA) was minimal. However, when cells were fixed with 4% paraformaldehyde, they became permeable to BCθ. Under this condition, BCθ mainly stained cholesterol-rich domains inside the cells, with the signal being much stronger in CT cells than in RA cells. The sensitivity of BCθ staining was superior to that of filipin staining. The staining of cholesterol-rich domain(s) inside RA cells was sensitive to β-cyclodextrin treatment, while most of the staining inside CT cells was relatively resistant to cyclodextrin treatment. Clear differences in intracellular BCθ staining were also seen between the normal and mutant NPC1 fibroblasts of human or mouse origin.Thus, BCθ is a powerful tool for visually monitoring cholesterol-rich domains inside normal and NPC cells.
abstract_unstemmed	BCθ is a proteolytically nicked and biotinylated derivative of a cholesterol binding protein perfringolysin O (θ-toxin), and has been used to detect cholesterol-rich domains at the plasma membrane (PM). Here we show that by modifying the cell fixation condition, BCθ can also be used to detect cholesterol-rich domains intracellularly. When cells were processed for PM cholesterol staining, the difference in BCθ signals between the CT43 (CT) cell, a mutant Chinese hamster ovary cell line lacking the Niemann-Pick type C1 (NPC1) protein, and its parental cell 25RA (RA) was minimal. However, when cells were fixed with 4% paraformaldehyde, they became permeable to BCθ. Under this condition, BCθ mainly stained cholesterol-rich domains inside the cells, with the signal being much stronger in CT cells than in RA cells. The sensitivity of BCθ staining was superior to that of filipin staining. The staining of cholesterol-rich domain(s) inside RA cells was sensitive to β-cyclodextrin treatment, while most of the staining inside CT cells was relatively resistant to cyclodextrin treatment. Clear differences in intracellular BCθ staining were also seen between the normal and mutant NPC1 fibroblasts of human or mouse origin.Thus, BCθ is a powerful tool for visually monitoring cholesterol-rich domains inside normal and NPC cells.
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title_short	Biotinylated θ-toxin derivative as a probe to examine intracellular cholesterol-rich domains in normal and Niemann-Pick type C1 cells
url	https://doi.org/10.1194/jlr.D200036-JLR200 https://doaj.org/article/126374b3ddf84e7dac98a60a4e3ae71d http://www.sciencedirect.com/science/article/pii/S0022227520311494 https://doaj.org/toc/0022-2275
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author2	Patrick C. Reid Nobutaka Ohgami Yukiko Shimada Robert A. Maue Haruaki Ninomiya Yoshiko Ohno-Iwashita Ta-Yuan Chang
author2Str	Patrick C. Reid Nobutaka Ohgami Yukiko Shimada Robert A. Maue Haruaki Ninomiya Yoshiko Ohno-Iwashita Ta-Yuan Chang
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doi_str	10.1194/jlr.D200036-JLR200
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up_date	2024-07-03T21:37:54.482Z
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Biotinylated θ-toxin derivative as a probe to examine intracellular cholesterol-rich domains in normal and Niemann-Pick type C1 cells

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