Molecular Trapping of a Fluorescent Ceramide Analogue at the Golgi Apparatus of Fixed Cells: Interaction with Endogenous Lipids Provides a trans-Golgi Marker for Both Light and Electron Microscopy
We have previously shown that a fluorescent derivative of ceramide, N-(ε-7-nitrobenz-2-oxa-1,3-diazol-4-yl-aminocaproyl)-D-erythro-sphingosine ( C6- NBD- Cer), vitally stains the Golgi apparatus of cells (Lipsky, N. G., and R. E. Pagano. 1985. Science (Wash. DC). 228:745-747). In the present paper w...
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| Veröffentlicht in: | The Journal of cell biology 1989-11, Vol.109 (5), p.2067-2079 |
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| creator | Pagano, Richard E. Sepanski, Michael A. Martin, Ona C. |
| description | We have previously shown that a fluorescent derivative of ceramide, N-(ε-7-nitrobenz-2-oxa-1,3-diazol-4-yl-aminocaproyl)-D-erythro-sphingosine ( C6- NBD- Cer), vitally stains the Golgi apparatus of cells (Lipsky, N. G., and R. E. Pagano. 1985. Science (Wash. DC). 228:745-747). In the present paper we demonstrate that C6- NBD- Cer also accumulates at the Golgi apparatus of fixed cells and we explore the mechanism by which this occurs. When human skin fibroblasts were fixed with glutaraldehyde and then incubated with C6- NBD- Cer at 2°C, the fluorescent lipid spontaneously transferred into the cells, labeling the Golgi apparatus as well as other intracellular membranes. Subsequent incubations with defatted BSA at 24°C removed excess C6- NBD- Cer from the cells such that fluorescence was then detected only at the Golgi apparatus. Similar results were obtained using other cell types. A method for visualizing the fluorescent lipid at the electron microscopic level, based on the photoconversion of a fluorescent marker to a diaminobenzidine product (Sandell, J. H., and R. H. Masland. 1988. J. Histochem. Cytochem. 36:555-559), is described and evidence is presented that C6- NBD- Cer was localized to the trans cisternae of the Golgi apparatus. While accumulation occurred in cells fixed in various ways, it was inhibited when fixation protocols that extract or modify cellular lipids were used. In addition, Filipin, which forms complexes with cellular cholesterol, labeled the Golgi apparatus of fixed cells and inhibited accumulation of C6- NBD- Cer at the Golgi apparatus. These results are discussed in terms of a simple model based on the physical properties of C6- NBD- Cer and its interactions with endogenous lipids of the Golgi apparatus. Possible implications of these findings for metabolism and transport of (fluorescent) sphingolipids in vivo are also presented. |
| doi_str_mv | 10.1083/jcb.109.5.2067 |
| format | Article |
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G., and R. E. Pagano. 1985. Science (Wash. DC). 228:745-747). In the present paper we demonstrate that C6- NBD- Cer also accumulates at the Golgi apparatus of fixed cells and we explore the mechanism by which this occurs. When human skin fibroblasts were fixed with glutaraldehyde and then incubated with C6- NBD- Cer at 2°C, the fluorescent lipid spontaneously transferred into the cells, labeling the Golgi apparatus as well as other intracellular membranes. Subsequent incubations with defatted BSA at 24°C removed excess C6- NBD- Cer from the cells such that fluorescence was then detected only at the Golgi apparatus. Similar results were obtained using other cell types. A method for visualizing the fluorescent lipid at the electron microscopic level, based on the photoconversion of a fluorescent marker to a diaminobenzidine product (Sandell, J. H., and R. H. Masland. 1988. J. Histochem. Cytochem. 36:555-559), is described and evidence is presented that C6- NBD- Cer was localized to the trans cisternae of the Golgi apparatus. While accumulation occurred in cells fixed in various ways, it was inhibited when fixation protocols that extract or modify cellular lipids were used. In addition, Filipin, which forms complexes with cellular cholesterol, labeled the Golgi apparatus of fixed cells and inhibited accumulation of C6- NBD- Cer at the Golgi apparatus. These results are discussed in terms of a simple model based on the physical properties of C6- NBD- Cer and its interactions with endogenous lipids of the Golgi apparatus. Possible implications of these findings for metabolism and transport of (fluorescent) sphingolipids in vivo are also presented.</description><identifier>ISSN: 0021-9525</identifier><identifier>EISSN: 1540-8140</identifier><identifier>DOI: 10.1083/jcb.109.5.2067</identifier><identifier>PMID: 2478562</identifier><identifier>CODEN: JCLBA3</identifier><language>eng</language><publisher>New York, NY: Rockefeller University Press</publisher><subject>4-Chloro-7-nitrobenzofurazan - analogs & derivatives ; 4-Chloro-7-nitrobenzofurazan - analysis ; Animal cells ; Animals ; Biological and medical sciences ; Cell Line ; Cell membranes ; Cell structures and functions ; Cells ; ceramide ; Ceramides ; Ceramides - analysis ; Cultured cells ; Fibroblasts ; Fibroblasts - enzymology ; Fluorescence ; Fundamental and applied biological sciences. Psychology ; Golgi apparatus ; Golgi Apparatus - ultrastructure ; Histocytochemistry ; Humans ; Indicators and Reagents ; Lipids ; Lipids - analysis ; Microscopy, Electron - methods ; Microscopy, Fluorescence - methods ; Molecular and cellular biology ; Oxadiazoles - analysis ; Room temperature ; Skin - enzymology ; Staining and Labeling ; Thiamine Pyrophosphatase - analysis</subject><ispartof>The Journal of cell biology, 1989-11, Vol.109 (5), p.2067-2079</ispartof><rights>Copyright 1989 The Rockefeller University Press</rights><rights>1990 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c466t-e997c74e849a43c5fc56e16cae1570620a0ac47ac04f49d8017b71b293d1f6d63</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>230,314,777,781,882,27906,27907</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=6645356$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/2478562$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Pagano, Richard E.</creatorcontrib><creatorcontrib>Sepanski, Michael A.</creatorcontrib><creatorcontrib>Martin, Ona C.</creatorcontrib><title>Molecular Trapping of a Fluorescent Ceramide Analogue at the Golgi Apparatus of Fixed Cells: Interaction with Endogenous Lipids Provides a trans-Golgi Marker for Both Light and Electron Microscopy</title><title>The Journal of cell biology</title><addtitle>J Cell Biol</addtitle><description>We have previously shown that a fluorescent derivative of ceramide, N-(ε-7-nitrobenz-2-oxa-1,3-diazol-4-yl-aminocaproyl)-D-erythro-sphingosine ( C6- NBD- Cer), vitally stains the Golgi apparatus of cells (Lipsky, N. G., and R. E. Pagano. 1985. Science (Wash. DC). 228:745-747). In the present paper we demonstrate that C6- NBD- Cer also accumulates at the Golgi apparatus of fixed cells and we explore the mechanism by which this occurs. When human skin fibroblasts were fixed with glutaraldehyde and then incubated with C6- NBD- Cer at 2°C, the fluorescent lipid spontaneously transferred into the cells, labeling the Golgi apparatus as well as other intracellular membranes. Subsequent incubations with defatted BSA at 24°C removed excess C6- NBD- Cer from the cells such that fluorescence was then detected only at the Golgi apparatus. Similar results were obtained using other cell types. A method for visualizing the fluorescent lipid at the electron microscopic level, based on the photoconversion of a fluorescent marker to a diaminobenzidine product (Sandell, J. H., and R. H. Masland. 1988. J. Histochem. Cytochem. 36:555-559), is described and evidence is presented that C6- NBD- Cer was localized to the trans cisternae of the Golgi apparatus. While accumulation occurred in cells fixed in various ways, it was inhibited when fixation protocols that extract or modify cellular lipids were used. In addition, Filipin, which forms complexes with cellular cholesterol, labeled the Golgi apparatus of fixed cells and inhibited accumulation of C6- NBD- Cer at the Golgi apparatus. These results are discussed in terms of a simple model based on the physical properties of C6- NBD- Cer and its interactions with endogenous lipids of the Golgi apparatus. Possible implications of these findings for metabolism and transport of (fluorescent) sphingolipids in vivo are also presented.</description><subject>4-Chloro-7-nitrobenzofurazan - analogs & derivatives</subject><subject>4-Chloro-7-nitrobenzofurazan - analysis</subject><subject>Animal cells</subject><subject>Animals</subject><subject>Biological and medical sciences</subject><subject>Cell Line</subject><subject>Cell membranes</subject><subject>Cell structures and functions</subject><subject>Cells</subject><subject>ceramide</subject><subject>Ceramides</subject><subject>Ceramides - analysis</subject><subject>Cultured cells</subject><subject>Fibroblasts</subject><subject>Fibroblasts - enzymology</subject><subject>Fluorescence</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Golgi apparatus</subject><subject>Golgi Apparatus - ultrastructure</subject><subject>Histocytochemistry</subject><subject>Humans</subject><subject>Indicators and Reagents</subject><subject>Lipids</subject><subject>Lipids - analysis</subject><subject>Microscopy, Electron - methods</subject><subject>Microscopy, Fluorescence - methods</subject><subject>Molecular and cellular biology</subject><subject>Oxadiazoles - analysis</subject><subject>Room temperature</subject><subject>Skin - enzymology</subject><subject>Staining and Labeling</subject><subject>Thiamine Pyrophosphatase - analysis</subject><issn>0021-9525</issn><issn>1540-8140</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1989</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFUsGO0zAQjRBoKQtXTiD5gLil2IntxByQStUuK7WCw3K2po6TurhxsJ2F_T8-DEetCpw4eaR57834zcuylwTPCa7Ldwe1S4WYs3mBefUomxFGcV4Tih9nM4wLkgtWsKfZsxAOGGNa0fIquypoVTNezLJfW2e1Gi14dOdhGEzfIdciQGs7Oq-D0n1ES-3haBqNFj1Y140aQURxr9GNs51Bi2EAD3EME3NtfuomMawN79FtHxNVReN69MPEPVr1jet07xJ2YwbTBPTFu_skHdLI6KEP-UlzC_6b9qh1Hn10ibgx3T4i6Bu0SvtGnwS3RnkXlBsenmdPWrBBvzi_19nX9epu-SnffL65XS42uaKcx1wLUamK6poKoKVirWJcE65AE1ZhXmDAoGgFCtOWiqbGpNpVZFeIsiEtb3h5nX046Q7j7qibyRsPVg7eHME_SAdG_tvpzV527l4WhLCa4STw9izg3fdRhyiPJllsLfQ6eSIrUdS8JuS_wHTkQnAiEnB-Ak5eBK_byzYEyykgMgUkFUIyOQUkEV7__YcL_JyI1H9z7kNQYNt0E2XCBcY5ZSWbrHh1gh1CdP7PUE7KZGX5GyOH0Qs</recordid><startdate>19891101</startdate><enddate>19891101</enddate><creator>Pagano, Richard E.</creator><creator>Sepanski, Michael A.</creator><creator>Martin, Ona C.</creator><general>Rockefeller University Press</general><general>The Rockefeller University Press</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>8FD</scope><scope>FR3</scope><scope>M7Z</scope><scope>P64</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>19891101</creationdate><title>Molecular Trapping of a Fluorescent Ceramide Analogue at the Golgi Apparatus of Fixed Cells: Interaction with Endogenous Lipids Provides a trans-Golgi Marker for Both Light and Electron Microscopy</title><author>Pagano, Richard E. ; Sepanski, Michael A. ; Martin, Ona C.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c466t-e997c74e849a43c5fc56e16cae1570620a0ac47ac04f49d8017b71b293d1f6d63</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1989</creationdate><topic>4-Chloro-7-nitrobenzofurazan - analogs & derivatives</topic><topic>4-Chloro-7-nitrobenzofurazan - analysis</topic><topic>Animal cells</topic><topic>Animals</topic><topic>Biological and medical sciences</topic><topic>Cell Line</topic><topic>Cell membranes</topic><topic>Cell structures and functions</topic><topic>Cells</topic><topic>ceramide</topic><topic>Ceramides</topic><topic>Ceramides - analysis</topic><topic>Cultured cells</topic><topic>Fibroblasts</topic><topic>Fibroblasts - enzymology</topic><topic>Fluorescence</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Golgi apparatus</topic><topic>Golgi Apparatus - ultrastructure</topic><topic>Histocytochemistry</topic><topic>Humans</topic><topic>Indicators and Reagents</topic><topic>Lipids</topic><topic>Lipids - analysis</topic><topic>Microscopy, Electron - methods</topic><topic>Microscopy, Fluorescence - methods</topic><topic>Molecular and cellular biology</topic><topic>Oxadiazoles - analysis</topic><topic>Room temperature</topic><topic>Skin - enzymology</topic><topic>Staining and Labeling</topic><topic>Thiamine Pyrophosphatase - analysis</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Pagano, Richard E.</creatorcontrib><creatorcontrib>Sepanski, Michael A.</creatorcontrib><creatorcontrib>Martin, Ona C.</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biochemistry Abstracts 1</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>The Journal of cell biology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Pagano, Richard E.</au><au>Sepanski, Michael A.</au><au>Martin, Ona C.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Molecular Trapping of a Fluorescent Ceramide Analogue at the Golgi Apparatus of Fixed Cells: Interaction with Endogenous Lipids Provides a trans-Golgi Marker for Both Light and Electron Microscopy</atitle><jtitle>The Journal of cell biology</jtitle><addtitle>J Cell Biol</addtitle><date>1989-11-01</date><risdate>1989</risdate><volume>109</volume><issue>5</issue><spage>2067</spage><epage>2079</epage><pages>2067-2079</pages><issn>0021-9525</issn><eissn>1540-8140</eissn><coden>JCLBA3</coden><abstract>We have previously shown that a fluorescent derivative of ceramide, N-(ε-7-nitrobenz-2-oxa-1,3-diazol-4-yl-aminocaproyl)-D-erythro-sphingosine ( C6- NBD- Cer), vitally stains the Golgi apparatus of cells (Lipsky, N. G., and R. E. Pagano. 1985. Science (Wash. DC). 228:745-747). In the present paper we demonstrate that C6- NBD- Cer also accumulates at the Golgi apparatus of fixed cells and we explore the mechanism by which this occurs. When human skin fibroblasts were fixed with glutaraldehyde and then incubated with C6- NBD- Cer at 2°C, the fluorescent lipid spontaneously transferred into the cells, labeling the Golgi apparatus as well as other intracellular membranes. Subsequent incubations with defatted BSA at 24°C removed excess C6- NBD- Cer from the cells such that fluorescence was then detected only at the Golgi apparatus. Similar results were obtained using other cell types. A method for visualizing the fluorescent lipid at the electron microscopic level, based on the photoconversion of a fluorescent marker to a diaminobenzidine product (Sandell, J. H., and R. H. Masland. 1988. J. Histochem. Cytochem. 36:555-559), is described and evidence is presented that C6- NBD- Cer was localized to the trans cisternae of the Golgi apparatus. While accumulation occurred in cells fixed in various ways, it was inhibited when fixation protocols that extract or modify cellular lipids were used. In addition, Filipin, which forms complexes with cellular cholesterol, labeled the Golgi apparatus of fixed cells and inhibited accumulation of C6- NBD- Cer at the Golgi apparatus. These results are discussed in terms of a simple model based on the physical properties of C6- NBD- Cer and its interactions with endogenous lipids of the Golgi apparatus. Possible implications of these findings for metabolism and transport of (fluorescent) sphingolipids in vivo are also presented.</abstract><cop>New York, NY</cop><pub>Rockefeller University Press</pub><pmid>2478562</pmid><doi>10.1083/jcb.109.5.2067</doi><tpages>13</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | 4-Chloro-7-nitrobenzofurazan - analogs & derivatives 4-Chloro-7-nitrobenzofurazan - analysis Animal cells Animals Biological and medical sciences Cell Line Cell membranes Cell structures and functions Cells ceramide Ceramides Ceramides - analysis Cultured cells Fibroblasts Fibroblasts - enzymology Fluorescence Fundamental and applied biological sciences. Psychology Golgi apparatus Golgi Apparatus - ultrastructure Histocytochemistry Humans Indicators and Reagents Lipids Lipids - analysis Microscopy, Electron - methods Microscopy, Fluorescence - methods Molecular and cellular biology Oxadiazoles - analysis Room temperature Skin - enzymology Staining and Labeling Thiamine Pyrophosphatase - analysis |
| title | Molecular Trapping of a Fluorescent Ceramide Analogue at the Golgi Apparatus of Fixed Cells: Interaction with Endogenous Lipids Provides a trans-Golgi Marker for Both Light and Electron Microscopy |
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