Role of phospholipid synthesis and phospholipase C in the regulation of diacylglycerol required for membrane trafficking at the Golgi complex
Diacylglycerol (DAG) is required for the generation of transport carriers at the Golgi complex. Several enzymatic reactions have been found to contribute to the maintenance of DAG levels at this organelle. However, the specific metabolic pathways that, under physiological conditions, are regulated t...
| Autor: | |
|---|---|
| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2014 |
| País: | España |
| Institución: | CBUC, CESCA |
| Repositorio: | TDR. Tesis Doctorales en Red |
| OAI Identifier: | oai:www.tdx.cat:10803/283432 |
| Acceso en línea: | http://hdl.handle.net/10803/283432 |
| Access Level: | acceso abierto |
| Palabra clave: | Transport biològic Transporte biológico Biological transport Aparell de Golgi Aparato de Golgi Golgi apparatus Fosfolípids Fosfolípidos Phospholipids Diacilglicerol Diacyl-glycerol Ciències de la Salut 576 |
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| dc.title.none.fl_str_mv |
Role of phospholipid synthesis and phospholipase C in the regulation of diacylglycerol required for membrane trafficking at the Golgi complex |
| title |
Role of phospholipid synthesis and phospholipase C in the regulation of diacylglycerol required for membrane trafficking at the Golgi complex |
| spellingShingle |
Role of phospholipid synthesis and phospholipase C in the regulation of diacylglycerol required for membrane trafficking at the Golgi complex Sicart Casellas, Adrià Transport biològic Transporte biológico Biological transport Aparell de Golgi Aparato de Golgi Golgi apparatus Fosfolípids Fosfolípidos Phospholipids Diacilglicerol Diacyl-glycerol Ciències de la Salut 576 |
| title_short |
Role of phospholipid synthesis and phospholipase C in the regulation of diacylglycerol required for membrane trafficking at the Golgi complex |
| title_full |
Role of phospholipid synthesis and phospholipase C in the regulation of diacylglycerol required for membrane trafficking at the Golgi complex |
| title_fullStr |
Role of phospholipid synthesis and phospholipase C in the regulation of diacylglycerol required for membrane trafficking at the Golgi complex |
| title_full_unstemmed |
Role of phospholipid synthesis and phospholipase C in the regulation of diacylglycerol required for membrane trafficking at the Golgi complex |
| title_sort |
Role of phospholipid synthesis and phospholipase C in the regulation of diacylglycerol required for membrane trafficking at the Golgi complex |
| dc.creator.none.fl_str_mv |
Sicart Casellas, Adrià |
| author |
Sicart Casellas, Adrià |
| author_facet |
Sicart Casellas, Adrià |
| author_role |
author |
| dc.contributor.none.fl_str_mv |
Egea Guri, Gustavo Sarri Plans, Elisabet Universitat de Barcelona. Departament de Biologia Cel·lular, Immunologia i Neurociències |
| dc.subject.none.fl_str_mv |
Transport biològic Transporte biológico Biological transport Aparell de Golgi Aparato de Golgi Golgi apparatus Fosfolípids Fosfolípidos Phospholipids Diacilglicerol Diacyl-glycerol Ciències de la Salut 576 |
| topic |
Transport biològic Transporte biológico Biological transport Aparell de Golgi Aparato de Golgi Golgi apparatus Fosfolípids Fosfolípidos Phospholipids Diacilglicerol Diacyl-glycerol Ciències de la Salut 576 |
| description |
Diacylglycerol (DAG) is required for the generation of transport carriers at the Golgi complex. Several enzymatic reactions have been found to contribute to the maintenance of DAG levels at this organelle. However, the specific metabolic pathways that, under physiological conditions, are regulated to produce the DAG required for the membrane trafficking at the Golgi complex are not known. In the first part of this work we worked on the hypothesis that phospholipid synthesis can control DAG levels at the Golgi complex for the generation of transport carriers at the Golgi complex. To study this, we altered phosphatidylcholine (PC) and phosphatidylinositol synthesis for a short period of time in CHO cells to evaluate the changes in DAG and its effects in membrane trafficking at the Golgi. We found that cellular DAG rapidly increased when PC synthesis was inhibited at the non-permissive temperature for the rate-limiting step of PC synthesis in CHO-MT58 cells. DAG also increased when choline and inositol were not supplied. The major phospholipid classes and triacylglycerol remained unaltered for both experimental approaches. The analysis of Golgi ultrastructure and membrane trafficking showed that 1) the accumulation of the budding vesicular profiles induced by propanolol was prevented by inhibition of PC synthesis, 2) the density of KDEL receptor-containing punctated structures at the endoplasmic reticulum-Golgi interface correlated with the amount of DAG, and 3) the post-Golgi transport of the yellow fluorescent temperature-sensitive G protein of stomatitis virus and the secretion of a secretory form of HRP were both reduced when DAG was lowered. We confirmed that DAG-consuming reactions of lipid synthesis were present in Golgi-enriched fractions. We conclude that phospholipid synthesis pathways play a significant role to regulate the DAG required in Golgi-dependent membrane trafficking. In the second part of this work we investigate the role of phospholipase Cγ1 (PLCγ1), a DAG-producing signalling enzyme, on the membrane traffic at the Golgi complex and in the maintenance of its structure in HeLa cells. We based our work on the effects of the silencing and overexpression of PLCγ1. We found that PLCγ1 is required for post-Golgi trafficking of transmembrane and soluble proteins, that the catalytic activity of PLCγ1 is necessary to maintain the morphology of the Golgi complex, in particular the trans-Golgi compartments, and that PLCγ1 contributes to DAG homeostasis at the Golgi, measured by the localization of the DAG-sensing construct C1-PKCθ-GFP to the Golgi complex. Finally we show that cargo arrival at the Golgi complex increases the DAG production and that PLCγ1 is required for this increase of DAG localized at the Golgi. Our results show for the first time that a physiologic event along the secretory pathway, such as cargo arrival at the Golgi complex, triggers DAG production at this organelle for membrane traffic. We also provide evidence for a pivotal role of PLCγ1 at the Golgi: PLCγ1 mediates the DAG production triggered by cargo arrival and is required for the Golgi structure and membrane traffic at the trans-Golgi compartment. The main conclusions of this work are that: 1- Metabolic pathways for the synthesis of phospholipids that consume DAG regulate its levels at the Golgi complex. 2.- Phospholipid synthesis controls the levels of DAG needed for both retrograde and anterograde trafficking at the Golgi complex. 3.- Cargo arrival at the Golgi complex promotes DAG production. 4.- PLCγ1 is involved in the production of DAG triggered by cargo arrival at the Golgi complex. 5.- PLCγ1 is needed for post-Golgi transport and maintenance of the structure of the Golgi complex. |
| publishDate |
2014 |
| dc.date.none.fl_str_mv |
2014 2014 2014 |
| dc.type.none.fl_str_mv |
info:eu-repo/semantics/doctoralThesis info:eu-repo/semantics/publishedVersion |
| format |
doctoralThesis |
| status_str |
publishedVersion |
| dc.identifier.none.fl_str_mv |
http://hdl.handle.net/10803/283432 |
| url |
http://hdl.handle.net/10803/283432 |
| dc.language.none.fl_str_mv |
Inglés |
| language_invalid_str_mv |
Inglés |
| dc.rights.none.fl_str_mv |
http://creativecommons.org/licenses/by/3.0/es/ info:eu-repo/semantics/openAccess |
| rights_invalid_str_mv |
http://creativecommons.org/licenses/by/3.0/es/ |
| eu_rights_str_mv |
openAccess |
| dc.format.none.fl_str_mv |
175 p. application/pdf application/pdf |
| dc.publisher.none.fl_str_mv |
Universitat de Barcelona |
| publisher.none.fl_str_mv |
Universitat de Barcelona |
| dc.source.none.fl_str_mv |
TDX (Tesis Doctorals en Xarxa) reponame:TDR. Tesis Doctorales en Red instname:CBUC, CESCA |
| instname_str |
CBUC, CESCA |
| reponame_str |
TDR. Tesis Doctorales en Red |
| collection |
TDR. Tesis Doctorales en Red |
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| _version_ |
1869422657239252992 |
| spelling |
Role of phospholipid synthesis and phospholipase C in the regulation of diacylglycerol required for membrane trafficking at the Golgi complexSicart Casellas, AdriàTransport biològicTransporte biológicoBiological transportAparell de GolgiAparato de GolgiGolgi apparatusFosfolípidsFosfolípidosPhospholipidsDiacilglicerolDiacyl-glycerolCiències de la Salut576Diacylglycerol (DAG) is required for the generation of transport carriers at the Golgi complex. Several enzymatic reactions have been found to contribute to the maintenance of DAG levels at this organelle. However, the specific metabolic pathways that, under physiological conditions, are regulated to produce the DAG required for the membrane trafficking at the Golgi complex are not known. In the first part of this work we worked on the hypothesis that phospholipid synthesis can control DAG levels at the Golgi complex for the generation of transport carriers at the Golgi complex. To study this, we altered phosphatidylcholine (PC) and phosphatidylinositol synthesis for a short period of time in CHO cells to evaluate the changes in DAG and its effects in membrane trafficking at the Golgi. We found that cellular DAG rapidly increased when PC synthesis was inhibited at the non-permissive temperature for the rate-limiting step of PC synthesis in CHO-MT58 cells. DAG also increased when choline and inositol were not supplied. The major phospholipid classes and triacylglycerol remained unaltered for both experimental approaches. The analysis of Golgi ultrastructure and membrane trafficking showed that 1) the accumulation of the budding vesicular profiles induced by propanolol was prevented by inhibition of PC synthesis, 2) the density of KDEL receptor-containing punctated structures at the endoplasmic reticulum-Golgi interface correlated with the amount of DAG, and 3) the post-Golgi transport of the yellow fluorescent temperature-sensitive G protein of stomatitis virus and the secretion of a secretory form of HRP were both reduced when DAG was lowered. We confirmed that DAG-consuming reactions of lipid synthesis were present in Golgi-enriched fractions. We conclude that phospholipid synthesis pathways play a significant role to regulate the DAG required in Golgi-dependent membrane trafficking. In the second part of this work we investigate the role of phospholipase Cγ1 (PLCγ1), a DAG-producing signalling enzyme, on the membrane traffic at the Golgi complex and in the maintenance of its structure in HeLa cells. We based our work on the effects of the silencing and overexpression of PLCγ1. We found that PLCγ1 is required for post-Golgi trafficking of transmembrane and soluble proteins, that the catalytic activity of PLCγ1 is necessary to maintain the morphology of the Golgi complex, in particular the trans-Golgi compartments, and that PLCγ1 contributes to DAG homeostasis at the Golgi, measured by the localization of the DAG-sensing construct C1-PKCθ-GFP to the Golgi complex. Finally we show that cargo arrival at the Golgi complex increases the DAG production and that PLCγ1 is required for this increase of DAG localized at the Golgi. Our results show for the first time that a physiologic event along the secretory pathway, such as cargo arrival at the Golgi complex, triggers DAG production at this organelle for membrane traffic. We also provide evidence for a pivotal role of PLCγ1 at the Golgi: PLCγ1 mediates the DAG production triggered by cargo arrival and is required for the Golgi structure and membrane traffic at the trans-Golgi compartment. The main conclusions of this work are that: 1- Metabolic pathways for the synthesis of phospholipids that consume DAG regulate its levels at the Golgi complex. 2.- Phospholipid synthesis controls the levels of DAG needed for both retrograde and anterograde trafficking at the Golgi complex. 3.- Cargo arrival at the Golgi complex promotes DAG production. 4.- PLCγ1 is involved in the production of DAG triggered by cargo arrival at the Golgi complex. 5.- PLCγ1 is needed for post-Golgi transport and maintenance of the structure of the Golgi complex.Universitat de BarcelonaEgea Guri, GustavoSarri Plans, ElisabetUniversitat de Barcelona. Departament de Biologia Cel·lular, Immunologia i Neurociències201420142014info:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/publishedVersion175 p.application/pdfapplication/pdfhttp://hdl.handle.net/10803/283432TDX (Tesis Doctorals en Xarxa)reponame:TDR. Tesis Doctorales en Redinstname:CBUC, CESCAInglésL'accés als continguts d'aquesta tesi queda condicionat a l'acceptació de les condicions d'ús establertes per la següent llicència Creative Commons: http://creativecommons.org/licenses/by/3.0/es/http://creativecommons.org/licenses/by/3.0/es/info:eu-repo/semantics/openAccessoai:www.tdx.cat:10803/2834322026-06-14T12:46:07Z |
| score |
15.301629 |