Effect of palmitoylated synaptic proteins and biologically relevant calcium concentrations on the compartmentalization of PI(4,5)P2 in membrane model systems and in the plasma membrane of PC12 cells

Detalhes bibliográficos
Autor(a) principal: Sarmento, Maria João Martins
Data de Publicação: 2011
Tipo de documento: Dissertação
Idioma: eng
Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
Texto Completo: http://hdl.handle.net/10451/8859
Resumo: Tese de mestrado em Bioquímica (Bioquímica Médica), apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2011
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spelling Effect of palmitoylated synaptic proteins and biologically relevant calcium concentrations on the compartmentalization of PI(4,5)P2 in membrane model systems and in the plasma membrane of PC12 cellsPI(4,5)P2DistributionExocytosisProtein palmitoylationCalciumGUV immobilizationTeses de mestrado - 2011Tese de mestrado em Bioquímica (Bioquímica Médica), apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2011Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a minor component of the plasma membrane of eukaryotic cells that is essential for several cellular mechanisms, including organization of the actin cytoskeleton, membrane trafficking, endocytosis and exocytosis. The regulation of these processes is thought to involve the local enrichment of PI(4,5)P2 in the plasma membrane at particular timings. In the first part of this work, we aimed to better characterize the mechanisms responsible for PI(4,5)P2 clustering in the plasma membrane of neuronal-like (PC12) and non-neuronal (HEK293) cells. Using FRET microscopy carried out with PH-domains labelled with fluorescent proteins (PH-CFP and PG-YFP), we detected a highly clustered distribution of PI(4,5)P2 in PC12 cells and some indication of clustering below the confocal resolution for HEK293 cells. Moreover, FRET efficiency decreased after inhibition of protein palmitoylation, emphasizing the importance of the interaction between palmitoylated proteins and PI(4,5)P2 for its lateral membrane organization. Additionally, since we were also interested in studying PI(4,5)P2 distribution in giant unilamellar vesicles (GUVs), we optimized a GUV immobilization method based on the interaction of biotinylated lipids with an avidin-coated surface. Three different biotinylated lipids were studied: DOPE-Cap-biotin, DPPE-Cap-biotin and DPPE-biotin. Since PI(4,5)P2 has been shown to partition into lipid rafts, studies with POPC:Chol:PSM (1:1:1) GUVs presenting a coexistence of liquid ordered/liquid disordered phases were performed. Our results showed that the immobilization conditions and the type of immobilizing lipid significantly influence the distribution of lipid domains within GUVs, potentially generating artefacts in lipid phase coexistence studies. By imaging GUVs with a fluorescent analogue of PI(4,5)P2, we observed a Ca2+ induced clustering of the fluorescent lipid at concentrations (>100 μM) known to occur in the proximity of calcium channels after synaptic stimulation. Then, PI(4,5)P2 can potentially act as a lipidic calcium sensor, regulating not only local plasma membrane charge and curvature but also synaptic protein organization.Fernandes, FábioCoutinho, Ana Isabel Abrantes, 1965-Repositório da Universidade de LisboaSarmento, Maria João Martins2013-07-24T10:08:15Z20112011-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10451/8859enginfo:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-11-08T15:52:53Zoai:repositorio.ul.pt:10451/8859Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T21:33:14.060428Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse
dc.title.none.fl_str_mv Effect of palmitoylated synaptic proteins and biologically relevant calcium concentrations on the compartmentalization of PI(4,5)P2 in membrane model systems and in the plasma membrane of PC12 cells
title Effect of palmitoylated synaptic proteins and biologically relevant calcium concentrations on the compartmentalization of PI(4,5)P2 in membrane model systems and in the plasma membrane of PC12 cells
spellingShingle Effect of palmitoylated synaptic proteins and biologically relevant calcium concentrations on the compartmentalization of PI(4,5)P2 in membrane model systems and in the plasma membrane of PC12 cells
Sarmento, Maria João Martins
PI(4,5)P2
Distribution
Exocytosis
Protein palmitoylation
Calcium
GUV immobilization
Teses de mestrado - 2011
title_short Effect of palmitoylated synaptic proteins and biologically relevant calcium concentrations on the compartmentalization of PI(4,5)P2 in membrane model systems and in the plasma membrane of PC12 cells
title_full Effect of palmitoylated synaptic proteins and biologically relevant calcium concentrations on the compartmentalization of PI(4,5)P2 in membrane model systems and in the plasma membrane of PC12 cells
title_fullStr Effect of palmitoylated synaptic proteins and biologically relevant calcium concentrations on the compartmentalization of PI(4,5)P2 in membrane model systems and in the plasma membrane of PC12 cells
title_full_unstemmed Effect of palmitoylated synaptic proteins and biologically relevant calcium concentrations on the compartmentalization of PI(4,5)P2 in membrane model systems and in the plasma membrane of PC12 cells
title_sort Effect of palmitoylated synaptic proteins and biologically relevant calcium concentrations on the compartmentalization of PI(4,5)P2 in membrane model systems and in the plasma membrane of PC12 cells
author Sarmento, Maria João Martins
author_facet Sarmento, Maria João Martins
author_role author
dc.contributor.none.fl_str_mv Fernandes, Fábio
Coutinho, Ana Isabel Abrantes, 1965-
Repositório da Universidade de Lisboa
dc.contributor.author.fl_str_mv Sarmento, Maria João Martins
dc.subject.por.fl_str_mv PI(4,5)P2
Distribution
Exocytosis
Protein palmitoylation
Calcium
GUV immobilization
Teses de mestrado - 2011
topic PI(4,5)P2
Distribution
Exocytosis
Protein palmitoylation
Calcium
GUV immobilization
Teses de mestrado - 2011
description Tese de mestrado em Bioquímica (Bioquímica Médica), apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2011
publishDate 2011
dc.date.none.fl_str_mv 2011
2011-01-01T00:00:00Z
2013-07-24T10:08:15Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/masterThesis
format masterThesis
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://hdl.handle.net/10451/8859
url http://hdl.handle.net/10451/8859
dc.language.iso.fl_str_mv eng
language eng
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.source.none.fl_str_mv reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
instacron:RCAAP
instname_str Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
instacron_str RCAAP
institution RCAAP
reponame_str Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
collection Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
repository.name.fl_str_mv Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
repository.mail.fl_str_mv
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