No public access
master's thesis
Cloning, mutagenesis and characterization of phosphatidylinositol 3-monophosphate binding domains in HEK293 cells and primary megakaryocytes

Paola Paškvan (2016)
Sveučilište u Rijeci
Odjel za biotehnologiju
Metadata
TitleKloniranje, mutageneza i karakterizacija fosfatidilinozitol 3-monofosfat vezujućih domena u HEK293T i primarnim megakariocitima
AuthorPaola Paškvan
Mentor(s)Antonija Jurak Begonja (thesis advisor)
Abstract
Fosfoinozitidi (PI) su skupina staničnih fosfolipida od iznimne važnosti za regulaciju stanične fiziologije i membranske dinamike. U stanicama eukariota nastaju fosforilacijom fosfatidilinozitola (PtdIns) aktivacijom niza kinaza i fosfataza. U ovom radu praćena je ekspresija i lokalizacija FYVE i PX domena koje specifično vežu fosfatidilinozitol 3-monofosfat [PI(3)P] te njihov utjecaj na razvoj megakariocita i produkciju protrombocita. U retroviralni vektor, klonirane su fluorescentno obilježene tandem FYVE i mutirana domene (eGFP-2x-FYVE i eGFP-2x-FYVE mut.). Također mutirana je i YFP-PX domena već klonirana u retroviralni vektor. Imunoflorescencijom je uočen specifičan obrazac lokalizacije eGFP-2x-FYVE proteina vidljiv kao diskretne točke u stanici koje su kolokalizirale sa EEA1 (markerom ranih endosoma), LAMP1 (markerom kasnih endosoma) te sa LysoTrackerom (markerom lizosoma). YFP-PX također je karakteriziran ekspresijom u vidu vezikula unutar stanice koje dijelom kolokaliziraju sa EEA1. Oba konstrukta mutiranih domena izgubila su karakterističan obrazac lokalizacije te se pojavljuju kao jednoliko obojenje koje se proteže kroz čitavu stanicu. Selektivna inhibicija PI3-kinaze klase III koja producira PI(3)P dovela je do poremećenog obrasca smještaja eGFP-2x-FYVE domene, čime je potvrđena specifičnost klonirane domene u vezanju PI(3)P. Megakariociti koji eksprimiraju eGFP-2x-FYVE stvaraju znatno manje protrombocita nego kada eksprimiraju samo eGFP ili mutantu. Naposljetku je utvrđena kolokalizacija kloniranih PI(3)P vezujućih domena (eGFP-2x-FYVE, YFP-PX) sa protein kinazom A, upućujući na potencijalno novu ulogu PI(3)P. Dobiveni rezultati upućuju na visoku značajnost FYVE i PX domena, otvaraju nova pitanja i potiču nove smjerove istraživanja.
Keywordsphosphatidylinositol 3-monophosphate eGFP-2x-FYVE YFP-PX phosphoinositide 3-kinase megakaryocyte
Parallel title (English)Cloning, mutagenesis and characterization of phosphatidylinositol 3-monophosphate binding domains in HEK293 cells and primary megakaryocytes
Committee MembersAntonija Jurak Begonja (committee chairperson)
Igor Jurak (committee member)
Miranda Mladinić Pejatović (committee member)
GranterSveučilište u Rijeci
Lower level organizational unitsOdjel za biotehnologiju
PlaceRijeka
StateCroatia
Scientific field, discipline, subdisciplineBIOTECHNICAL SCIENCES
Biotechnology
Study programme typeuniversity
Study levelgraduate
Study programmeBiotechnology in medicine
Academic title abbreviationmag. biotech. in med.
Genremaster's thesis
Language Croatian
Defense date2016-09-26
Parallel abstract (English)
Phosphoinositides (PI) are a group of cellular phospholipids of the utmost importance for the regulation of cellular physiology and membrane dynamics. In eukaryotic cells they are generated by phosphorylation of phosphatidylinositol (PtdIns) by activation of a series of kinases and phosphatases. In this study we have investigated the expression and localization of FYVE and PX domains that specifically bind phosphatidylinositol 3-monophosphate [PI(3)P] and their impact on the development of megakaryocytes and production of proplatelets. We have cloned fluorescently labeled tandem FYVE and mutated domain (eGFP-2x-FYVE and eGFP-2x-FYVE mut.) into a retroviral vector. YFP-PX domain already cloned in retroviral vector was also mutated. By immunofluorescence, a specific localization pattern of eGFP-2x-FYVE was observed in form of discrete dots throughout the cell. These dots colocalized with EEA1 (early endosome marker), LAMP1 (late endosome marker) and LysoTracker (lysosome marker). YFP-PX was also characterized by the appearance of discrete vesicles partially colocalizing with EEA1. Both mutated domains lost the distinctive pattern of localization and appeared as a uniform signal throughout the cell. Selective inhibition of PI3-kinase class III which produces PI(3)P led to a disturbed pattern of eGFP-2x-FYVE localization, and thus confirmed specificity of the cloned domain for PI(3)P. Megakaryocytes that express GFP-2x-FYVE produce significantly less proplatelets than when expressing only eGFP or mutated domain. Finally, a colocalization of PI(3)P binding domains (eGFP-2x-FYVE, YFP-PX) with protein kinase A was determined, implicating a potentially new role of PI(3)P. These results indicate a high significance of FYVE and PX domains, create new questions, and elicit new research directions.
Parallel keywords (Croatian)fosfatidilinozitol 3-monofosfat eGFP-2x-FYVE YFP-PX PI3 kinaza klase III megakariocit
Resource typetext
Access conditionNo public access
URN:NBNhttps://urn.nsk.hr/urn:nbn:hr:193:331332
CommitterIvana Dorotić