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Znotrajcelična porazdelitev in mobilnost proteina Nef virusa HIV-1 v kulturi humanih celic mikroglije
Gabrovec, Ana (Author), Stenovec, Matjaž (Mentor) More about this mentor... This link opens in a new window, Kreft, Marko (Co-mentor)

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Abstract
Po okužbi z virusom HIV-1, mikroglija množično proizvaja virusni protein Nef, ki nadalje spodbudi proizvodnjo virusa in poveča njegovo infektivnost. Kljub temu je malo znanega o znotrajcelični porazdelitvi tega proteina, njegovem transportu in izločanju iz mikroglije. V raziskavi smo zato transfecirali imortalizirano mikroglijo človeka s plazmidom za Nef označenim z zelenim fluorescentnim proteinom (Nef.GFP) in optofiziološko raziskali z Nef.GFP-obogatene celične predelke, njihovo mobilnost in izločanje Nef iz mikroglije v kulturi. Z analizo konfokalnih mikrografij smo potrdili, da so Nef.GFP-pozitivne strukture manjše od dekstran-pozitivnih mešičkov in se počasi ter neusmerjeno premikajo v primerjavi s hitro in usmerjeno mobilnimi dekstran-pozitivnimi mešički. Nef.GFP se je neznatno vključil v membrane različnih endosomov vključno z dekstran-pozitivnimi mešički in znatno lokaliziral v membranske predelke imunopozitivne za tetraspanina CD9 (36±4 %) in CD81 (22±1 %). Povečano izražanje virusnega proteina je sovpadalo tudi z zmanjšanjem števila CD9- in CD81-pozitivnih struktur v periplazmalemalnem predelu mikroglije. Z ionomicinom izzvano povečanje citosolne aktivnosti kalcija ni vplivalo na mobilnost Nef.GFP-pozitivnih struktur, a je močno in nepovratno zavrlo mobilnost dekstran-pozitivnih mešičkov. Počasno zmanjšanje števila Nef.GFP-pozitivnih struktur (5±1 struktur/min), ki je indikativno za izločanje Nef iz mikroglije smo opazili le v nedraženih, kontrolnih celicah in ne v celicah draženih z ionomicinom, v katerih se število struktur ni zmanjšalo v času (0±2 strukturi /min; P<0.05). Povečana znorajcelična koncentracija prostih Ca2+ ([Ca2+]i) je tako zavrla in ne spodbudila izločanje Nef.GFP iz celic, kar kaže, da se Nef verjetno izloča z ektosomi po brstenju iz plazmaleme in ne z uravnavano eksocitozo eksosomov iz svetline multivezikularnih telesc mikroglije.

Language:Slovenian
Keywords:mikroglija, HIV-1 Nef.GFP, lokalizacija, mobilnost mešičkov, kalcijeva signalizacija, eksocitoza
Work type:Master's thesis/paper (mb22)
Organization:BF - Biotechnical Faculty
Year:2019
COBISS.SI-ID:5327439 This link opens in a new window
Views:540
Downloads:176
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Secondary language

Language:Unknown
Title:Intracellular localization and mobility of HIV-1 protein Nef in cultured human microglia
Abstract:
Once infected by HIV-1, microglia abundantly produce accessory protein Nef that enhances virus production and infectivity, but little is known about its intracellular compartmentalization, trafficking mode(s), and release from microglia. Here, we transfected immortalized human microglia with a plasmid encoding Nef tagged with green fluorescent protein (Nef.GFP) to optophysiologically identify Nef.GFP-associated cellular compartments and examine their mobility and Nef release from cultured cells. As revealed by analysis of confocal micrographs, Nef.GFP-positive structures were smaller than dextran-laden vesicles, and displayed slow and non-directional mobility, in contrast to the faster and directional mobility of dextran-laden vesicles. Nef.GFP negligibly co-localized with different endosomes including Dextran-laden vesicles, but significantly co-localized with membranes immuno-positive for tetraspanins CD9 (36±4 %) and CD81 (22±1 %). Expression of Nef was further accompanied by a decrease in number of CD9- and CD81-positive structures in the periplasmalemmal space of microglia. Ionomycin-evoked elevation in cytosolic calcium activity negligibly affected mobility of Nef.GFP structures, but strongly and irrecoverably attenuated mobility of dextran-laden vesicles. A slow time-dependent decrease in number of Nef.GFP-positive structures was observed in non-stimulated controls (5±1 structures/min), but not in ionomycin-stimulated cells (0±2 structures/min; P<0.05), indicating that elevated free intracellular Ca2+ concentration ([Ca2+]i) inhibits the release of Nef.GFP structures. Our results thus suggest that Nef is likely released by ectosomes after budding from the plasmalemma and not by regulated exocytosis of exosomes from the lumen of multivesicular bodies in microglia.

Keywords:microglia, HIV-1 Nef.GFP, localization, mobility, Ca2+ signaling, exocytosis

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