| Abstract | The maturation of megakaryocytes (MKs) implies the polyploidization of the nucleus and the formation of a demarcation membrane system (DMS), an extensive internal membrane reservoir necessary for platelet production. While the initiation of the DMS starts with plasma membrane invagination and continues in the perinuclear region by the insertion of Golgiderived vesicles, the membrane source(s) and mechanism for further DMS expansion, cell growth and proplatelet formation remain elusive.
In the present study, we investigated the role of endolysosomal system during MKs maturation and platelet production. Vps34 kinase and its product lipid phosphatidylinositol 3-monophosphate (PI3P) are key components of early and late endosomes where they regulate vesicular trafficking. We demonstrate that, during maturation, Vps34-derived PI3P translocates
from the perinuclear early endosomes (EE) in immature, to peripheral late endosomes/lysosomes (LE/Lys) in mature MKs. Moreover, PI3P was found in close contact with PI(4,5)P2 (plasma membrane/DMS) and LE/Lys in mature MKs. Sequestration of PI3P by expression of 2xFYVE domain, specific pharmacological inhibition of Vps34-mediated PI3P production, or depletion of PI3P by expression of PI3P-phosphatase (MTM1) decreased proplatelet formation. At the same time, inhibition of PI3P production led to the intracellular accumulation of LE/Lys markers preventing their surface expression, suggesting an essential
role for PI3P in LE/Lys translocation toward the plasma membrane. Moreover, inhibition of Vps34 at earlier stages of MK maturation caused aberrant DMS development, decreased the size of MKs and the expression of GPIb, a DMS marker. In line with the above was targeting of downstream events in endosome maturation: overexpression of the dominant-negative late
endosomal GTPase Rab7 T22N or pharmacological inhibition of PI3P conversion to PI(3,5)P2 led to the enlarged LE/Lys, reduced surface levels of LE/Lys markers, and decreased proplatelet formation. In parallel, we investigated the role of early endosomal GTPase Rab5 which regulates endocytic uptake and represents the starting point for endosome maturation. In addition, Rab5 mediates the recruitment of Vps34 on the membrane of EEs, which is a key step during early-to-late endosome maturation. Rab5 mutants expressed from retroviruses showed the opposing effects on EE morphology and the amount of endocytosis in MKs. Active Rab5 Q79L induced the formation of large EEs that accumulated GPIb, increased transferrin internalization and at the same time increased proplatelet production. In contrast, inactive Rab5 N133L caused EE
fragmentation, GPIb retention at the plasma membrane, impaired transferrin endocytosis and strongly blocked proplatelet formation. Furthermore, active Rab5 Q79L increased membrane labeling by lipophilic dye PKH26 suggesting that the increased levels of the endocytosis might contribute to the internal membrane content in MKs. Together, these results suggest that PI3Ppositive LE/Lys contribute to the membrane growth and proplatelet formation by their translocation to the cell periphery and fusion with the plasma membrane, while early endosomal Rab5 regulates GPIb trafficking and represents the limiting step during endocytosis in MKs, the amount of which we believe is a major determinant of membrane growth and proplatelet
production. We also demonstrated for the first time that Vps34 localizes in the nucleus of primary MKs and other cells (BALB3T3, human mononuclear cells), as shown by immunofluorescence and cell fractionation experiments. Further analysis by confocal microscopy revealed that Vps34 is confined to the nucleolus, where it colocalizes with upstream binding factor (UBF), a
transcription factor required for rRNA expression. Inhibition of RNA polymerase I (Pol I) with actinomycin D, but not RNA Pol II with α-amanitin, abolished Vps34 nucleolar localization and Vps34 binding to UBF. In addition, pull-down of green fluorescent protein (GFP)-UBF coimmunoprecipitated Vps34 from BALB3T3 cells. Inhibition of Vps34 decreased mTORmediated S6K1 and S6 phosphorylation in MKs and blocked rRNA synthesis in BALB3T3, suggesting that it might play an important, still undescribed, role in the ribosome biogenesis and nucleolar organization and function. Our attempts to produce a stable cell line with Vps34
deletion by CRISPR/Cas9 gene-editing technology resulted in the successful generation of knockout (KO) cells for a single Vps34 isoform containing exon 2. Targeting other exons was unsuccessful, most likely because Vps34 is an essential gene and cells with complete Vps34 deletion do not survive. Taken together, the results of this thesis contribute to a better understanding of the involvement of the small lipid PI3P and related kinase Vps34 in the MK maturation and platelet production in addition to, until now, undescribed nucleolar localization and function of Vps34. |
| Abstract (english) | Maturacija megakariocita (MK) uključuje poliploidizaciju jezgre te formiranje demarkacijskog sustava membrana (DMS), unutarstaničnog membranskog rezervoara neophodnog za stvaranje trombocita. Dok DMS započinje invaginacijom plazmatske membrane i dalje raste u perinuklearnoj regiji ugradnjom vezikula podrijetlom iz Golgijevog aparata, izvor(i) membrana potrebnih za daljnje širenje DMS-a i mehanizam nastajanja protrombocita još uvijek nisu dovoljno razjašnjeni.
U ovom radu istraživali smo ulogu endolizosomalnog sustava tijekom maturacije MK i stvaranja krvnih pločica. Vps34 kinaza i njen lipidni produkt fosfatidilinozitol 3-monofosfat (PI3P) su ključne komponente ranih i kasnih endosoma gdje reguliraju promet vezikula. Pokazali smo da se tijekom sazrijevanja, PI3P kojeg stvara Vps34, translocira s perinuklearnih
ranih endosoma u nezrelim, na kasne endosome i lizosome u zrelim MK. Također, PI3P kolokalizira s PI(4,5)P2 (marker plazmatske mebrane i DMS-a) i kasnim endosomima/lizosomima u zrelim MK. Sekvestracija PI3P zbog prekomjerne ekspresije 2xFYVE domene (veže PI3P), inhibicija proizvodnje PI3P putem farmakološke inhibicije Vps34 ili smanjenje PI3P zbog ekspresije PI3P-fosfataze (MTM1), značajno je smanjila produkciju protrombocita. U isto vrijeme, inhibicija stvaranja PI3P dovela je do unutarstaničnog nakupljanja markera kasnih endosoma/lizosoma sprječavajući njihovu
površinsku ekspresiju te sugerirajući esencijalnu ulogu PI3P tijekom translokacije kasnih endosoma/lizosoma prema plazmatskoj membrani. Nadalje, inhibicija Vps34 tijekom ranijih stadija sazrijevanja MK narušila je razvoj DMS-a, smanjila veličinu MK te razinu ekspresije GPIb, glavnog receptora MK i markera DMS-a. U skladu s prethodnim rezultatima bilo je i
ciljanje nizvodnih događaja u maturaciji endosoma: prekomjerna ekspresija dominantno negativne GTPaze Rab7 T22N na kasnim endosomima ili farmakološka inhibicija konverzije PI3P u PI(3,5)P2 uzrokovala je povećanje kasnih endosoma/lizosoma, smanjila površinsku ekspresiju markera kasnih endosoma/lizosoma te proizvodnju protrombocita.
Paralelno smo istraživali ulogu Rab5 GTPaze na ranim endosomima koja regulira endocitozu te predstavlja početnu točku za sazrijevanje endosoma. Također, Rab5 posreduje u vezanju Vps34 na membranu ranih endosoma što je ključni korak tijekom maturacije ranih endosoma u kasne. Ekspresija Rab5 mutanti sa retrovirusa pokazala je suprotne učinke na morfologiju ranih endosoma i endocitozu u MK. Aktivni Rab5 Q79L potaknuo je stvaranje velikih ranih endosoma koji su sadržavali GPIb, povisio je internalizaciju transferina te je u isto vrijeme povećao stvaranja protrombocita. Nasuprot tome, inaktivni Rab5 N133L prouzročio je fragmentaciju ranih endosoma, zadržavanje GPIb na plazmatskoj membrani, te je značajno snizio endocitozu transferina i produkciju protrombocita. Nadalje, aktivni Rab5 Q79L povećao je obilježavanje membrana sa lipofilnom bojom PKH26 sugerirajući kako bi povećana razina endocitoze mogla doprinijeti sadržaju unutrašnjih membrana u MK. Ovi rezultati sugeriraju da PI3P-pozitivni kasni endosomi/lizosomi doprinose rastu membrana i stvaranju protrombocita translokacijom na periferiju stanice i fuzijom s plazmatskom membranom, dok Rab5 regulira
unutarstanični promet GPIb te predstavlja ograničavajući korak tijekom endocitoze u MK, za koju vjerujemo da predstavlja glavnu odrednicu za rast membrana i stvaranje protrombocita. Također smo, pomoću imunofluorescencije i stanične frakcionacije, po prvi put pokazali lokalizaciju Vps34 u jezgri primarnih MK i ostalih stanica (BALB3T3, ljudski mononukleari). Daljnja analiza konfokalnom mikroskopijom otkrila je prisutnost Vps34 u jezgrici gdje kolokalizira s uzvodnim faktorom vezanja (engl. upstream binding factor, UBF), transkripcijskim faktorom potrebnim za ekspresiju rRNA. Inhibicija RNA polimeraze I (Pol I) s aktinomicinom D, ali ne i inhibicija Pol II s α-amanitinom, narušila je lokalizaciju Vps34 u
jezgrici te kontakt s UBF-om. Dodatno, imunoprecipitacija UBF-a obilježenog zelenim fluorescentnim proteinom (GFP) u BALB3T3 staničnoj liniji potvrdila je prisutnost Vps34 u istom kompleksu. Inhibicija Vps34 snizila je mTOR-posredovanu fosforilaciju S6K1 i S6 u MK te inhibirala sintezu rRNA u BALB3T3, sugerirajući kako bi Vps34 mogao imati važnu,
dosad neistraženu, ulogu u biogenezi ribosoma te organizaciji i funkciji jezgrice. Pokušaji da proizvedemo stabilnu staničnu liniju s deletiranim Vps34 pomoću CRISPR/Cas9 tehnologije za uređivanje gena bili su uspješni samo za jednu Vps34 izoformu koja sadrži egzon 2. Ciljanje drugih egzona nije bilo uspješno, vrlo vjerojatno zbog toga što je Vps34 esencijalan gen te stanice s potpunom delecijom Vps34 ne preživljavaju. Zajedno, rezultati ovog istraživanja doprinose boljem razumijevanju uloge male lipidne molekule PI3P i njene kinaze Vps34 u sazrijevanja MK i stvaranju krvnih pločica, uz do sada neopisanu lokalizaciju i funkciju Vps34 kinaze u jezgrici. |
