Pregled bibliografske jedinice broj: 1246310
The role of PI(4,5)P2 and PI4P in megakaryocyte maturation, proplatelet formation, and platelet function
The role of PI(4,5)P2 and PI4P in megakaryocyte maturation, proplatelet formation, and platelet function, 2022., doktorska disertacija, Odjel za biotehnologiju, Rijeka
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Naslov
The role of PI(4,5)P2 and PI4P in megakaryocyte
maturation, proplatelet formation, and platelet
function
Autori
Ana Bura
Vrsta, podvrsta i kategorija rada
Ocjenski radovi, doktorska disertacija
Fakultet
Odjel za biotehnologiju
Mjesto
Rijeka
Datum
20.12
Godina
2022
Stranica
169
Mentor
Antonija Jurak Begonja
Ključne riječi
megakaryocytes, platelets, OCRL, PI4P, SACM1L
Sažetak
During megakaryocyte (MK) maturation there is an extensive formation of the demarcation membrane system (DMS) which serves as a membrane reservoir for future platelets (PLTs). During the DMS formation, there is an active transport of vesicles from the Golgi apparatus to the DMS suggesting that lipids and proteins that are necessary for DMS growth originate from the Golgi apparatus. Once mature, MKs release PLTs into the bloodstream where upon encounter with a vessel wall injury PLTs adhere, activate, and aggregate which results in clot formation. The most important lipid that regulates the anterograde Golgi trafficking is the phosphatidylinositol-4- monophosphate (PI4P) and its levels at the Golgi apparatus are controlled to a great extent by the SACM1L phosphatase. In this study, we show that PI4P localizes to the Golgi apparatus and the plasma membrane (PM) of immature MKs while in mature MKs is mostly localized to the PM. We demonstrate that the Golgi pool of PI4P that is controlled by the SACM1L phosphatase is necessary for MK maturation and proplatelet formation because the exogenous expression of wild-type, but not catalytically inactivated SACM1L, retains the dispersion of the Golgi apparatus during MK maturation and results in a decrease of proplatelet formation. In addition, we show that the PM pool of PI4P that is controlled by the PI4KIIIα is also necessary for proplatelet formation since pharmacological inhibition of this kinase decreases proplatelet formation. PLT activation is accompanied by massive shape change, and an important role in actin reorganization has phosphatidylinositol-4, 5-bisphosphate [PI(4, 5)P2] that is, among other enzymes, regulated by the OCRL phosphatase. Mutations in OCRL cause Lowe syndrome (LS) and it has been shown that, among other symptoms, LS patients can have bleeding problems. Here, we firstly show that for the visualization of PI4P and PI(4, 5)P2 in PLTs, a modification of the existing staining protocol is needed and that the best staining of the PM pool of these lipids is obtained when permeabilizing the cells with 0.5% saponin for 5 minutes. Next, we show that the pharmacological inhibition of OCRL in human PLTs leads to an impaired spreading on three different matrices (glass, fibrinogen, and collagen) and that OCRL-inhibited PLTs spread in fibrinogen instead of actin stress fibres, form actin nodules. These actin nodules colocalize with proteins important for actin dynamics (ARP2/3 complex, vinculin, SNX9) and are sites of active signalling. The impaired actin reorganization is mediated by the myosin light chain (MLC) signalling since OCRL-inhibited PLTs have decreased MLC phosphorylation. Furthermore, OCRL- inhibited PLTs have impaired microtubular reorganization, shown by a retained microtubular coil during PLT activation which is accompanied by higher levels of acetylated tubulin. Interestingly, OCRL inhibition does not affect PLT degranulation or integrin activation. Finally, we show that V the OCRL KO mice also have impaired PLT spreading shown by an increased number of PLTs forming filopodia, and a decreased number of PLTs forming lamellipodia. Taken together, these results contribute to a better understanding of the role of PI4P during MK maturation and PI(4, 5)P2 during PLT activation. They also show the importance of different phosphoinositide pools in MK maturation and proplatelet formation, and they give an insight into the molecular mechanism of impaired PLT activation in LS patients.
Izvorni jezik
Engleski
Znanstvena područja
Biologija, Biotehnologija u biomedicini (prirodno područje, biomedicina i zdravstvo, biotehničko područje)
POVEZANOST RADA
Projekti:
HRZZ-DOK-2018-01 - Projekt razvoja karijera mladih istraživača – izobrazba novih doktora znanosti (Jurak Begonja, Antonija, HRZZ - Projekt razvoja karijera mladih istraživača – izobrazba novih doktora znanosti) ( CroRIS)
Ostalo-CRP/HRV15-04_EC - Deciphering the role of phosphoinositol lipids in thrombopoiesis (Jurak Begonja, Antonija, Ostalo - CRP-ICGEB Early Career Return Grant) ( CroRIS)
HRZZ-UIP-2014-09-2400 - Uloga fosfoinozitida u nastanku trombocita (MkPI) (Jurak Begonja, Antonija, HRZZ - 2014-09) ( CroRIS)
NadSve-Sveučilište u Rijeci-uniri-biomed-18-188-1343 - Identifikacija novih interakcijskih partnera Vps34 u megakariopoezi (Jurak Begonja, Antonija, NadSve - UNIRI projekti 2018) ( CroRIS)
Ustanove:
Sveučilište u Rijeci - Odjel za biotehnologiju