Title The role of PI(4,5)P2 and PI4P in megakaryocyte maturation, proplatelet formation, and platelet function Title (croatian) Uloga PI(4,5)P2 i PI4P u sazrijevanju megakariocita, stvaranju protrombocita i funkciji trombocita Author Ana Bura Mentor Antonija Jurak Begonja (mentor) Committee member Christian Andrew Reynolds (predsjednik povjerenstva) Committee member Bojan Polić (član povjerenstva) Committee member Jelena Ban (član povjerenstva) Granter University of Rijeka Defense date and country 2022-12-20, Croatia Scientific / art field, INTERDISCIPLINARY AREAS OF KNOWLEDGE Universal decimal classificationUDC ) 576 - Cellular and subcellular biology. Cytology Abstract 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
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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.
Abstract (croatian) Megakariociti (MK) prilikom sazrijevanja stvaranju demarkacijski sustav membrana (DMS) koji služi kao rezervoar membrana za buduće trombocite. Za vrijeme stvaranja DMS-a, vezikule iz Golgijevog aparata konstantno putuju prema DMS-u, navodeći na zaključak da lipidi i proteini koji su potrebni za rast DMS-a nastaju u Golgijevom aparatu. Kad MK sazriju, u krvotok otpuštaju trombocite. Trombociti, u kontaktu s oštećenim endotelom krvne žile, adheriraju na mjesto ozljede, aktiviraju se te agregiraju što dovodi do stvaranja krvnog ugruška i zaustavljanja krvarenja. Jedan od najvažnijih lipida koji reguliraju anterogradni transport vezikula od Golgijevog aparata je fosfatidilinozitol-4-monofosfat (PI4P) te su njegove razine u Golgijevom aparatu u velikoj mjeri regulirane djelovanjem SACM1L fosfataze. U ovom istraživanju, pokazali smo da se PI4P u nezrelim MK nalazi u Golgijevom aparatu i na plazma membrani (PM) dok se u zrelim MK uglavnom nalazi na PM. Pokazali smo da PI4P koji se nalazi na Golgijevom aparatu, a čije su razine regulirane djelovanjem SACM1L fosfataze, je potreban za sazrijevanje MK i stvaranje protrombocita jer egzogena ekspresija divljeg tipa, ali ne katalitički ne-aktivnog tipa SACM1L fosfataze ne dozvoljava raspršenje Golgijevog aparata prilikom sazrijevanja MK i dovodi do smanjenog stvaranja protrombocita. Također, pokazali smo da PI4P koji se nalazi na PM, a čije je stvaranje regulirano djelovanjem PI4KIIIα je isto nužan za stvaranje protrombocita jer farmakološka inhibicija ove kinaze smanjuje postotak MK koji stvaraju protrombocite.
Prilikom aktivacije, trombociti mijenjaju svoj oblik, a u procesu reorganizacije aktina važnu ulogu ima fosfatidilinozitol-4,5-bisfosfat [PI(4,5)P2] kojeg, između ostalih enzima, regulira OCRL fosfataza. Mutacije OCRL fosfataze dovode do Lowe sindroma (LS) te je pokazano da, između ostalih simptoma, LS pacijenti mogu imati problema s krvarenjem. U ovom istraživanju smo pokazali da je za vizualizaciju PI4P i PI(4,5)P2 u trombocitima potrebno modificirati postojeći protokol te da je za optimalnu vizualizaciju ovih lipida na PM za permeabilizaciju potrebno koristiti 0.5% saponin 5 minuta. Nadalje, pokazali smo da farmakološka inhibicija OCRL-a u ljudskim trombocitima dovodi do smanjene aktivacije trombocita na različitim matricama (staklo, fibrinogen i kolagen) te da OCRL inhibirani trombociti aktivirani na fibrinogenu umjesto aktinskih stres niti stvaraju aktinske nodule. Ovi aktinski noduli kolokaliziraju s proteinima koji su važni za dinamiku aktina (ARP2/3 kompleks, vinculin, SNX9) te su mjesta aktivne signalizacije. Poremećena reorganizacija aktina je posredovana putem signalizacije lakog lanca miozina (MLC) jer OCRL inhibirani trombociti imaju smanjenu fosforilaciju MLC. Također, OCRL inhibirani trombociti ne reorganiziraju mikrotubule prilikom aktivacije već zadržavaju mikrotubularnu zavojnicu te imaju povišene razine acetiliranog tubulina. Unatoč nepotpunoj aktivaciji, OCRL inhibicija ne utječe na degranulaciju i aktivaciju integrina. Konačno, pokazali smo da se trombociti
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OCRL KO miševa također ne aktiviraju u potpunosti, odnosno da stvaraju više filopodija, a manje lamelipodija.
Zajedno, ovi rezultati doprinose boljem razumijevanju uloge PI4P-a tijekom sazrijevanja MK i PI(4,5)P2 tijekom aktivacije trombocita. Također, ovi rezultati ukazuju na važnost različitih izvora fosfoinozitida tijekom sazrijevanja MK i stvaranja protrombocita i daju uvid u molekularni mehanizam nepotpune aktivacije trombocita u LS pacijenata.
Keywords
megakaryocytes
platelets
PI4P
PI(4
5)P2
SACM1L
OCRL
Keywords (croatian)
megakariociti
trombociti
PI4P
PI(4
5)P2
SACM1L
OCRL
Language english URN:NBN urn:nbn:hr:193:664740 Promotion 2022-12-20 Study programme Title: Medicinal chemistry Type of resource Text Extent 169 str. File origin Born digital Access conditions Embargoed access Terms of use Repository Repository of the University of Rijeka, Faculty of Biotechnology and Drug Development Created on 2022-12-22 12:25:41
