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Origin and development of the demarcation membrane system in megakaryocytes

Research unit

UMR_S 949 - Biologie et pharmacologie des plaquettes sanguines : hémostase, thrombose, transfusion
10, rue Spielmann - BP 36 67065 Strasbourg Cedex

Group

Name: Biologie et Pharmacologie des plaquettes: athérothrombose expérimentale et clinique, transfusion

Group leader: GACHET Christian - christian.gachet@efs-sante.fr

Group leader's phone: 03.88.21.25.25

Website: Visit website

Group organization:
- Chercheurs: 5
- ITA: 5
- Doctorants: 2
- Post-Docs: 3
- Autres: 2

Publications of the team linked to the topic (3 last years):
1) 1.       Eckly A, Heijnen H, Pertuy F, Geerts W, Proamer F, Rinckel JY, Léon C, Lanza F, Gachet C.(2013) Biogenesis of the demarcation membrane system (DMS) in megakaryocytes. Blood. 123(6):921-30.
2) 2.       Eckly A, Strassel C, Cazenave JP, Lanza F, Leon C, Gachet C. (2012) Characterization of megakaryocyte development in the native bone marrow environment. Methods Mol Biol 788: 175-92.
3) 3.       Eckly A, Rinckel J-Y, Laeuffer P, Cazenave J-P, Lanza F, Gachet C and Léon C. (2010) Proplatelet formation deficit and megakaryocyte death contribute to thrombocytopenia in Myh9 knockout mice. J. Thromb. Haemost. 8 (10):2243-51.

About PhD

PhD Director: GACHET Christian - christian.gachet@efs-sante.fr

Phone: 03.88.21.25.25

Junior advisor: ECKLY Anita

Co-tutely: non

Co-Director: non

About PhD topic :

Title: Origin and development of the demarcation membrane system in megakaryocytes

Project: Background: Blood platelets play a crucial role in the maintenance of a hemostatic balance. They derive from megakaryocytes (MKs) in the bone marrow. MKs have the unique property to duplicate their DNA without cellular division (endomitosis). The cytoplasm of a mature MK contains an elaborate membrane system known as the demarcation membrane system (DMS). This DMS segregates the MK cytoplasm into distinct platelet territories that will become the future circulating platelets. The DMS ultimately forms the cell membrane of nascent platelets and plays a crucial role in platelet production. The exact origin of this unique membrane system and the molecular mechanisms driving its explosive growth are unknown. A current view is that the DMS develops from the invagination of the MK plasma membrane (PM). We recently identified an intracellular membrane structure that represents a putative precursor form of the DMS (the pre-DMS). This pre-DMS is precisely positioned between the nuclear lobes. This localization of the pre-DMS suggests a relationship with endomitosis and cleavage furrow formation, a connection that has not yet been explored.
Aim: Considering the importance of the DMS as the major membrane supply for platelet production, we will investigate the molecular mechanisms of DMS biogenesis. In particular, the link between DMS development and the endomitotic process and aborted cytokinesis will be explored.
Methods: The experiments will be performed on bone marrow megakaryocytes from mice and on cultured megakaryocytes differentiated from human or murine stem cells. We will use time-lapse videomicroscopy to visualize the first steps of pre-DMS formation at the earliest stages of MK development, i.e. at the first endomitotic cycle (2N-4N). Specific labeling of the nucleus, of the microtubules and of the plasma membrane using GFP-tagged markers or antibodies will be used to identify the MKs undergoing cytokinesis. The mechanisms involved in the formation of the pre-DMS will be explored by focusing on the role of some Rab GTPases which are known to regulate endomitosis (RhoA, Rab11), cytoskeletal proteins necessary for the formation of the cleavage furrow (actin, tubulins) and cytokinesis-specific proteins (Aurora B, PRC-1). Their implication will be evaluated using shRNA extinction and specific inhibitors.
This study will be applied to normal and pathological MKs. Since we previously observed abnormal DMS development in GPIb and MYH9 deficient mice, we will take advantage of these models and of the diseases to identify the abnormal DMS stages and to precise the role of these proteins in DMS formation.
Perspectives: This work, which aims to elucidate the mechanisms of DMS formation, is of particular importance since this step regulates the efficient production of mature platelets. (2790)

Approaches: They will include the isolation of megakaryocytes from the bone marrow and their culture from bone marrow progenitors, the use of innovative imaging techniques (time-lapse confocal microscopy and high resolution electron microscopy such as FIB-SEM, Tomography, Correlative microscopy), the use of genetically modified mice, insertion of GFP-tagged DMS markers using lentiviral vectors.

Feasibility: Megakaryocyte culture, microscopy and lentiviral technologies are mastered in the laboratory.


Mentorship : 1 DR1, 1 « chargé de recherche EFS »

Wished skills: Theoritical and practical experience in molecular and cellular biology. Hands-on experience in cell culture and fluorescence microscopy. Precision, thoroughness and motivation

Expertises which will be acquired during the training: Confocal and electron microscopy, culture of hematopoietic progenitors, mouse bone marrow transplantation.