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Membrane dynamics and cytoskeleton regulation in megakaryocyte differentiation

Thesis

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Abstract

Megakaryocytes (MK) are giant cells located in the bone marrow that produce blood platelets. Their differentiation is characterized by an increase in proteins and membranes that leads to the elongation of cytoplasmic protrusions called proplatelets (PPTs). PPTs will cross the medullar endothelium to release platelets in the blood flow. Tubulin plays a major role in PPT elongation. However, the involvement of the actin cytoskeleton remains unclear and controversial. In this context, the purpose of my thesis was to study the role of actin and its regulators, namely the Rho GTPases and phosphoinositides, in PPTs formation. In a first part, I demonstrated that the F-actin was the main regulator of the formation of the demarcation membranes system (DMS), which represents a membrane reservoir for PPTs. Using complementary mouse and human models, I demonstrated that the Cdc42 GTPase and its effector Pak2 were key regulators of this process. My data set the bases of the internal maturation process. I demonstrated that MKs display the intrinsic property of polarizing in response to thrombopoietin, which is the main cytokine regulating megakaryopoiesis, independently of contacts with the hematopoietic niche. Polarization results in the formation of the DMS territory at the future PPTs emission site, facing the nuclei territory. 3D confocal and FRET imaging demonstrated that active Cdc42 was associated with endomembranes. Interestingly, Cdc42 pharmacological inhibition resulted in total DMS disorganization, which completely abolished PPTs formation. In this phenomenon, Pak2 acted downstream of Cdc42 and its inhibition also abrogated platelets production. These data shed new light on MK physiological maturation that ends in normal platelet production and points to new potential molecular targets that might be dysregulated in thrombocytopenia. We then set up a collaboration with Dr B. Nieswandt's group (University Clinic of Würzburg, Germany) to evaluate the impact of megakaryocytic specific knockout of the Rac1, Cdc42 and RhoA GTPases, alone or in combination, on murine MKs differentiation. My results with these models validate the central role of Cdc42 and demonstrated an unexpected cross-regulation between Cdc42 and RhoA. In a second part, I studied the role of the phosphatidylinositol 5-phosphate (PtdIns5P), which the team demonstrated to activate Rac1 and Cdc42, and identified as a central regulator of lymphoma invasion through elaboration of invasive podosomes. Contacts with the extracellular matrix (ECM) within the bone marrow are instrumental into MKs terminal differentiation. I demonstrated that MKs form linear podosomes along collagen fibers while they form regular dotted structures on fibrinogen. Interestingly, we observed a difference in podosome-associated GTPases according to the matrix. Podosomes formed on collagen were enriched in Rac1, while Cdc42 localized in podosomes when cells were cultivated on fibrinogen. In this case, PtdIns5P that was vesicular in MKs progenitors concentrated in the actin core of those invasive podosomes. Taken together, these data suggest that when the matrix is similar to the vascular environment (fibrinogen), concentration of PtdIns5P and Cdc42 in podosomes might lead to the emergence of the invasive phenotype seen in vitro on fibrinogen-containing gels. An interesting hypothesis regarding the in vivo outcome of this observation is that the PtdIns5P/Cdc42 pathway might be involved in the regulation of MKs contacts with endothelial cells to drive endothelium crossing. Overall, these data shed new lights on MKs maturation and its relationship with medullar and vascular environments that results in production of functional platelets.

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