Role of actomyosin system in normal and pathological megakaryopoiesis – MyoMeg

The maturation of megakaryocytes (MK), which leads to the formation of platelets, is a unique phenomenon characterized by three different processes: Polyploidization (MK become polyploid by a mechanism called endomitosis); the formation of proplatelets (at the end of differentiation, MK form cytoplasmic extensions by unfolding of the demarcation membranes due to the cytoskeleton dynamic) and migration (maturing MK migrate from the bone marrow microenvironment to the capillary-rich vascular niche to form proplatelets and release platelets). All these processes are accompanied by important changes of the cytoskeleton structures.
My team has been involved in the studies of the role of small Rho GTPases during the late steps of MK differentiation. Our results have shown that: - inhibition of Rho/Rock pathway favors MK polyploidization, probably by controlling the contractile ring function; - activation of the RhoA/Rock pathway inhibits proplatelet formation by phosphorylating the myosin light chain 2 (MLC2/MYL9); -through the MAL/SRF transcription complex, Rho GTPases control the transcription of both the myosin light chain and the metalloprotease MMP9, which are necessary for MK migration and proplatelet formation, and also of numerous compounds of the actomyosin complex. These data have underscored the key role of actomyosin system in platelet production, whereas it was believed that microtubules were the main cytoskeleton determinant in this process. Indeed, mutations or deletions of certain members of the actomyosin cytoskeleton are associated with several platelet disorders, such as the MYH9 or WASP syndrome.
To go further in this demonstration, the goal of this new project is trying to understand directly the role of the actomyosin system in the three remarkable aspects of late stages of megakaryopoiesis (endomitosis, formation of proplatelets and migration) under the pathological and physiological condition. Particularly, the specific or redundant roles of MYH9 and another non muscle myosin II (NMII) isoform MYH10, which is identified recently by our team in MK. We will test the hypothesis that MYH10 is implicated in the endomitosis whereas MYH9 is more involved in the proplatelet formation process. Moreover, the participation of mDia, a major actin nucleation-polymerization system, in these processes will be also highlighted by this project since it may link the microtubule and actomyosin cytoskeletons. To achieve this goal, we propose to study three complementary models for molecular and functional analyses in vivo and in vitro: One model is to study a mouse line with a deletion of myh10 in hematopoietic tissue to understand the role of MYH10 in mouse hematopoiesis, especially megakaryopoiesis; the second model is also a mouse line, in which the myh9 first coding exon has been disrupted by a cDNA encoding GFP-tagged myh10. The third one is to silence genes of interest with different shRNA in human MK which are derived from CD34+ cells isolated either from adult or cord blood. These three models will allow addressing four main goals in our project: i) Role of MYH10, the new identified NMII isoform, in normal and pathological megakaryopoiesis; 2) Regulation of MYH10 in normal and pathological megakaryopoiesis; 3) Identification of the specific and redundant roles of MYH9 and MYH10 during megakaryopoiesis; 4) Role of mDia (mDia1 and mDia2) on early and late stages of megakaryopoiesis.
This study will have general implications for understanding how the actomyosin system (NMII and mDia) influences the megakaryopoiesis. The primary consequences of this work are fundamental enabling us to better comprehend the mechanism of platelet production, one unique phenomenon in cellular biology. On the other hand, this work is a prerequisite to better understand the mechanism of some hereditary or acquired thrombopocytopenia including those resulting from new targetted therapies used in cancerology or other disorders.