Megakaryocytes use in vivo podosome‐like structures working collectively to penetrate the endothelial barrier of bone marrow sinusoids

Abstract

Background: Blood platelets are anucleate cell fragments that prevent bleeding and minimize blood vessel injury. They are formed from the cytoplasm of megakaryocytes located in the bone marrow. For successful platelet production, megakaryocyte fragments must pass through the sinusoid endothelial barrier by a cell biology process unique to these giant cells as compared with erythrocytes and leukocytes. Currently, the mechanisms by which megakaryocytes interact and progress through the endothelial cells are not understood, resulting in a significant gap in our knowledge of platelet production. Objective: The aim of this study was to investigate how megakaryocytes interact and progress through the endothelial cells of mouse bone marrow sinusoids. Methods: We used a combination of fluorescence, electron, and three-dimensional microscopy to characterize the cellular events between megakaryocytes and endothelial cells. Results: We identified protrusive, F-actin-based podosome-like structures, called in vivo-MK podosomes, which initiate the formation of pores through endothelial cells. These structures present a collective and spatial organization through their interconnection via a contractile network of actomyosin, essential to regulate the endothelial openings. This ensures proper passage of megakaryocyte-derived processes into the blood circulation to promote thrombopoiesis. Conclusion: This study provides novel insight into the in vivo function of podosomes of megakaryocytes with critical importance to platelet production.

Transcriptome analysis of the effect of AHR on productive and unproductive pathways of in vitro megakaryocytopoiesis

“…GSEA identified 26 genes participating in the "Platelet activation, signaling and aggregation" reactome pathway, reflecting megakaryocytic commitment between D0 and D7 and overexpression during D10-D14 MK maturation. The genes of this group were also involved in the organization of cellular structures important in MK maturation: (i) the trans-Golgi network, which has previously been shown to expand and to lie in the proximity of the demarcation membrane system (DMS) of mature MKs (13)-, (ii) secretory vesicles, to which platelet granules are related, and (iii) membrane organization (podosomes, ruffles, the leading edge of cells), which highlights the role of plasma membrane dynamics in mature MKs and is in agreement with the importance of podosomes in intravasation (14).…”

“…In addition, mTORC2 signaling participates in the signal transduction initiated by tyrosine kinase receptors or GPCR (35), which in the context of megakaryocytopoiesis might be triggered by stromal cell products. It is also involved in mechanical membrane stretching (16), an event potentially encountered during podosome formation, before crossing of the endothelial barrier (14), or during proplatelet elongation in vitro or in the blood stream. The differences between in vitro and in vivo conditions might lead to differences in the activation pathways of mature MKs, which could result in different mechanisms of proplatelet generation, as recently documented (38).…”

RhoA/Cdc42 signaling drives cytoplasmic maturation but not endomitosis in megakaryocytes

“…RhoA/Cdc42 deficiency nearly abrogated PPF in vitro and in vivo. Recent studies have highlighted a critical role of MK podosome formation for PPF in vivo by allowing MK fragments or proplatelets to penetrate the endothelial barrier at the vascular sinusoids (Brown et al, 2018;Eckly et al, 2020;Schachtner et al, 2013). Impaired podosome formation of DKO MKs may thus contribute to the severe platelet production defect in these mice.…”

Tissue remodeling by invadosomes

“…The presence of podosomes in mice aortas was also determined in vascular smooth muscle cells lacking microRNA-143/145, in which circular structures composed of cortactin and Tks5 were visualized by immunoelectron microscopy 71 . More recently, a combination of fluorescence, electron, and three‐dimensional microscopy was used to image the contacts between megakaryocytes and endothelial cells in mouse bone marrow sections, revealing that megakaryocytes use in vivo podosome‐like structures that collectively allow the penetration into the endothelium of bone marrow sinusoids 72 . Finally, elegant work in living Caenorhabditis elegans larvae showed the formation of invadosomes in the anchor cell and their role in basement membrane remodeling during vulval development 5 , 73 .…”