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.
Bone marrow megakaryocytes (MKs) undergo a maturation involving contacts with the microenvironment before extending proplatelets through sinusoids to deliver platelets in the bloodstream. We ...demonstrated that MKs assemble linear F-actin-enriched podosomes on collagen I fibers. Microscopy analysis evidenced an inverse correlation between the number of dot-like versus linear podosomes over time. Confocal videomicroscopy confirmed that they derived from each-other. This dynamics was dependent on myosin IIA. Importantly, MKs progenitors expressed the Tks4/5 adaptors, displayed a strong gelatinolytic ability and did not form linear podosomes. While maturing, MKs lost Tks expression together with digestive ability. However, those MKs were still able to remodel the matrix by exerting traction on collagen I fibers through a collaboration between GPVI, ß1 integrin and linear podosomes. Our data demonstrated that a change in structure and composition of podosomes accounted for the shift of function during megakaryopoiesis. These data highlight the fact that members of the invadosome family could correspond to different maturation status of the same entity, to adapt to functional responses required by differentiation stages of the cell that bears them.
Abstract
The biological responses that control the development of Transfusion-Related Acute Lung Injury (TRALI), a serious post-transfusion respiratory syndrome, still need to be clarified. Since ...extracellular nucleotides and their P2 receptors participate in inflammatory processes as well as in cellular responses to stress, we investigated the role of the ATP-gated P2X
1
cation channel in antibody-mediated TRALI. The effects of NF449, a selective P2X1 receptor (P2RX1) antagonist, were analyzed in a mouse two-hit model of TRALI. Mice were primed with lipopolysaccharide (LPS) and 24 h later challenged by administrating an anti-MHC I antibody. The selective P2RX1 antagonist NF449 was administrated before the administration of LPS and/or the anti-MHC I antibody. When given before antibody administration, NF449 improved survival while maximal protection was achieved when NF449 was also administrated before the sensitization step. Under this later condition, protein contents in bronchoalveolar lavages were dramatically reduced. Cell depletion experiments indicated that monocytes/macrophages, but not neutrophils, contribute to this effect. In addition, the reduced lung periarteriolar interstitial edemas in NF449-treated mice suggested that P2RX1 from arteriolar smooth muscle cells could represent a target of NF449. Accordingly, inhibition of TRPC6, another cation channel expressed by smooth muscle cells, also reduced TRALI-associated pulmonary interstitial and alveolar edemas. These data strongly suggest that cation channels like P2RX1 or TRPC6 participate to TRALI pathological responses.
Complexes between CD1 molecules and self or microbial glycolipids represent important immunogenic ligands for specific subsets of T cells. However, the function of one of the CD1 family members, ...CD1e, has yet to be determined. Here, we show that the mycobacterial antigens hexamannosylated phosphatidyl-myo-inositols (PIM₆) stimulate CD1b-restricted T cells only after partial digestion of the oligomannose moiety by lysosomal alpha-mannosidase and that soluble CD1e is required for this processing. Furthermore, recombinant CD1e was able to bind glycolipids and assist in the digestion of PIM₆. We propose that, through this form of glycolipid editing, CD1e helps expand the repertoire of glycolipidic T cell antigens to optimize antimicrobial immune responses.
Previous work has suggested that the peptide‐carrier, heat‐shock protein (hsp)70, could directly activate APC. Here we show that this ability is related to endotoxin contamination of the humanrhsp70 ...produced in Escherichia coli. Hence, the ability of 1–3 μg/ml of rhsp70 to induce the maturation of human monocyte‐derived DC is abrogated in the presence of the LPS‐antagonist polymyxin B or when the rhsp70 contains less than 60 IU/mg endotoxin. Such a level of contamination of the rhsp70 is, however, sufficient — in the presence of soluble rCD14, the LPS co‐receptor — toinduce cytokine secretion from monocytes and DC, despite the presence of polymyxin B. However, when endotoxin contamination is below 10 IU/mg, rhsp70 does not induce cytokine secretion — even in the presence of soluble rCD14 — or activate p38 mitogen‐activated protein kinase signaling pathways, thus showing that an "endotoxin free" hsp70 does not activate APC.
A large body of knowledge relating to the constitution of Rab GTPase/Rab effector complexes and their impact on both membrane domain organization and overall membrane trafficking has been built up in ...recent years. However in the context of the live cell there are still many questions that remain to be answered, such as where and when these complexes assemble and where they perform their primary function(s). We describe here the dynamic processes that take place in the final steps of the Rab11A dependent recycling pathway, in the context of the membrane platform constituted by Myosin Vb, Rab11A, and Rab11‐FIP2. We first confirm that a series of previously reported observations obtained during the study of a number of trafficking cargoes also apply to langerin. Langerin is a cargo molecule that traffics through Rab11A‐positive membrane domains of the endosomal recycling pathway. In order to explore the relative dynamics of this set of partners, we make extensive use of a combinatory approach of Live‐FRET, fast FRAP video, fast confocal and TIRF microscopy modalities. Our data show that the Myosin Vb/Rab11A/Rab11‐FIP2 platform is spatially involved in the regulation of langerin trafficking at two distinct sites within live cells, first at the sorting site in the endosomal recycling compartment (ERC) where transport vesicles are formed, and subsequently, in a strict time‐defined order, at the very late stage of docking/tethering and fusion of these langerin recycling vesicles to the plasma membrane.
Previous investigations have indicated that RNAs are mostly present in the minor population of the youngest platelets, whereas translation in platelets could be biologically important. To attempt to ...solve this paradox, we studied changes in the RNA content of reticulated platelets, i.e., young cells brightly stained by thiazole orange (TObright), a fluorescent probe for RNAs. We provoked in mice strong thrombocytopenia followed by dramatic thrombocytosis characterized by a short period with a vast majority of reticulated platelets. During thrombocytosis, the TObright platelet count rapidly reached a maximum, after which TOdim platelets accumulated, suggesting that most of the former were converted into the latter within 12 h. Experiments on platelets, freshly isolated or incubated ex vivo at 37°C, indicated that their "RNA content", here corresponding to the amounts of extracted RNA, and the percentage of TObright platelets were positively correlated. The "RNA Content" normalized to the number of platelets could be 20 to 40 fold higher when 80-90% of the cells were reticulated (20-40 fg/platelet), than when only 5-10% of control cells were TObright (less than 1fg/platelet). TObright platelets, incubated ex vivo at 37°C or transfused into mice, became TOdim within 24 h. Ex vivo at 37°C, platelets lost about half of their ribosomal and beta actin RNAs within 6 hours, and more than 98% of them after 24 hours. Accordingly, fluorescence in situ hybridization techniques confirmed the presence of beta actin mRNAs in most reticulated-enriched platelets, but detected them in only a minor subset of control platelets. In vitro, constitutive translation decreased considerably within less than 6 hours, questioning how protein synthesis in platelets, especially in non-reticulated ones, could have a biological function in vivo. Nevertheless, constitutive transient translation in young platelets under pathological conditions characterized by a dramatic increase in circulating reticulated platelets could deserve to be investigated.
Birbeck granules are unusual rod-shaped structures specific to epidermal Langerhans cells, whose origin and function remain undetermined. We investigated the intracellular location and fate of ...Langerin, a protein implicated in Birbeck granule biogenesis, in human epidermal Langerhans cells. In the steady state, Langerin is predominantly found in the endosomal recycling compartment and in Birbeck granules. Langerin internalizes by classical receptor-mediated endocytosis and the first Birbeck granules accessible to endocytosed Langerin are those connected to recycling endosomes in the pericentriolar area, where Langerin accumulates. Drug-induced inhibition of endocytosis results in the appearance of abundant open-ended Birbeck granule-like structures appended to the plasma membrane, whereas inhibition of recycling induces Birbeck granules to merge with a tubular endosomal network. In mature Langerhans cells, Langerin traffic is abolished and the loss of internal Langerin is associated with a concomitant depletion of Birbeck granules. Our results demonstrate an exchange of Langerin between early endosomal compartments and the plasma membrane, with dynamic retention in the endosomal recycling compartment. They show that Birbeck granules are not endocytotic structures, rather they are subdomains of the endosomal recycling compartment that form where Langerin accumulates. Finally, our results implicate ADP-ribosylation factor proteins in Langerin trafficking and the exchange between Birbeck granules and other endosomal membranes.
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