Platelets are released by megakaryocytes (MKs) via cytoplasmic extensions called proplatelets, which require profound changes in the microtubule and actin organization. Here, we provide evidence that the Rho/ROCK pathway, a well-known regulator of actin cytoskeleton, acts as a negative regulator of proplatelet formation (PPF). Rho is expressed at a high level during the entire MK differentiation including human CD34 ؉ cells. Thrombopoietin stimulates its activity but at a higher extent in immature than in mature MKs. Overexpression of a dominantnegative or a spontaneously active RhoA leads to an increase or a decrease in PPF indicating that Rho activation inhibits PPF. This inhibitory effect is mediated through the main Rho effector, Rho kinase (ROCK), the inhibition of which also increases PPF. Furthermore, inhibition of Rho or ROCK in MKs leads to a decrease in myosin light chain 2 (MLC2) phosphorylation, which is required for myosin contractility. Interestingly, inhibition of the MLC kinase also decreases MLC2 phosphorylation while increasing PPF. Taken together, our results suggest that MLC2 phosphorylation is regulated by both ROCK and MLC kinase and plays an important role in platelet biogenesis by controlling PPF and fragmentation. IntroductionMegakaryocytes (MKs) are the highly specialized precursor cells that lead to platelet production. MK differentiation is a continuous process characterized by sequential steps. 1 First, MKs increase their ploidy via endomitosis and begin to increase their size. 2 Then, the synthesis of storage organelles is enhanced, as well as the synthesis of plasma membrane to form the demarcation membranes. This cytoplasmic maturation is associated with a marked increase in the MK size. Finally, mature MKs release platelets probably through cytoplasmic fragmentation at the tips of long and thin extensions called proplatelets (PPTs) that contain all the platelet organelles. 3,4 The mechanisms controlling proplatelet formation (PPF) are still incompletely understood. However, PPF is associated with remarkable morphologic changes that require a profound reorganization of the cytoskeleton. 5 Increasing evidence indicates that PPTs arise from the unfolding of demarcation membranes. The microtubule cytoskeleton provides the sliding power to unfold demarcation membranes and thus to induce the pseudopodial elongations corresponding to PPTs. 6 In addition, microtubules permit the organelle transport in the PPTs and maintain the platelet discoid shape. [7][8][9][10] Although not studied in detail, the actin cytoskeleton may also participate in PPF because cytoplasmic polymerized actin is associated with demarcation membranes and actin is highly aggregated in cultured MKs when PPF occurs. 11 In addition, a crucial role of the actin cytoskeleton has been reported in platelet functions since it regulates platelet shape in unstimulated and activated platelets. 12 Evidence suggests that actin cytoskeleton may play important roles during PPT formation at 2 different stages: (1) at early stages,...

Plasmodium sp. are obligate intracellular parasites that derive most of their nutrients from their host meaning the metabolic circuitry of both are intricately linked. We employed untargeted, global mass spectrometry to identify metabolites present in the culture supernatants of P . falciparum- infected red blood cells synchronized at ring, trophozoite and schizont developmental stages. This revealed a temporal regulation in release of a distinct set of metabolites compared with supernatants of non-infected red blood cells. Of the distinct metabolites we identified pipecolic acid to be abundantly present in parasite lysate, infected red blood cells and infected culture supernatant. Further, we performed targeted metabolomics to quantify pipecolic acid concentrations in both the supernatants of red blood cells infected with P . falciparum , as well as in the plasma and infected RBCs of P . berghei -infected mice. Measurable and significant hyperpipecolatemia suggest that pipecolic acid has the potential to be a diagnostic marker for malaria.

No abstract

Megakaryopoiesis is a highly specialized cellular process which sustains platelet production. At the end of megakaryopoiesis, megakaryocyte (MKs) fragments into platelets via long and thin cytoplasmic extensions called proplatelets. Proplatelet formation (PPF) is associated essentially with cytoskeleton changes, including actin dynamics. The Rho/Rock pathway is a well characterized regulator of the actin reorganization. In the present study, we have tried to understand the precise role of the Rho/Rock pathway in PPF from human CD34+ derived MKs. Our results show that Rho is expressed in MKs and that its expression and activity remain stable during megakaryopoiesis. Overexpression of a RhoA dominant negatif (RhoA N19) in MKs leads to an increase in PPF. Conversely overexpression of a RhoA spontaneous active (RhoA V14) in MKs leads to a decrease in PPF. These results indicate that Rho activation could inhibit PPF in vitro. It is known that Rho/ROCK promotes actin cytoskeleton dynamics by regulating myosin light chain 2 (MLC2) phosphorylation. To demonstrate that Rho/Rock inhibits PPF through MLC2 phosphorylation, we added MLC kinase inhibitor (P18), Rho inhibitor (TatC3) and ROCK inhibitor (Y27362) in MKs culture just before PPF. Western blot analysis shows that MLC2 phosphorylation was inhibited by these 3 compounds, in contrast, PPF was significantly increased. Moreover, the platelet produced have an identical size and ultrastructure as control platelets and could be normally activated. These results suggest that Rho/ROCK could inhibit PPF through MLC phosphorylation during megakaryopoiesis.

Small microRNA levels are altered in many diseases, including those caused by parasites. Here, we describe how infection by two important animal and human parasites, Theileria annulata and Plasmodium falciparum , induce changes in infected host cell miR-34c-3p levels to regulate host cell PKA kinase activity by targeting mammalian prkar2b .

402 The site of platelet production in the human body is still unclear, but several evidence, including the recent demonstration by our team that shear stress in vitro accelerates platelet formation, favour the hypothesis of an intravascular location of the platelet release process. Therefore we have undertaken the following study to compare the effect of two cell types from the human bone marrow microenvironment, stromal medullary cells and endothelial cells, on the final steps of megakaryocyte (MK) maturation and platelet production. Firstly, supporting the goal of our study, we show that entire mature MK can be encountered in the lumen of normal bone marrow sinusoids and therefore enter the circulation of flowing blood; Secondly, we have used a new microcapillary device coated with von Willebrand factor and were able to confirm by videomicroscopy that exposure of human mature MK to shear forces accelerates proplatelet extension and facilitates platelet liberation from the tips of proplatelets. During this process, the nuclear lobes of polyploid MK tend to separate and to form distinct proplatelet units ready to deliver platelets: this observation might explain why massive senescent MK nuclei are only rarely found in human tissues, bone marrow or lung. Then, we used flow cytometry, optical and electron microscopy, to document and visualise the interaction of human medullary stromal cells (HS5), murine medullary stromal cells (MS5), and human endothelial cells (HUVEC) with the final steps of human MK maturation, namely proplatelet formation. MK were grown from umbilical cord blood CD34+ cells in the presence of Stem Cell Factor (SCF) and thrombopoietin and co-cultured with stromal cells between day 10 and day 13 of culture. Stromal cells virtually completely inhibited proplatelet formation from MK, whereas endothelial cells showed no effect and allowed the emission of proplatelets. EM and flow cytometry confirmed that stromal cells blocked proplatelet and platelet formation and showed that this was accompanied with a considerable development of demarcation membranes which coincided with a 60% increase of CD41 expression by MK. In parallel, stromal cells induced a reduction of apoptosis signs with a reduction of annexin V fixation by maturing MK. Since the step of proplatelet formation is accompanied with cytoplasmic apoptotic signs, among which increased phosphatidyl serine exposure, this result is in accordance with the observed blockade of proplatelet extension. Stromal cell secretion include several growth factors, namely SCF and GMCSF. MK were cultured in the presence of stromal cell culture supernatants, and with these growth factors either individually or mixed together, but this had no effects on proplatelet production, suggesting that physical contacts between the two cell types are necessary. In conclusion, our data show that human bone marrow microenvironment (stromal cells) has an inhibitory effect on proplatelet and platelet formation whereas the vascular microenvironment (endothelial cells) is permissive. This may explain the absence of proplatelets in the bone marrow parenchyma and be an additional evidence that platelet formation and release has to mainly occur in the intravascular compartment. Disclosures: No relevant conflicts of interest to declare.

A fungal metabolite, FR235222, specifically inhibits a histone deacetylase of the apicomplexan parasite Toxoplasma gondii and TgHDAC3 has emerged as a key factor regulating developmental stage transition in this species. Here, we exploited FR235222 to ask if changes in histone acetylation regulate developmental stage transition of Theileria annulata, another apicomplexan species. We found that FR235222 treatment of T. annulata-infected transformed leukocytes induced a proliferation arrest. The blockade in proliferation was due to drug-induced conversion of intracellular schizonts to merozoites that lack the ability to maintain host leukocyte cell division. Induction of merogony by FR235222 leads to an increase in expression of merozoite-marker (rhoptry) proteins. RNA-seq of FR235222-treated T. annulata-infected B cells identified deregulated expression of 468 parasite genes including a number encoding parasite ApiAP2 transcription factors. Thus, similar to T. gondii, FR235222 inhibits T. annulata HDAC (TaHDAC1) activity and places parasite histone acetylation as a major regulatory event of the transition from schizonts to merozoites.

A fungal metabolite, FR235222, specifically inhibits a histone deacetylase of the apicomplexan parasite Toxoplasma gondii and TgHDAC3 has emerged as a key factor regulating developmental stage transition in this species. Here, we exploited FR235222 to ask if changes in histone acetylation regulate developmental stage transition of Theileria annulata another apicomplexan species. We found that FR235222 treatment of T. annulata-infected transformed leukocytes induced a proliferation arrest. The blockade in proliferation was due to drug-induced conversion of intracellular schizonts to merozoites that lack the ability to maintain host leukocyte cell division. Induction of merogony by FR235222 leads to an increase in expression of merozoite-marker (rhoptry) proteins. RNA-seq of FR235222-treated T. annulata-infected B cells identified deregulated expression of 468 parasite genes, which included a number encoding parasite ApiAP2 transcription factors. Thus, similar to T. gondii, FR235222 inhibits T. annulata HDAC (TaHDAC1) activity and places parasite histone acetylation as a major regulatory event of the transition from schizonts to merozoites and hence pathogenesis of tropical theileriosis.