Impaired microtubule dynamics contribute to microthrombocytopenia in RhoB-deficient mice.

Megakaryocytes are large cells in the bone marrow that give rise to blood platelets. Platelet biogenesis involves megakaryocyte maturation, the localization of the mature cells in close proximity to bone marrow sinusoids, and the formation of protrusions, which are elongated and shed within the circulation. Rho GTPases play important roles in platelet biogenesis and function. RhoA-deficient mice display macrothrombocytopenia and a striking mislocalization of megakaryocytes into bone marrow sinusoids and a specific defect in G-protein signaling in platelets. However, the role of the closely related protein RhoB in megakaryocytes or platelets remains unknown. In this study, we show that, in contrast to RhoA deficiency, genetic ablation of RhoB in mice results in microthrombocytopenia (decreased platelet count and size). RhoB-deficient platelets displayed mild functional defects predominantly upon induction of the collagen/glycoprotein VI pathway. Megakaryocyte maturation and localization within the bone marrow, as well as actin dynamics, were not affected in the absence of RhoB. However, in vitro-generated proplatelets revealed pronouncedly impaired microtubule organization. Furthermore, RhoB-deficient platelets and megakaryocytes displayed selective defects in microtubule dynamics/stability, correlating with reduced levels of acetylated α-tubulin. Our findings imply that the reduction of this tubulin posttranslational modification results in impaired microtubule dynamics, which might contribute to microthrombocytopenia in RhoB-deficient mice. Importantly, we demonstrate that RhoA and RhoB are localized differently and have selective, nonredundant functions in the megakaryocyte lineage.

G6b-B regulates an essential step in megakaryocyte maturation.

G6b-B is a megakaryocyte lineage-specific immunoreceptor tyrosine-based inhibition motif-containing receptor, essential for platelet homeostasis. Mice with a genomic deletion of the entire Mpig6b locus develop severe macrothrombocytopenia and myelofibrosis, which is reflected in humans with null mutations in MPIG6B. The current model proposes that megakaryocytes lacking G6b-B develop normally, whereas proplatelet release is hampered, but the underlying molecular mechanism remains unclear. We report on a spontaneous recessive single nucleotide mutation in C57BL/6 mice, localized within the intronic region of the Mpig6b locus that abolishes G6b-B expression and reproduces macrothrombocytopenia, myelofibrosis, and osteosclerosis. As the mutation is based on a single-nucleotide exchange, Mpig6bmut mice represent an ideal model to study the role of G6b-B. Megakaryocytes from these mice were smaller, displayed a less-developed demarcation membrane system, and had a reduced expression of receptors. RNA sequencing revealed a striking global reduction in the level of megakaryocyte-specific transcripts, in conjunction with decreased protein levels of the transcription factor GATA-1 and impaired thrombopoietin signaling. The reduced number of mature MKs in the bone marrow was corroborated on a newly developed Mpig6b-null mouse strain. Our findings highlight an unexpected essential role of G6b-B in the early differentiation within the megakaryocytic lineage.

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

Megakaryocytes (MKs), the precursors of blood platelets, are large, polyploid cells residing mainly in the bone marrow. We have previously shown that balanced signaling of the Rho GTPases RhoA and Cdc42 is critical for correct MK localization at bone marrow sinusoids in vivo. Using conditional RhoA/Cdc42 double-knockout (DKO) mice, we reveal here that RhoA/Cdc42 signaling is dispensable for the process of polyploidization in MKs but essential for cytoplasmic MK maturation. Proplatelet formation is virtually abrogated in the absence of RhoA/Cdc42 and leads to severe macrothrombocytopenia in DKO animals. The MK maturation defect is associated with downregulation of myosin light chain 2 (MLC2) and ß1-tubulin, as well as an upregulation of LIM kinase 1 and cofilin-1 at both the mRNA and protein level and can be linked to impaired MKL1/SRF signaling. Our findings demonstrate that MK endomitosis and cytoplasmic maturation are separately regulated processes, and the latter is critically controlled by RhoA/Cdc42.

Isolation of murine bone marrow by centrifugation or flushing for the analysis of hematopoietic cells - a comparative study.

Investigation of the bone marrow as the main compartment of hematopoiesis is critical in many research fields. Here, we adapted a centrifugation-based method for the isolation of murine bone marrow and compared it to the traditional flushing method. Analysis of primary hematopoietic stem cells, immune cells, and megakaryocytes revealed a comparable distribution of cellular (sub)populations. Furthermore, in vitro differentiated megakaryocytes displayed unaltered proplatelet formation. Strikingly, bone marrow isolation by centrifugation was considerably faster than the flushing method and significantly increased the cell yield. Thus, the centrifugation-based isolation method is highly suitable for the study of murine bone marrow cells.

Estimation of the Adhesion Interface Performance in Aluminum-PLA Joints by Thermographic Monitoring of the Material Extrusion Process.

Using additive manufacturing to generate a polymer-metal structure offers the potential to achieve a complex customized polymer structure joined to a metal base of high stiffness and strength. A tool to evaluate the generated interface during the process is of fundamental interest, as the sequential deposition of the polymer as well as temperature gradients within the substrate lead to local variations in adhesion depending on the local processing conditions. On preheated aluminum substrates, 0.3 and 0.6 mm high traces of polylactic acid (PLA) were deposited. Based on differential scanning calorimetry (DSC) and rheometry measurements, the substrate temperature was varied in between 150 and 200 °C to identify an optimized manufacturing process. Decreasing the layer height and increasing the substrate temperature promoted wetting and improved the adhesion interface performance as measured in a single lap shear test (up to 7 MPa). Thermographic monitoring was conducted at an angle of 25° with respect to the substrate surface and allowed a thermal evaluation of the process at any position on the substrate. Based on the thermographic information acquired during the first second after extrusion and the preset shape of the polymer trace, the resulting wetting and shear strength were estimated.

Actin/microtubule crosstalk during platelet biogenesis in mice is critically regulated by Twinfilin1 and Cofilin1.

Rearrangements of the microtubule (MT) and actin cytoskeleton are pivotal for platelet biogenesis. Hence, defects in actin- or MT-regulatory proteins are associated with platelet disorders in humans and mice. Previous studies in mice revealed that loss of the actin-depolymerizing factor homology (ADF-H) protein Cofilin1 (Cof1) in megakaryocytes (MKs) results in a moderate macrothrombocytopenia but normal MK numbers, whereas deficiency in another ADF-H protein, Twinfilin1 (Twf1), does not affect platelet production or function. However, recent studies in yeast have indicated a critical synergism between Twf1 and Cof1 in the regulation of actin dynamics. We therefore investigated platelet biogenesis and function in mice lacking both Twf1 and Cof1 in the MK lineage. In contrast to single deficiency in either protein, Twf1/Cof1 double deficiency (DKO) resulted in a severe macrothrombocytopenia and dramatically increased MK numbers in bone marrow and spleen. DKO MKs exhibited defective proplatelet formation in vitro and in vivo as well as impaired spreading and altered assembly of podosome-like structures on collagen and fibrinogen in vitro. These defects were associated with aberrant F-actin accumulation and, remarkably, the formation of hyperstable MT, which appears to be caused by dysregulation of the actin- and MT-binding proteins mDia1 and adenomatous polyposis coli. Surprisingly, the mild functional defects described for Cof1-deficient platelets were only slightly aggravated in DKO platelets suggesting that both proteins are largely dispensable for platelet function in the peripheral blood. In summary, these findings reveal critical redundant functions of Cof1 and Twf1 in ensuring balanced actin/microtubule crosstalk during thrombopoiesis in mice and possibly humans.

A Cdc42/RhoA regulatory circuit downstream of glycoprotein Ib guides transendothelial platelet biogenesis.

Blood platelets are produced by large bone marrow (BM) precursor cells, megakaryocytes (MKs), which extend cytoplasmic protrusions (proplatelets) into BM sinusoids. The molecular cues that control MK polarization towards sinusoids and limit transendothelial crossing to proplatelets remain unknown. Here, we show that the small GTPases Cdc42 and RhoA act as a regulatory circuit downstream of the MK-specific mechanoreceptor GPIb to coordinate polarized transendothelial platelet biogenesis. Functional deficiency of either GPIb or Cdc42 impairs transendothelial proplatelet formation. In the absence of RhoA, increased Cdc42 activity and MK hyperpolarization triggers GPIb-dependent transmigration of entire MKs into BM sinusoids. These findings position Cdc42 (go-signal) and RhoA (stop-signal) at the centre of a molecular checkpoint downstream of GPIb that controls transendothelial platelet biogenesis. Our results may open new avenues for the treatment of platelet production disorders and help to explain the thrombocytopenia in patients with Bernard-Soulier syndrome, a bleeding disorder caused by defects in GPIb-IX-V.