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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Platelets are produced by bone marrow megakaryocytes through cytoplasmic protrusions, named native proplatelets (nPPT), into blood vessels. Proplatelets also refer to protrusions observed in ...megakaryocyte culture (cPPT) that are morphologically different. Contrary to cPPT, the mechanisms of nPPT formation are poorly understood. We show here in living mice that nPPT elongation is in equilibrium between protrusive and retraction forces mediated by myosin-IIA. We also found, using WT and β1-tubulin-deficient mice, that microtubule behavior differs between cPPT and nPPT, being absolutely required in vitro, while less critical in vivo. Remarkably, microtubule depolymerization in myosin-deficient mice did not affect nPPT elongation. We then calculated that blood Stokes'forces may be sufficient to promote nPPT extension, independently of myosin and microtubules. Together, we propose a new mechanism for nPPT extension that might explain contradictions between severely affected cPPT production and moderate platelet count defects in some patients and animal models.
Abstract
Zika virus (ZIKV) invades and persists in the central nervous system (CNS), causing severe neurological diseases. However the virus journey, from the bloodstream to tissues through a mature ...endothelium, remains unclear. Here, we show that ZIKV-infected monocytes represent suitable carriers for viral dissemination to the CNS using human primary monocytes, cerebral organoids derived from embryonic stem cells, organotypic mouse cerebellar slices, a xenotypic human-zebrafish model, and human fetus brain samples. We find that ZIKV-exposed monocytes exhibit higher expression of adhesion molecules, and higher abilities to attach onto the vessel wall and transmigrate across endothelia. This phenotype is associated to enhanced monocyte-mediated ZIKV dissemination to neural cells. Together, our data show that ZIKV manipulates the monocyte adhesive properties and enhances monocyte transmigration and viral dissemination to neural cells. Monocyte transmigration may represent an important mechanism required for viral tissue invasion and persistence that could be specifically targeted for therapeutic intervention.
Platelet δ-Storage Pool Disease: An Update Dupuis, Arnaud; Bordet, Jean-Claude; Eckly, Anita ...
Journal of clinical medicine,
08/2020, Letnik:
9, Številka:
8
Journal Article
Recenzirano
Odprti dostop
Platelet dense-granules are small organelles specific to the platelet lineage that contain small molecules (calcium, adenyl nucleotides, serotonin) and are essential for the activation of blood ...platelets prior to their aggregation in the event of a vascular injury. Delta-storage pool diseases (δ-SPDs) are platelet pathologies leading to hemorrhagic syndromes of variable severity and related to a qualitative (content) or quantitative (numerical) deficiency in dense-granules. These pathologies appear in a syndromic or non-syndromic form. The syndromic forms (Chediak–Higashi disease, Hermansky–Pudlak syndromes), whose causative genes are known, associate immune deficiencies and/or oculocutaneous albinism with a platelet function disorder (PFD). The non-syndromic forms correspond to an isolated PFD, but the genes responsible for the pathology are not yet known. The diagnosis of these pathologies is complex and poorly standardized. It is based on orientation tests performed by light transmission aggregometry or flow cytometry, which are supplemented by complementary tests based on the quantification of platelet dense-granules by electron microscopy using the whole platelet mount technique and the direct determination of granule contents (ADP/ATP and serotonin). The objective of this review is to present the state of our knowledge concerning platelet dense-granules and the tools available for the diagnosis of different forms of δ-SPD.
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.
The demarcation membrane system (DMS) in megakaryocytes forms the plasma membrane (PM) of future platelets. Using confocal microscopy, electron tomography, and large volume focused ion beam/scanning ...electron microscopy (FIB/SEM), we determined the sequential steps of DMS formation. We identified a pre-DMS that initiated at the cell periphery and was precisely located between the nuclear lobes. At all developmental stages, the DMS remained continuous with the cell surface. The number of these connections correlated well with the nuclear lobulation, suggesting a relationship with cleavage furrow formation and abortive cytokinesis. On DMS expansion, Golgi complexes assembled around the pre-DMS, and fusion profiles between trans-golgi network–derived vesicles and the DMS were observed. Brefeldin-A reduced DMS expansion, indicating that the exocytic pathway is essential for DMS biogenesis. Close contacts between the endoplasmic reticulum (ER) and the DMS were detected, suggesting physical interaction between the 2 membrane systems. FIB/SEM revealed that the DMS forms an intertwined tubular membrane network resembling the platelet open canalicular system. We thus propose the following steps in DMS biogenesis: (1) focal membrane assembly at the cell periphery; (2) PM invagination and formation of a perinuclear pre-DMS; (3) expansion through membrane delivery from Golgi complexes; and (4) ER-mediated lipid transfer.
•Using state-of-the-art three-dimensional electron microscopy approaches, we show that the onset of the DMS formation is at the megakaryocyte plasma membrane.•A pre-DMS structure is formed in the perinuclear region, through a PM invagination process that resembles cleavage furrow formation.
Electron microscopy (EM) has a long history in megakaryocyte (MK) cellular biology. This chapter shows how the electron microscope, since its first appearance almost 90 years ago, has occupied center ...stage in the studies of MK morphology and function. It describes some of the more productive EM techniques that have shaped our understanding of the physiology of thrombopoiesis. These include the standard transmission and scanning EM techniques as well as the new imaging methods, correlative microscopy and volume EM which provide information on the 3D organization of MKs on different scales: single organelles, whole cells and tissues. For each technique, we list the advantages and limitations, the resolution that can be achieved, the technical difficulties and the applications in MK biology.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Blood platelets are anuclear cell fragments that are essential for blood clotting. Platelets are produced by bone marrow megakaryocytes (MKs), which extend protrusions, or so-called proplatelets, ...into bone marrow sinusoids. Proplatelet formation requires a profound reorganization of the MK actin and tubulin cytoskeleton. Rho GTPases, such as RhoA, Rac1, and Cdc42, are important regulators of cytoskeletal rearrangements in platelets; however, the specific roles of these proteins during platelet production have not been established. Using conditional knockout mice, we show here that Rac1 and Cdc42 possess redundant functions in platelet production and function. In contrast to a single-deficiency of either protein, a double-deficiency of Rac1 and Cdc42 in MKs resulted in macrothrombocytopenia, abnormal platelet morphology, and impaired platelet function. Double-deficient bone marrow MKs matured normally in vivo but displayed highly abnormal morphology and uncontrolled fragmentation. Consistently, a lack of Rac1/Cdc42 virtually abrogated proplatelet formation in vitro. Strikingly, this phenotype was associated with severely defective tubulin organization, whereas actin assembly and structure were barely affected. Together, these results suggest that the combined action of Rac1 and Cdc42 is crucial for platelet production, particularly by regulating microtubule dynamics.
•Rac1 and Cdc42 have redundant functions in platelet biogenesis.•Deficiency of Rac1 and Cdc42 results in highly abnormal megakaryocyte morphology associated with severely defective tubulin organization.
Platelet activation at sites of vascular injury is crucial for hemostasis, but it may also cause myocardial infarction or stroke. Cytoskeletal reorganization is essential for platelet activation and ...secretion. The small GTPase Cdc42 has been implicated as an important mediator of filopodia formation and exocytosis in various cell types, but its exact function in platelets is not established. Here, we show that the megakaryocyte/platelet-specific loss of Cdc42 leads to mild thrombocytopenia and a small increase in platelet size in mice. Unexpectedly, Cdc42-deficient platelets were able to form normally shaped filopodia and spread fully on fibrinogen upon activation, whereas filopodia formation upon selective induction of GPIb signaling was reduced compared with wild-type platelets. Furthermore, Cdc42-deficient platelets showed enhanced secretion of α granules, a higher adenosine diphosphate (ADP)/adenosine triphosphate (ATP) content, increased aggregation at low agonist concentrations, and enhanced aggregate formation on collagen under flow. In vivo, lack of Cdc42 resulted in faster occlusion of ferric chloride–injured arterioles. The life span of Cdc42-deficient platelets was markedly reduced, suggesting increased clearing of the cells under physiologic conditions. These data point to novel multiple functions of Cdc42 in the regulation of platelet activation, granule organization, degranulation, and a specific role in GPIb signaling.
In this chapter, we describe the study of bone marrow megakaryocytes (MKs) using a high-resolution 3D imaging approach known as focused ion beam-scanning electron microscopy (FIB-SEM). The apparatus ...consists of a scanning electron microscope equipped with a focused gallium ion beam, used to sequentially mill away the sample surface, and an electron beam, used to image the milled surfaces. This produces a series of ultrastructural images which can be computationally reconstructed into three-dimensional (3D) volume images. Using this approach it is possible to characterize the 3D ultrastructure of MKs in their native bone marrow environment, to study subcellular organelle interactions in the context of a complete cell and to quantify specific features. This chapter provides protocols for sample preparation, image acquisition and 3D reconstruction, the whole procedure requiring about 7-8 days. It also describes a method combining light microscopy (LM) with FIB-SEM, a procedure called correlative light electron microscopy (CLEM), which allows the site-specific 3D imaging of MKs in tissues.
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