In mammals, megakaryocytes (MKs) in the bone marrow (BM) produce blood platelets, required for hemostasis and thrombosis. MKs originate from hematopoietic stem cells and are thought to migrate from an endosteal niche towards the vascular sinusoids during their maturation. Through imaging of MKs in the intact BM, here we show that MKs can be found within the entire BM, without a bias towards bone-distant regions. By combining in vivo two-photon microscopy and in situ light-sheet fluorescence microscopy with computational simulations, we reveal surprisingly slow MK migration, limited intervascular space, and a vessel-biased MK pool. These data challenge the current thrombopoiesis model of MK migration and support a modified model, where MKs at sinusoids are replenished by sinusoidal precursors rather than cells from a distant periostic niche. As MKs do not need to migrate to reach the vessel, therapies to increase MK numbers might be sufficient to raise platelet counts.

RNA duplex formation between U1 snRNA and a splice donor (SD) site can protect pre-mRNA from degradation prior to splicing and initiates formation of the spliceosome. This process was monitored, using sub-genomic HIV-1 expression vectors, by expression analysis of the glycoprotein env, whose formation critically depends on functional SD4. We systematically derived a hydrogen bond model for the complementarity between the free 5¢ end of U1 snRNA and 5¢ splice sites and numerous mutations following transient transfection of HeLa-T4 + cells with 5¢ splice site mutated vectors. The resulting model takes into account number, interdependence and neighborhood relationships of predicted hydrogen bond formation in a region spanning the three most 3¢ base pairs of the exon (±3 to ±1) and the eight most 5¢ base pairs of the intron (+1 to +8). The model is represented by an algorithm classifying U1 snRNA binding sites which can or cannot functionally substitute SD4 with respect to Rev-mediated env expression. In a data set of 5¢ splice site mutations of the human ATM gene we found a signi®cant correlation between the algorithmic classi®cation and exon skipping (P = 0.018, c 2 -test), showing that the applicability of the proposed model reaches far beyond HIV-1 splicing. However, the algorithmic classi®cation must not be taken as an absolute measure of SD usage as it may be modi®ed by upstream sequence elements. Upstream to SD4 we identi®ed a fragment supporting ASF/SF2 binding. Mutating GAR nucleotide repeats within this site decreased the SD4-dependent Rev-mediated env expression, which could be balanced simply by arti®cially increasing the complementarity of SD4.

Key Points• Rac1 and Cdc42 have redundant functions in platelet biogenesis. • Deficiency of Rac1 andCdc42 results in highly abnormal megakaryocyte morphology associated with severely defective tubulin organization.Blood platelets are anuclear cell fragments that are essential for blood clotting. Platelets are produced by bone marrow megakaryocytes (MKs), which extend protrusions, or socalled 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 singledeficiency 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. (Blood. 2013;122(18):3178-3187)

Microtubule spindle assembly in mitosis is stimulated by Ran⅐GTP, which is generated along condensed chromosomes by the guanine nucleotide exchange factor (GEF) RCC1. This relationship suggests that similar activities might modulate other microtubule structures. Interphase microtubules usually extend from the centrosome, although noncentrosomal microtubules function in some differentiated cells, including megakaryocytes. In these cells, platelet biogenesis requires massive mobilization of microtubules in the cell periphery, where they form proplatelets, the immediate precursors of platelets, in the apparent absence of centrioles. Here we identify a cytoplasmic Ranbinding protein, RanBP10, as a factor that binds ␤-tubulin and associates with megakaryocyte microtubules. Unexpectedly, RanBP10 harbors GEF activity toward Ran. A point mutation in the candidate GEF domain abolishes exchange activity, and our results implicate RanBP10 as a localized cytoplasmic Ran-GEF. RNA interference-mediated loss of RanBP10 in cultured megakaryocytes disrupts microtubule organization. These results lead us to propose that spatiotemporally restricted generation of cytoplasmic Ran⅐GTP may influence organization of the specialized microtubules required in thrombopoiesis and that RanBP10 might serve as a molecular link between Ran and noncentrosomal microtubules.Microtubules (MTs) 3 are essential components of the cytoskeleton in all cells, although specific fiber types and nucleation mechanisms differ according to cell lineage and over the cell cycle. Interphase cells typically contain a single MT-organizing center (MTOC) located within the centrosome. This structure contains two perpendicularly oriented centrioles and the ␥-tubulin ring complex, which initiates assembly of radial MT arrays (1, 2). Centrioles duplicate during S phase and subsequently migrate to opposite cell poles, where they anchor the mitotic spindle and enable chromosome segregation (3-5). After cytokinesis, the centrioles serve again as origins for interphase MTs, which are relatively stable structures. In contrast, the considerably more dynamic MTs in the mitotic spindle are assembled through the activity of the small GTPase Ran (6 -9).Ran controls at least three essential functions at different stages in the cell cycle: nucleocytoplasmic traffic in interphase, spindle formation at mitosis, and nuclear envelope assembly during telophase (10). Each function requires GTP hydrolysis and exchange of GDP for GTP. Small GTPases have low intrinsic rates of nucleotide exchange, which are enhanced over 10 5 -fold by guanine nucleotide exchange factors (GEFs), a diverse group of enzymes that show limited sequence similarity and considerable target protein specificity (11)(12)(13)(14). RCC1, the only known Ran-GEF, is a chromatin-associated nuclear protein that activates Ran by loading GTP (15). Conversely, the cytoplasmic Ran-GTPase-activating protein (GAP) RanGAP1 acts to produce Ran⅐GDP (16), and the resulting interphase Ran⅐GTP concentration gradient drives macromolecul...

Collagen receptors GPVI (also known as GP6) and integrin α2β1 are highly expressed on blood platelets and megakaryocytes, their immediate precursors. After vessel injury, subendothelial collagen becomes exposed and induces platelet activation to prevent blood loss. Collagen types I and IV are thought to have opposite effects on platelet biogenesis, directing proplatelet formation (PPF) towards the blood vessels to prevent premature release within the marrow cavity. We used megakaryocytes lacking collagen receptors or treated megakaryocytes with blocking antibodies, and could demonstrate that collagen-I-mediated inhibition of PPF is specifically controlled by GPVI. Other collagen types competed for binding and diminished the inhibitory signal, which was entirely dependent on receptor-proximal Src family kinases, whereas Syk and LAT were dispensable. Adhesion assays indicate that megakaryocyte binding to collagens is mediated by α2β1, and that collagen IV at the vascular niche might displace collagen I from megakaryocytes and thus contribute to prevention of premature platelet release into the marrow cavity and thereby directionally promote PPF at the vasculature.

The co-operation of specialized organ systems in complex multicellular organisms depends on effective chemical communication. Thus, body fluids (like blood, lymph or intraspinal fluid) contain myriads of signaling mediators apart from metabolites. Moreover, these fluids are also of crucial importance for immune and wound responses. Compositional analyses of human body fluids are therefore of paramount diagnostic importance. Further improving their comprehensiveness should increase our understanding of inter-organ communication. In arthropods, which have trachea for gas exchange and an open circulatory system, the single dominating interstitial fluid is the hemolymph. Accordingly, a detailed analysis of hemolymph composition should provide an especially comprehensive picture of chemical communication and defense in animals. Therefore we used an extensive protein fractionation workflow in combination with a discovery-driven proteomic approach to map out the detectable protein composition of hemolymph isolated from Drosophila larvae. Combined mass spectrometric analysis revealed more than 700 proteins extending far beyond the previously known Drosophila hemolymph proteome. Moreover, by comparing hemolymph isolated from either fed or starved larvae, we provide initial provisional insights concerning compositional changes in response to nutritional state. Storage proteins in particular were observed to be strongly reduced by starvation. Our hemolymph proteome catalog provides a rich basis for data mining, as exemplified by our identification of potential novel cytokines, as well as for future quantitative analyses by targeted proteomics.

Terminally mature megakaryocytes undergo dramatic cellular reorganization to produce hundreds of virtually identical platelets. A hallmark feature of this process is the generation of an elaborate system of branched protrusions called proplatelets. We recently identified RanBP10 as a tubulin-binding protein that is concentrated along polymerized microtubules in mature megakaryocytes. RanBP10 depletion in vitro caused the disturbance of polymerized filaments.Here we study the function of RanBP10 in vivo by generating deficient mice using a gene-trap approach. Mutant mice show normal platelet counts, and fetal liverderived megakaryocytes reveal only slightly reduced proplatelet formation. However, ultrastructural analysis unveiled a significantly increased geometric axis ratio for resting platelets, and many platelets exhibited disorders in microtubule filament numbers and localization. Mutant mice showed a markedly prolonged bleeding time. Granule release, a process that depends on internal contraction of the microtubule marginal coil, also was reduced. Flow cytometry analysis revealed reduced expression of CD62P and CD63 after PAR4-peptide stimulation. These data suggest that RanBP10 plays an essential role in hemostasis and in maintaining microtubule dynamics with respect to both platelet shape and function. (Blood. 2009;114:5532-5540) IntroductionPlatelets develop from megakaryocytes (MKs), large polyploid cells localized in the bone marrow. Mature MKs rearrange their entire cytoplasm into pseudopod protrusions (designated proplatelets), a process that is driven by and dependent on microtubules (MTs). 1,2 MT filaments emanate from the cell cortex into the proplatelet tips, where they coil into a loop and later build the platelet marginal band. In wild-type platelets, this MT loop is normally coiled 8 to 12 times. 3 Ultrastructural data previously suggested that the platelet marginal bundle contains a single coiled filament, [3][4][5][6] and isolation of MT coils by platelet extraction affirmed this idea. However, the study by Patel-Hett et al 7 on living platelets revealed up to 7 free plus-ends, suggesting that several shorter MT filaments associate dynamically with one longer filament, forming a bipolar array.Each MT filament is composed of 13 protofilament heteropolymers of ␣-and ␤-tubulins. Mammalian genomes encode 6 ␣-tubulin and 5 ␤-tubulin genes. ␤1-tubulin (also referred to as class VI) is the most diverse isotype 8 and is found exclusively in cells that harbor a marginal band: MKs, platelets, and erythroblasts. 9 More than 90% of proplatelet MT filaments are composed of this ␤-tubulin isotype, 10 whose expression depends on the MK-specific transcription factor NF-E2. Genetically engineered mice that lack a functional Tubb1 gene are thrombocytopenic, and Tubb1 Ϫ/Ϫ platelets lack lentiform shape; the marginal band consists of only 2 to 3 coils, resulting in platelet spherocytosis. 11,12 Although ubiquitously expressed tubulin isotypes ␤2 and ␤5 are up-regulated, the equilibrium is shifted toward monomer...

Objective To assess the clinical effectiveness of oral hydroxychloroquine (HCQ) with or without azithromycin (AZI) in preventing death or leading to hospital discharge. Design Retrospective cohort study. Setting An analysis of data from electronic medical records and administrative claim data from the French Assistance Publique - Hopitaux de Paris (AP-HP) data warehouse, in 39 public hospitals, Ile-de-France, France. Participants All adult inpatients with at least one PCR-documented SARS-CoV-2 RNA from a nasopharyngeal sample between February 1st, 2020 and April 6th, 2020 were eligible for analysis. The study population was restricted to patients who did not receive COVID-19 treatments assessed in ongoing trials, including antivirals and immunosuppressive drugs. End of follow-up was defined as the date of death, discharge home, day 28 after admission, whichever occurred first, or administrative censoring on May 4, 2020. Intervention Patients were further classified into 3 groups: (i) receiving HCQ alone, (ii) receiving HCQ together with AZI, and (iii) receiving neither HCQ nor AZI. Exposure to a HCQ/AZI combination was defined as a simultaneous prescription of the 2 treatments (more or less one day). Main outcome measures The primary outcome was all-cause 28-day mortality as a time-to-event endpoint under a competing risks survival analysis framework. The secondary outcome was 28-day discharge home. Augmented inverse probability of treatment weighted (AIPTW) estimates of the average treatment effect (ATE) were computed to account for confounding. Results A total of 4,642 patients (mean age: 66.1 +/- 18; males: 2,738 (59%)) were included, of whom 623 (13.4%) received HCQ alone, 227 (5.9%) received HCQ plus AZI, and 3,792 (81.7%) neither drug. Patients receiving "HCQ alone" or "HCQ plus AZI" were more likely younger, males, current smokers and overall presented with slightly more co-morbidities (obesity, diabetes, any chronic pulmonary diseases, liver diseases), while no major difference was apparent in biological parameters. After accounting for confounding, no statistically significant difference was observed between the "HCQ" and "Neither drug" groups for 28-day mortality: AIPTW absolute difference in ATE was +1.24% (-5.63 to 8.12), ratio in ATE 1.05 (0.77 to 1.33). 28-day discharge rates were statistically significantly higher in the "HCQ" group: AIPTW absolute difference in ATE (+11.1% [3.30 to 18.9]), ratio in ATE (1.25 [1.07 to 1.42]). As for the "HCQ+AZI" vs neither drug, trends for significant differences and ratios in AIPTW ATE were found suggesting higher mortality rates in the former group (difference in ATE +9.83% [-0.51 to 20.17], ratio in ATE 1.40 [0.98 to 1.81];p=0.062). Conclusions Using a large non-selected population of inpatients hospitalized for COVID-19 infection in 39 hospitals in France and robust methodological approaches, we found no evidence for efficacy of HCQ or HCQ combined with AZI on 28-day mortality. Our results suggested a possible excess risk of mortality associated with HCQ combined with AZI, but not with HCQ alone. Significantly higher rates of discharge home were observed in patients treated by HCQ, a novel finding warranting further confirmation in replicative studies. Altogether, our findings further support the need to complete currently undergoing randomized clinical trials.