Dynein-dynactin complex mediates protein kinase A-dependent clustering of Weibel-Palade bodies in endothelial cells

Perinuclear clustering is observed for several different organelles and illustrates dynamic regulation of the secretory pathway and organelle distribution. Previously, we observed that a subset of Weibel-Palade bodies (WPBs), endothelial cell-specific storage organelles, undergo centralization when endothelial cells are stimulated with cAMP-raising agonists of von Willebrand factor (vWF) secretion. In this study, we investigated this phenomenon of WPB clustering in more detail. Our results demonstrate that the clustered WPBs are localized at the microtubule organizing center and that cluster formation depends on an intact microtubule network. Disruption of the microtubules by nocodazole completely abolished clustering, whereas treatment with the actin depolymerizing compound cytochalasin B had no effect on WPB clustering. Interfering with the dynein-dynactin interaction by overexpression of the p50 dynamitin subunit or the CC1 domain of the p150glued subunit of the dynactin complex completely inhibited perinuclear clustering of WPBs, suggesting that dynein activity mediates this process. Furthermore, we found that inhibition of dephosphorylation resulted in an increase in clustering, whereas inhibition of protein kinase A (PKA) markedly reduced WPB clustering. These results suggest that perinuclear clustering of WPBs involves PKA-dependent regulation of the dynein-dynactin complex. Endothelial cell stimulation with epinephrine results in retrograde movement of a subset of WPBs to the microtubule organizing center. This minus-end directed transport requires an intact microtubular network and is mediated by the motor protein dynein. Together, our results suggest that epinephrine-induced clustering of WPBs involves PKA-dependent regulation of the dynein-dynactin comple

Large-scale in vitro production of red blood cells from human peripheral blood mononuclear cells

Transfusion of donor-derived red blood cells (RBC) is the most common form of cellular therapy. Donor availability and the potential risk of alloimmunization and other transfusion-related complications may, however, limit the availability of transfusion units, especially for chronically transfused patients. In vitro cultured, customizable RBC would negate these concerns and further increase precision medicine. Large-scale, cost-effective production depends on optimization of culture conditions. We developed a defined medium and adapted our protocols to good manufacturing practice (GMP) culture requirements, which reproducibly provided pure erythroid cultures from peripheral blood mononuclear cells without prior CD341 isolation, and a 3 3 107-fold increase in erythroblasts in 25 days (or from 100 million peripheral blood mononuclear cells, 2 to 4 mL packed red cells can be produced). Expanded erythroblast cultures could be differentiated to CD71dimCD235a1CD441CD1172DRAQ52 RBC in 12 days. More than 90% of the cells enucleated and expressed adult hemoglobin as well as the correct blood group antigens. Deformability and oxygen-binding capacity of cultured RBC was comparable to in vivo reticulocytes. Daily RNA sampling during differentiation followed by RNA-sequencing provided a high-resolution map/resource of changes occurring during terminal erythropoiesis. The culture process was compatible with upscaling using a G-Rex bioreactor with a capacity of 1 L per reactor, allowing transition toward clinical studies and small-scale applications

Von Willebrand factor targets IL-8 to Weibel-Palade bodies in an endothelial cell line

Vascular endothelial cells are able to store the chemotactic cytokine interleukin-8 (IL-8) in specialized storage vesicles, Weibel-Palade bodies, together with von Willebrand factor (VWF) and P-selectin. We investigated whether VWF plays a role in the sorting of IL-8 into these organelles. We examined the effect of VWF expression on IL-8 targeting in an endothelial cell line (EC-RF24). This cell line has retained the typical phenotypic characteristics of primary endothelial cells but has lost the capacity to produce VWF in appreciable amounts. EC-RF24 cells were retrovirally transduced with a vector encoding a VWF-green fluorescent protein chimera (VWF-GFP). This approach enables direct visualization of the cellular distribution and secretory behavior of the VWF-GFP hybrid. Expression of VWF-GFP resulted in the generation of Weibel-Palade body-like organelles as shown by the colocalization of VWF-GFP and P-selectin. VWF-GFP expressing EC-RF24 cells also showed significant colocalization of VWF-GFP with IL-8 in these storage vesicles. Live cell imaging revealed that the number of VWF-GFP-containing granules decreased upon cell stimulation. These observations indicate that VWF plays an active role in sequestering IL-8 into Weibel-Palade bodie

A Homozygous Mutation on the HBA1 Gene Coding for Hb Charlieu (HBA1: c.320T>C) Together with β-Thalassemia Trait Results in Severe Hemolytic Anemia

A 4-year-old boy, a β-thalassemia (β-thal) carrier, with an unexplained severe chronic microcytic anemia was referred to us. Sequencing of the α-globin genes revealed a Hb Charlieu [α106(G13)Leu→Pro, HBA1: c.320T>C, p.Leu107Pro] mutation present on both HBA1 genes. Quantitative polymerase chain reaction (qPCR) confirmed αCharlieu mRNA in the proband and his parents, showing that the mutation does not affect mRNA stability. However, we were unable to detect the Hb Charlieu protein by capillary electrophoresis (CE), reverse phase electrophoresis, cation exchange electrophoresis or isoelectric focusing. Mass spectrometry (MS) allowed us to confirm the presence of the Hb Charlieu peptide in erythrocyte progenitors. These findings suggest that the mutation affects the stability of αCharlieu. As hemoglobin (Hb) heat stability tests showed no abnormalities in erythrocytes, we speculated that αCharlieu is already degraded during red blood cell (RBC) development. The clinical severity in the proband and the presence of new methylene blue-stained aggregates in his reticulocytes indicates that incorporation of αCharlieu destabilizes Hb. This, combined with an excess of unstable free α-globins as the result of β-thal minor, results in severely impaired erythropoiesis and, as a consequence, severe and chronic microcytic anemia in the proband

A Homozygous Mutation on the HBA1 Gene Coding for Hb Charlieu (HBA1 : c.320T>C) Together with β-Thalassemia Trait Results in Severe Hemolytic Anemia

A 4-year-old boy, a β-thalassemia (β-thal) carrier, with an unexplained severe chronic microcytic anemia was referred to us. Sequencing of the α-globin genes revealed a Hb Charlieu [α106(G13)Leu→Pro, HBA1: c.320T>C, p.Leu107Pro] mutation present on both HBA1 genes. Quantitative polymerase chain reaction (qPCR) confirmed αCharlieu mRNA in the proband and his parents, showing that the mutation does not affect mRNA stability. However, we were unable to detect the Hb Charlieu protein by capillary electrophoresis (CE), reverse phase electrophoresis, cation exchange electrophoresis or isoelectric focusing. Mass spectrometry (MS) allowed us to confirm the presence of the Hb Charlieu peptide in erythrocyte progenitors. These findings suggest that the mutation affects the stability of αCharlieu. As hemoglobin (Hb) heat stability tests showed no abnormalities in erythrocytes, we speculated that αCharlieu is already degraded during red blood cell (RBC) development. The clinical severity in the proband and the presence of new methylene blue-stained aggregates in his reticulocytes indicates that incorporation of αCharlieu destabilizes Hb. This, combined with an excess of unstable free α-globins as the result of β-thal minor, results in severely impaired erythropoiesis and, as a consequence, severe and chronic microcytic anemia in the proband