Alpha hemolysin of Escherichia coli induces a necrotic-like procoagulant state in platelets.

Uropathogenic strains of E. coli (UPEC) is a leading cause of sepsis, deploying multiple virulence factors to evade host immune responses. Notably, alpha-hemolysin (HlyA) produced by UPEC is implicated in septic symptoms associated with bacteremia, correlating with thrombocytopenia, a critical indicator of organ dysfunction and a predictor of poorer patient prognosis. This study meticulously explores the impact of sublytic concentrations of HlyA on platelets. Findings reveal that HlyA triggers an increase in intracellular calcium, activating calpain and exposing phosphatidylserine to the cell surface, as validated by flow cytometric experiments. Electron microscopy reveals a distinctive balloon-like shape in HlyA-treated platelets, indicative of a procoagulant state. The toxin induces the release of procoagulant extracellular vesicles and the secretion of alpha and dense granules. Overall, the results point to HlyA inducing a necrotic-like procoagulant state in platelets. The effects of sublytic concentrations of HlyA on both erythrocytes and platelets could have a potential impact on capillary microcirculation. Targeting HlyA emerges as a viable therapeutic strategy to mitigate the adverse effects of UPEC infections, especially in South American countries where these infections are endemic, underscoring its significance as a potential therapeutic target.

Homeostasis of extracellular ATP in uninfected RBCs from a Plasmodium falciparum culture and derived microparticles.

Plasmodium falciparum, a dangerous parasitic agent causing malaria, invades human red blood cells (RBCs), causing hemolysis and microvascular obstruction. These and other pathological processes of malaria patients are due to metabolic and structural changes occurring in uninfected RBCs. In addition, infection activates the production of microparticles (MPs). ATP and byproducts are important extracellular ligands modulating purinergic signaling within the intravascular space. Here, we analyzed the contribution of uninfected RBCs and MPs to the regulation of extracellular ATP (eATP) of RBCs, which depends on the balance between ATP release by specific transporters and eATP hydrolysis by ectonucleotidases. RBCs were cultured with P. falciparum for 24-48 h prior to experiments, from which uninfected RBCs and MPs were purified. On-line luminometry was used to quantify the kinetics of ATP release. Luminometry, colorimetry and radioactive methods were used to assess the rate of eATP hydrolysis by ectonucleotidases. Rates of ATP release and eATP hydrolysis were also evaluated in MPs. Uninfected RBCs challenged by different stimuli displayed a strong and transient activation of ATP release, together with an elevated rate of eATP hydrolysis. MPs contained ATP in their lumen, which was released upon vesicle rupture, and were able to hydrolyze eATP. Results suggest that uninfected RBCs and MPs can act as important determinants of eATP regulation of RBCs during malaria. The comparison of eATP homeostasis in infected RBCs, ui-RBCs, and MPs allowed us to speculate on the impact of P. falciparum infection on intravascular purinergic signaling and the control of the vascular caliber by RBCs.

Human erythroid differentiation requires VDAC1-mediated mitochondrial clearance.

Erythroblast maturation in mammals is dependent on organelle clearance throughout terminal erythropoiesis. We studied the role of the outer mitochondrial membrane protein voltage-dependent anion channel-1 (VDAC1) in human terminal erythropoiesis. We show that short hairpin (shRNA)-mediated downregulation of VDAC1 accelerates erythroblast maturation. Thereafter, erythroblasts are blocked at the orthochromatic stage, exhibiting a significant decreased level of enucleation, concomitant with an increased cell death. We demonstrate that mitochondria clearance starts at the transition from basophilic to polychromatic erythroblast, and that VDAC1 downregulation induces the mitochondrial retention. In damaged mitochondria from non-erythroid cells, VDAC1 was identified as a target for Parkin-mediated ubiquitination to recruit the phagophore. Here, we showed that VDAC1 is involved in phagophore's membrane recruitment regulating selective mitophagy of still functional mitochondria from human erythroblasts. These findings demonstrate for the first time a crucial role for VDAC1 in human erythroblast terminal differentiation, regulating mitochondria clearance.

Transmission Electron Microscopy to Follow Ultrastructural Modifications of Erythroblasts Upon ex vivo Human Erythropoiesis.

Throughout mammal erythroid differentiation, erythroblasts undergo enucleation and organelle clearance becoming mature red blood cell. Organelles are cleared by autophagic pathways non-specifically targeting organelles and cytosolic content or by specific mitophagy targeting mitochondria. Mitochondrial functions are essential to coordinate metabolism reprogramming, cell death, and differentiation balance, and also synthesis of heme, the prosthetic group needed in hemoglobin assembly. In mammals, mitochondria subcellular localization and mitochondria interaction with other structures as endoplasmic reticulum and nucleus might be of importance for the removal of the nucleus, that is, the enucleation. Here, we aim to characterize by electron microscopy the changes in ultrastructure of cells over successive stages of human erythroblast differentiation. We focus on mitochondria to gain insights into intracellular localization, ultrastructure, and contact with other organelles. We found that mitochondria are progressively cleared with a significant switch between PolyE and OrthoE stages, acquiring a rounded shape and losing contact sites with both ER (MAM) and nucleus (NAM). We studied intracellular vesicle trafficking and found that endosomes and MVBs, known to be involved in iron traffic and heme synthesis, are increased during BasoE to PolyE transition; autophagic structures such as autophagosomes increase from ProE to OrthoE stages. Finally, consistent with metabolic switch, glycogen accumulation was observed in OrthoE stage.

Effect of Autophagy Modulators on Vascular, Glial, and Neuronal Alterations in the Oxygen-Induced Retinopathy Mouse Model.

Hypoxia is one of the main insults in proliferative retinopathies, leading to neovascularization and neurodegeneration. To maintain homeostasis, neurons require efficient degradation and recycling systems. Autophagy participates in retinal cell death, but it is also a cell survival mechanism. Here, we analyzed the role of autophagy at the three characteristic time periods in the oxygen-induced retinopathy (OIR) mouse model and determined if its modulation can improve vascular and non-vascular alterations. Experiments were performed with chloroquine (CQ) in order to monitor autophagosome accumulation by lysosomal blockade. Post natal day (P)17 OIR mouse retinas showed a significant increase in autophagy flux. In particular, an intense LC3B and p62 staining was observed in inner layers of the retina, mainly proliferating endothelial cells. After a single intraocular injection of Rapamycin at P12 OIR, a decreased neovascular area and vascular endothelial growth factor (VEGF) protein expression were observed at P17 OIR. In addition, whereas the increased expression of glial fibrillary acidic protein (GFAP) was reversed at P26 OIR, the functional alterations persisted. Using a similar therapeutic schedule, we analyzed the effect of anti-VEGF therapy on autophagy flux. Like Rapamycin, VEGF inhibitor treatment not only reduced the amount of neovascular tufts, but also activated autophagy flux at P17 OIR, mainly in ganglion cell layer and inner nuclear layer. Finally, the effects of the disruption of autophagy by Spautin-1, were evaluated at vascular, glial, and neuronal levels. After a single dose of Spautin-1, Western blot analysis showed a significant decrease in LC3B II and p62 protein expression at P13 OIR, returning both autophagy markers to OIR control levels at P17. In addition, neither gliosis nor functional alterations were attenuated. In line with these results, TUNEL staining showed a slight increase in the number of positive cells in the outer nuclear layer at P17 OIR. Overall, our results demonstrate that all treatments of induction or inhibition of the autophagic flux reduced neovascular area but were unable to completely reverse the neuronal damage. Besides, compared to current treatments, rapamycin provides a more promising therapeutic strategy as it reduces both neovascular tufts and persistent gliosis.

Insulin-induced exocytosis regulates the cell surface level of low-density lipoprotein-related protein-1 in Müller Glial cells.

Low-density lipoprotein (LDL) receptor-related protein-1 (LRP1) is expressed in retinal Müller glial cells (MGCs) and regulates intracellular translocation to the plasma membrane (PM) of the membrane proteins involved in cellular motility and activity. Different functions of MGCs may be influenced by insulin, including the removal of extracellular glutamate in the retina. In the present work, we investigated whether insulin promotes LRP1 translocation to the PM in the Müller glial-derived cell line MIO-M1 (human retinal Müller glial cell-derived cell line). We demonstrated that LRP1 is stored in small vesicles containing an approximate size of 100 nm (mean diameter range of 100-120 nm), which were positive for sortilin and VAMP2, and also incorporated GLUT4 when it was transiently transfected. Next, we observed that LRP1 translocation to the PM was promoted by insulin-regulated exocytosis through intracellular activation of the IR/PI3K/Akt axis and Rab-GTPase proteins such as Rab8A and Rab10. In addition, these Rab-GTPases regulated both the constitutive and insulin-induced LRP1 translocation to the PM. Finally, we found that dominant-negative Rab8A and Rab10 mutants impaired insulin-induced intracellular signaling of the IR/PI3K/Akt axis, suggesting that these GTPase proteins as well as the LRP1 level at the cell surface are involved in insulin-induced IR activation.

Autophagy: A necessary event during erythropoiesis.

Autophagy is a well-known cellular process involved in many physiological and pathological processes. During erythropoiesis, autophagy plays an important role participating in the clearance of unnecessary organelles such as ribosomes and mitochondria (mitophagy) allowing the correct formation of mature red blood cells. The dysfunction of autophagy proteins hamper the correct erythroid maturation, leading to anemia, the release of immature cells from the bone marrow and other hematological abnormalities. Autophagy plays different roles depending on the type of pathology. In leukemia cells, it has been demonstrated that autophagy could be either detrimental, leading to an increase of the apoptosis rate, or protective, acting as a key process that augments proliferation and survival of cancer cells. Thus, understanding the relationship between autophagy and erythropoiesis opens new avenues for the discovery of biochemical and pharmacological targets and for the development of novel therapeutic approaches.

Alpha-hemolysin is required for the activation of the autophagic pathway in Staphylococcus aureus-infected cells.

Staphylococcus aureus is a pathogen that causes serious infectious diseases eventually leading to septic and toxic shock. Classically S. aureus has been considered an extracellular pathogen, but cumulative evidence indicates that it invades cells and replicates intracellularly leading to staphylococcal persistence and chronic disease. It has been previously shown that this pathogen localizes to LC3-labeled compartments and subverts the autophagy pathway. One of the key features of S. aureus infection is the production of a series of virulence factors, including secreted enzymes and toxins. In the present report we present evidence that the pore-forming toxin alpha-hemolysin (Hla) is a S. aureus secreted factor which participates in the activation of the autophagic pathway. In addition, our results indicate that although the toxin elicits an autophagic response this pathway is dysfunctional as indicated by the accumulation of the LC3-II form in cell lysates obtained from intoxicated cells. In addition, not only the purified Hla toxin but also the toxin-secreting pathogen prevented the maturation of autophagosomes. Interestingly, in cells infected with the wild-type strain of S. aureus the bacteria-containing compartments which recruited LC3 onto the limiting membrane did not accumulate the acidotropic probe LysoTracker. In contrast, those phagosomes containing the Hla(-) mutant (unable to produce the toxin) localized in an acidic compartment unlabeled by LC3. These results suggest that the LC3 protein is recruited only to those damaged vacuoles (i.e., perforated by the toxin), perhaps as an attempt to protect the cells. Furthermore, we have demonstrated that the toxin-dependent activation of autophagy (although it is regulated by calcium and requires Atg5) is independent of both PI3Kinase activity and Beclin 1 suggesting the involvement of a non-canonical autophagy pathway.

Induction of autophagy promotes fusion of multivesicular bodies with autophagic vacuoles in k562 cells.

Morphological and biochemical studies have shown that autophagosomes fuse with endosomes forming the so-called amphisomes, a prelysosomal hybrid organelle. In the present report, we have analyzed this process in K562 cells, an erythroleukemic cell line that generates multivesicular bodies (MVBs) and releases the internal vesicles known as exosomes into the extracellular medium. We have previously shown that in K562 cells, Rab11 decorates MVBs. Therefore, to study at the molecular level the interaction of MVBs with the autophagic pathway, we have examined by confocal microscopy the fate of MVBs in cells overexpressing green fluorescent protein (GFP)-Rab11 and the autophagosomal protein red fluorescent protein-light chain 3 (LC3). Autophagy inducers such as starvation or rapamycin caused an enlargement of the vacuoles decorated with GFP-Rab11 and a remarkable colocalization with LC3. This convergence was abrogated by a Rab11 dominant negative mutant, indicating that a functional Rab11 is involved in the interaction between MVBs and the autophagic pathway. Interestingly, we presented evidence that autophagy induction caused calcium accumulation in autophagic compartments. Furthermore, the convergence between the endosomal and the autophagic pathways was attenuated by the Ca2+ chelator acetoxymethyl ester (AM) of the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), indicating that fusion of MVBs with the autophagosome compartment is a calcium-dependent event. In addition, autophagy induction or overexpression of LC3 inhibited exosome release, suggesting that under conditions that stimulates autophagy, MVBs are directed to the autophagic pathway with consequent inhibition in exosome release.

Multivesicular bodies and autophagy in erythrocyte maturation.

During reticulocyte maturation, hematopoietic progenitors undergo numerous changes to reach the final functional stage which concludes with the release of reticulocytes and erythrocytes into circulation. During this process some proteins, which are not required in the mature stage, are sequestered in the internal vesicles present in multivesicular bodies (MVBs). These small vesicles are known as exosomes because they are released into the extracellular medium by fusion of the MVB with the plasma membrane. Interestingly, during this maturation process some organelles, such as mitochondria and endoplasmic reticulum, are wrapped in double membrane vacuoles and degraded via autophagy. We have demonstrated in human leukemic K562 cells a role for calcium and Rab11 in the biogenesis of MVBs and exosome release. Here we discuss evidence indicating that K562 cells present a high basal level of autophagy, and that there is an association between MVBs and autophagosomes, suggesting a role for the autophagic pathway in the maturation process of this cell type.

Exosome secretion and red cell maturation: Exploring molecular components involved in the docking and fusion of multivesicular bodies in K562 cells.

During reticulocyte maturation, some membrane proteins and organelles that are not required in the mature red cell are lost. These proteins are released into the extracellular medium associated with vesicles present in multivesicular bodies (MVBs). Fusion of MVBs with the plasma membrane results in secretion of the small internal vesicles, termed exosomes. By studying MVBs fusion and exosome release in K562 cells, a human erythroleukemic cell line, we have determined the functional significance of Rab11 and calcium in these events. Additionally, in the transformation process that occurs during erythrocyte maturation, intracellular organelles are likely removed as a consequence of autophagic sequestration and degradation. We propose K562 cells as a useful tool to analyze, at the molecular level, the role of autophagy in the terminal differentiation of red cells.

Rab11 promotes docking and fusion of multivesicular bodies in a calcium-dependent manner.

Multivesicular bodies (MVBs) are membranous structures within 60-100 nm diameter vesicles accumulate. MVBs are generated after invagination and pinching off of the endosomal membrane in the lumen of the vacuole. In certain cell types, fusion of MVBs with the plasma membrane results in the release of the internal vesicles called exosomes. In this report we have examined how an increase in cytosolic calcium affects the development of MVBs and exosome release in K562 cells overexpressing GFP-Rab11 wt or its mutants. In cells overexpressing the Rab11Q70 L mutant or Rab11 wt, an increase in the cytosolic calcium concentration induced by monensin caused a marked enlargement of the MVBs. This effect was abrogated by the membrane permeant calcium chelator BAPTA-AM. We also examined the behavior of MVBs in living cells by time lapse confocal microscopy. Many MVBs, decorated by wt or Q70L mutant GFP-Rab11, were docked and ready to fuse in the presence of a calcium chelator. This observation suggests that Rab11 is acting in the tethering/docking of MVBs to promote homotypic fusion, but that the final fusion reaction requires the presence of calcium. Additionally, a rise in intracellular calcium concentration enhanced exosome secretion in Rab11 wt overexpressing cells and reversed the inhibition of the mutants. The results suggest that both Rab11 and calcium are involved in the homotypic fusion of MVBs.