Lipoxygenase mRNA in rabbit reticulocytes. Its isolation, characterization and translational repression

The synthesis of the erythroid lipoxygenase, an enzyme which is of importance for the degradation of mitochondria during the maturation of reticulocytes to erythrocytes, was studied in reticulocytes from bone marrow and in density-separated fractions from peripheral blood of anemic rabbits. Lipoxygenase mRNA was enriched to about 75% by digestion of polysomes with protease K, poly(U)-Sepharose chromatography and repeated sucrose gradient centrifugation. From sucrose gradient centrifugation, electrophoresis and electron microscopy a molecular weight of about 10(6) was calculated. Synthesis of lipoxygenase is absent in erythroblasts, in very young reticulocytes obtained from bone marrow, or in the lightest fractions of reticulocytes from the peripheral blood. More mature blood reticulocytes show a considerable synthesis of the enzyme. The induction of the synthesis of the lipoxygenase seems to be initiated when reticulocytes have reached the peripheral blood. It is shown that lipoxygenase mRNA is present in reticulocytes as a translationally inactive free cytoplasmic messenger ribonucleoprotein (mRNP) particle. After deproteinization isolated mRNA obtained from masked mRNP codes for authentic lipoxygenase in a cell-free protein-synthesizing system of reticulocytes.     

Selective mitochondrial autophagy during erythroid maturation

Accumulating evidence suggests that autophagy can be selective in the clearance of organelles in yeast and in mammalian cells. We have observed that the sequestration of mitochondria by autophagosomes was defective in reticulocytes in the absence of Nix. Nix is required for the dissipation of mitochondrial membrane potential (ΔΨm) during erythroid maturation. Moreover, pharmacological agents that induce the loss of ΔΨm can restore the sequestration of mitochondria by autophagosomes and promote mitochondrial clearance in Nix-/- erythroid cells. Our data suggest that mitochondrial depolarization induces recognition and sequestration of mitochondria by autophagosomes. Elucidating the mechanisms underlying selective mitochondrial autophagy not only will help us to understand the mechanisms for erythroid maturation, but also may provide insights into mitochondrial quality control by autophagy in the protection against aging, cancer, and neurodegenerative diseases.

Addendum to: Sandoval H, Thiagarajan P, Dasgupta SK, Schumacher A, Prchal JT, Chen M, Wang J. Essential role for Nix in autophagic maturation of erythroid cells. Nature 2008; 454:232-5.     

Selective mitochondrial autophagy during erythroid maturation

Accumulating evidence suggests that autophagy can be selective in the clearance of organelles in yeast and in mammalian cells. We have observed that the sequestration of mitochondria by autophagosomes was defective in reticulocytes in the absence of Nix. Nix is required for the dissipation of mitochondrial membrane potential (DeltaPsim) during erythroid maturation. Moreover, pharmacological agents that induce the loss of DeltaPsim can restore the sequestration of mitochondria by autophagosomes and promote mitochondrial clearance in Nix(-/-) erythroid cells. Our data suggest that mitochondrial depolarization induces recognition and sequestration of mitochondria by autophagosomes. Elucidating the mechanisms underlying selective mitochondrial autophagy not only will help us to understand the mechanisms for erythroid maturation, but also may provide insights into mitochondrial quality control by autophagy in the protection against aging, cancer and neurodegenerative diseases.     

Distribution and activity of certain dehydrogenases in the erythropoietic process in pigeons]

Cytochemical methods were applied for detecting of distribution and dynamics of dehydrogenase activity (H- and M-subunits of lactate dehydrogenase, malate and succinate dehydrogenase) during maturation of pigeon erythrocytes. In the erythroblasts the above enzymes were seen in the whole cell; in reticulocytes - only around the nucleus; in erythrocytes - on the border line between the nucleus and the cytoplasm. The cytophotometric data show a decrease in enzymatic activity during maturation being more significant in the period from the erythroblast to reticulocyte development than from the reticulocyte to erythrocyte development.     

Intranuclear and cytoplasmic hemoglobin in human erythroblasts during maturation. Electron microscopic immunocytochemistry

Changes in the hemoglobin level in human bone marrow erythroblasts associated with cell maturation were studied by the electron microscopic immunocytochemical technique using protein A-gold. Intense reaction of gold to hemoglobin was observed diffusely in the cytoplasm, but the reaction was weak in the Golgi zone. No reaction was observed in mitochondria or granules. Cytoplasmic hemoglobin was noted in basophilic erythroblasts and increased with maturation. Hemoglobin was also noted in the nucleus, especially in the euchromatin, though in smaller amounts than in the cytoplasm. Since intranuclear hemoglobin tended to increase in the euchromatin but to decrease in the heterochromatin with erythroblast maturation, the ratio of the amount of hemoglobin in the euchromatin to that in the heterochromatin increased with maturation.     

Intranuclear and cytoplasmic hemoglobin in human erythroblasts during maturation

Summary Changes in the hemoglobin level in human bone marrow erythroblasts associated with cell maturation were studied by the electron microscopic immunocytochemical technique using protein A-gold.Intense reaction of gold to hemoglobin was observed diffusely in the cytoplasm, but the reaction was weak in the Golgi zone. No reaction was observed in mitochondria or granules. Cytoplasmic hemoglobin was noted in basophilic erythroblasts and increased with maturation. Hemoglobin was also noted in the nucleus, especially in the euchromatin, though in smaller amounts than in the cytoplasm. Since intranuclear hemoglobin tended to increase in the euchromatin but to decrease in the heterochromatin with erythroblast maturation, the ratio of the amount of hemoglobin in the euchromatin to that in the heterochromatin increased with maturation.     

Rabbit red blood cell hexokinase:intracellular distribution during reticulocytes maturation

Summary The intracellular localization and isozyme distribution of hexokinase were studied during rabbit reticulocyte maturation and aging. In reticulocytes 50% of the enzyme was particulate while in the mature erythrocytes all the hexokinase activity was soluble. The bound enzyme co-sediments with mitochondria and by column chromatography it was found to be hexokinase Ia. The cytosol of reticulocytes contains hexokinase Ia (38%) and hexokinase Ib (62%) while the mature erythrocytes contain only hexokinase Ia. The amount of bound hexokinase decreases very quickly during cell maturation and aging as was shown by following in vivo reticulocyte maturation or by analysis of hexokinase compartmentation in cells of different ages, obtained by density gradient ultracentrifugations. A role for this intracellular distribution of hexokinase is suggested.     

NIX induces mitochondrial autophagy in reticulocytes

The controlled elimination of defective mitochondria is necessaryfor the health of long-lived post-mitotic cells, like cardiomyocytesand neurons. Mitochondrial elimination also occurs during thecourse of normal development, in lens epithelial and erythroidcells. Strikingly, at the final stage of erythroid cell maturation,newly formed erythrocytes, also known as reticulocytes, eliminatetheir entire cohort of mitochondria. We have employed thismodel to investigate the mechanism of programmed mitochondrialclearance. NIX (BNIP3L) is a Bcl-2-related protein that is upregulatedduring terminal erythroid differentiation.^1,2 NIX-deficientreticulocytes have a significant defect of mitochondrial clearance.Consistent with the ability of NIX to cause mitochondrial depolarization, ^3,4 we show that mitochondria are depolarized in wildtype but not NIX deficient reticulocytes. NIX does not functionthrough established proapoptotic pathways, nor does it mediate theinduction of autophagy in erythroid cells. Rather, NIX is requiredfor the selective incorporation of mitochondria into autophagosomes.Elucidation of the mechanism of this effect will improveour understanding of the role of autophagy in the maintenance ofcellular homeostasis.

Addendum to: Schweers RL, Zhang J, Randall MS, Loyd MR, Li W, Dorsey FC, Kundu M, Opferman JT, Cleveland JL, Miller JL, Ney PA. NIX is required for programmed mitochondrial clearance during reticulocyte maturation. Proc Natl Acad Sci USA 2007; 104:19500-5.     

NIX induces mitochondrial autophagy in reticulocytes

The controlled elimination of defective mitochondria is necessary for the health of long-lived post-mitotic cells, like cardiomyocytes and neurons. Mitochondrial elimination also occurs during the course of normal development, in lens epithelial and erythroid cells. Strikingly, at the final stage of erythroid cell maturation, newly formed erythrocytes, also known as reticulocytes, eliminate their entire cohort of mitochondria. We have employed this model to investigate the mechanism of programmed mitochondrial clearance. NIX (BNIP3L) is a Bcl-2-related protein that is upregulated during terminal erythroid differentiation. NIX-deficient reticulocytes have a significant defect of mitochondrial clearance. Consistent with the ability of NIX to cause mitochondrial depolarization, we show that mitochondria are depolarized in wild type but not NIX deficient reticulocytes. NIX does not function through established proapoptotic pathways, nor does it mediate the induction of autophagy in erythroid cells. Rather, NIX is required for the selective incorporation of mitochondria into autophagosomes. Elucidation of the mechanism of this effect will improve our understanding of the role of autophagy in the maintenance of cellular homeostasis.     

Maternal inheritance of mitochondrial DNA

Maternal inheritance of mitochondrial DNA (mtDNA) is generally observed in many eukaryotes. Sperm-derived paternal mitochondria and their mtDNA enter the oocyte cytoplasm upon fertilization and then normally disappear during early embryogenesis. However, the mechanism underlying this clearance of paternal mitochondria has remained largely unknown. Recently, we showed that autophagy is required for the elimination of paternal mitochondria in Caenorhabditis elegans embryos. Shortly after fertilization, autophagosomes are induced locally around the penetrated sperm components. These autophagosomes engulf paternal mitochondria, resulting in their lysosomal degradation during early embryogenesis. In autophagy-defective zygotes, paternal mitochondria and their genomes remain even in the larval stage. Therefore, maternal inheritance of mtDNA is accomplished by autophagic degradation of paternal mitochondria. We also found that another kind of sperm-derived structure, called the membranous organelle, is degraded by zygotic autophagy as well. We thus propose to term this allogeneic (nonself) organelle autophagy as allophagy.     

AN ELECTRON MICROSCOPIC STUDY OF NUCLEAR ELIMINATION FROM THE LATE ERYTHROBLAST

The process of expulsion of the nucleus during the transformation of the late erythroblast to reticulocyte is described. Erythroid clones taken from the spleen of lethally irradiated mice transplanted with syngeneic bone marrow were used. 10–12-day old isolated clones were fixed in glutaraldehyde, then in osmium tetroxide. Ultra-thin sections were stained with uranyl acetate and/or lead citrate before examination. Late (orthochromatic) erythroblasts develop pseudopod-like cytoplasmic protrusions into one of which the nucleus gradually penetrates, being deformed by the extrusion through the relatively narrow passage. During the whole process, mitochondria and vesicular and membranous elements are concentrated in the cytoplasm. Once outside the cell, the nucleus reassumes its rounded form. It is surrounded by a narrow rim of cytoplasm and structurally altered plasma membrane and is connected to the rest of the cell by a bridge. Elongated vacuoles appear within this bridge, with a resulting release of the enveloped nucleus which is soon phagocytized by macrophages; this leaves behind the newly formed reticulocyte. During this process, the cytoplasmic protrusions, the agglomeration of mitochondria, and the mode of separation of the nucleus from the rest of the cell are similar to those occurring in mitotic division.     

Characteristics of in vivo murine erythropoietic response to sodium orthovanadate

Current knowledge about the effects of vanadium compounds on erythropoiesis is still reduced and even contradictory. The aim of this work was to evaluate the in vivo effects of a single dose of sodium orthovanadate (OV, 33 mg/kg i.p.) on CF-1 mice in a time course study (0-8 days). Murine erythropoiesis was assessed through a combinatory of experimental approaches. Classical peripheral and bone marrow (BM) hematological parameters were determined. Erythroid maturation in blood stream and hemopoietic tissues (59Fe uptake assays), BM erythroid progenitor frequency (clonogenic assays) and erythroid crucial protein expressions for commitment and survival: GATA-1, erythropoietin receptor (Epo-R) and Bcl-xL (immunoblottings) were evaluated. Neither BM cellularities nor BM viabilities changed noticeably during the study. Peripheral reticulocytes showed a biphasic increment on days 2 and 8 post-OV. hematocrits enhanced transiently between days 2 and 4. 59Fe uptake percentages enhanced in peripheral blood nearly two-fold over control values between 4 and 8 days (p<0.01) without changes in BM and spleen. Additionally, mature erythroid BM compartments: polychromatophilic erythroblasts and orthochromatic normoblasts increased by the eighth day. BFU-E colonies remained near basal values during the whole experience, whilst CFU-E colonies raised 60% over control at 8 days post-OV (p<0.05). GATA-1 and Epo-R were significantly over-expressed from the third until the end of the experimental protocol (p<0.01). Surprisingly, Bcl-xL showed a constitutive expression pattern without changes during the experience. Experimental data let us suggest that OV does not to cause bone marrow cytotoxicity and that it accelerates maturation of BM committed erythroid precursors. Moreover, there are significant correlations among erythroid-related protein expressions: GATA-1 and Epo-R and the frequency of CFU-E. In addition, Bcl-xL expression invariance during the time course study would indicate that the stimulatory effect of OV treatment on erythropoiesis was mainly exerted on the maturation of red cell precursors rather than on the antiapoptosis of erythroid terminal progenitors.     

Proliferation kinetics of bone marrow cells in congenital dyserythropoietic anemia type II

The proliferation kinetics of erythropoiesis and of myelopoiesis have been studied in a case of congenital dyserythropoietic anemia type II (HEMPAS) by means of quantitative 14-C autoradiography, Feulgen cytophotometry and 59-Fe ferrokinetics. Increased total erythropoietic activity and ineffective erythropoiesis was demonstrated by ferrokinetics. Quantitative 14-C autoradiography showed a generally delayed proliferation rate of erythroid cells, most evident in the polychromatic compartment. A deficiency of cell production of 25% was detected among the polychromatic erythroblasts. Part of this fraction is represented by cells still capable of passing to the successive stages of maturation. We conclude that only part of the deficiency of cell production in the polychromatic compartment represents real cell destruction. Most of the measured ineffectiveness is confined to later stages of maturation, such as orthochromatic erythroblasts and marrow reticulocytes.     

Surface multivesicular structures associated with maturing erythrocytes in rats

Summary Surface multivesicular structures associated with the plasmalemma of erythrocytes were observed in the peripheral blood of rats which have a significant number of circulating reticulocytes. These surface structures appear as ovoid evaginations (0.2 to 0.7 in diameter) of the plasma membrane and contain numerous small vesicles ranging from 0.05 to 0.1 in diameter. The structures were present during the final stages of maturation of erythrocytes, after nuclei and mitochondria had been extruded and only a few polysomes and small vesicles remained. They appear quite distinct from the autophagic vacuoles which have been described in association with degeneration and extrusion of mitochondria from erythrocytes. The exact origin of the small internal vesicles of these surface multivesicular structures is unknown; however, similar vesicles have been observed in the cytoplasm of the maturing erythrocyte especially in the vicinity of the Golgi body. These structures suggest a process by which Golgi elements and other small cytoplasmic vesicles are extruded during the late stages of maturation of rat erythrocytes.Supported by U.S. Public Health Service Research Grant AM 12950.The author is indebted to Dr. Edward G. Rennels and Dr. William B. Winborn for their guidance.     

Cytoskeletal distribution and function during the maturation and enucleation of mammalian erythroblasts

We have used murine splenic erythrolasts infected with the anemia- inducing strain of Friend virus (FVA cells), as an in vitro model to study cytoskeletal elements during erythroid maturation and enucleation. FVA cells are capable of enucleating in suspension culture in vitro, indicating that associations with an extracellular matrix or accessory cells are not required for enucleation to occur. The morphology of FVA cells undergoing enucleation is nearly identical to erythroblasts enucleating in vivo. The nucleus is segregated to one side of the cell and then appears to be pinched off resulting in an extruded nucleus and reticulocyte. The extruded nucleus is surrounded by an intact plasma membrane and has little cytoplasm associated with it. Newly formed reticulocytes have an irregular shape, are vacuolated and contain all cytoplasmic organelles. The spatial distribution of several cytoskeletal proteins was examined during the maturation process. Spectrin was found associated with the plasma membrane of FVA cells at all stages of maturation but was segregated entirely to the incipient reticulocyte during enucleation. Microtubules formed cages around nuclei in immature FVA cells and were found primarily in the incipient reticulocyte in cells undergoing enucleation. Reticulocytes occasionally contained microtubules, but a generalized diffuse distribution of tubulin was more common. Vimentin could not be detected at any time in FVA cell maturation. Filamentous actin (F-actin) had a patchy distribution at the cell surface in the most immature erythroblasts, but F-actin bundles could be detected as the cells matured. F-actin was found concentrated between the extruding nucleus and incipient reticulocyte in enucleating erythroblasts. Newly formed reticulocytes exhibited punctate actin fluorescence whereas extruded nuclei lacked F-actin. Addition of colchicine, vinblastine, or taxol to cultures of FVA cells did not affect enucleation. In contrast, cytochalasin D caused a complete inhibition of enucleation that could be reversed by washing out the cytochalasin D. These results demonstrate that F-actin plays a role in enucleation while the complete absence of microtubules or excessive numbers of polymerized microtubules do not affect enucleation.     

Altering microtubule stability affects microtubule clearance and nuclear extrusion during erythropoiesis

Mammalian erythrocytes are highly specialized cells that have adapted to lose their nuclei and cellular components during maturation to ensure oxygen delivery. Nuclear extrusion, the most critical event during erythropoiesis, represents an extreme case of asymmetric partitioning that requires a dramatic reorganization of the cytoskeleton. However, the precise role of the microtubule cytoskeleton in the enucleation process remains controversial. In this study, we show that microtubule reorganization is critical for microtubule clearance and nuclear extrusion during erythropoiesis. Using a rodent anemia model, we found that microtubules were present in erythroblasts and reticulocytes but were undetectable in erythrocytes. Further analysis demonstrated that microtubules became disordered in reticulocytes and revealed that microtubule stabilization was critical for tubulin degradation. Disruption of microtubule dynamics using the microtubule-stabilizing agent paclitaxel or the microtubule-destabilizing agent nocodazole did not affect the efficiency of erythroblast enucleation. However, paclitaxel treatment resulted in the retention of tubulin in mature erythrocytes, and nocodazole treatment led to a defect in pyrenocyte morphology. Taken together, our data reveals a critical role for microtubules in erythrocyte development. Our findings also implicate the disruption of microtubule dynamics in the pathogenesis of anemia-associated diseases, providing new insight into the pathogenesis of the microtubule-targeted agent-associated anemia frequently observed during cancer chemotherapy.     

Mitochondrial clearance by autophagy in developing erythrocytes: Clearly important,but just how much so?

Erythrocytes are anucleated cells devoid of organelles. Expulsion of the nucleus from erythroblasts leads to the formation of reticulocytes, which still contain organelles. The mechanisms responsible for the final removal of organelles from developing erythroid cells are still being elucidated. Mitochondria are the most abundant organelles to be cleared for the completion of erythropoiesis. Macroautophagy, referred to as autophagy, is a regulated catabolic pathway consisting of the engulfment of cytoplasmic cargo by a double membraned-vesicle, the autophagosome, which typically then fuses to lysosomal compartments for the degradation of the sequestered material. Early electron microscopic observations of reticulocytes suggested the autophagic engulfment of mitochondria (mitophagy) as a possible mechanism for mitochondrial clearance in these. Recently, a number of studies have backed this hypothesis with molecular evidence. Indeed, the absence of Nix, which targets mitochondria to autophagosomes, or the deficiency of proteins in the autophagic pathway lead to impaired mitochondrial clearance from developing erythroid cells. Importantly, however, the extent to which the absence of mitophagy affects erythroid development differs depending on the model and gene investigated. This review will therefore focus on comparing the different studies of mitophagy in erythroid development and highlight some of the remaining controversial points.     

Shiga toxin of enterohaemorrhagic Escherichia coli directly injures developing human erythrocytes

Haemolytic anaemia is one of the characteristics of life‐threatening extraintestinal complications in humans during infection with enterohaemorrhagic Escherichia coli (EHEC). Shiga toxins (Stxs) of EHEC preferentially damage microvascular endothelial cells of the kidney and the brain, whereby occluded small blood vessels may elicit anaemia through mechanical erythrocyte disruption. Here we show for the first time that Stx2a, the major virulence factor of EHEC, is also capable of direct targeting developing human erythrocytes. We employed an ex vivo erythropoiesis model using mobilized CD34⁺ haematopoietic stem/progenitor cells from human blood and monitored expression of Stx receptors and Stx2a‐mediated cellular injury of developing erythrocytes. CD34⁺ haematopoietic stem/progenitor cells were negative for Stx2a receptors and resistant towards the toxin. Expression of Stx2a‐binding glycosphingolipids and toxin sensitivity was apparent immediately after initiation of erythropoietic differentiation, peaked for basophilic and polychromatic erythroblast stages and declined during maturation into orthochromatic erythroblasts and reticulocytes, which became highly refractory to Stx2a. The observed Stx‐mediated toxicity towards erythroblasts during the course of erythropoiesis might contribute, although speculative at this stage of research, to the anaemia caused by Stx‐producing pathogens.