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Reticulocyte remodeling has emerged as an important model for the understanding of vesicular trafficking and selective autophagy in mammalian cells. This review covers recent advances in our understanding of these processes in reticulocytes and the implications for erythroid development.
Enucleation is a key event, caused by chromatin condensation, vesicular trafficking, and microfilaments. Mitochondrial elimination is achieved through selective autophagy, in which mitochondria are targeted to autophagosomes, and undergo subsequent degradation and exocytosis. The mechanism involves an integral mitochondrial outer membrane protein, and general autophagy pathways. Plasma membrane remodeling, and the elimination of certain intracellular organelles, is achieved through the exosomal pathway.
Vesicular trafficking and selective autophagy have emerged as central processes in cellular remodeling. In reticulocytes, this includes enucleation and the elimination of all membrane-bound organelles and ribosomes. In contrast to yeast where certain ubiqutin-like conjugation pathways are required for all types of autophagy, in reticulocytes inactivation of these pathways has only a partial effect. Thus, in higher eukaryotes, there appears to be redundancy between these pathways and other processes. Future studies will address the relationship between autophagy and vesicular trafficking, and the significance of both for cellular remodeling.
Erythrocytes are highly specialized cells, which are dedicated to hemoglobin transport and gas exchange in the lungs and the peripheral tissues. Erythrocytes develop from reticulocytes, which are defined (in mammals) as erythroid cells that have lost their nucleus but still retain residual RNA. Reticulocytes in turn are generated in the bone marrow from mature erythroblasts by enucleation. After a brief period of time in the bone marrow, reticuloctyes are released into circulation, where they eliminate their residual RNA, and mature into erythrocytes. Under homeostatic conditions, maturation of rodent reticulocytes takes 2–3 days, equally divided between the bone marrow compartment and circulation [1–4]. Compared to the average life-span of an erythrocyte (120 days in humans), the reticulocyte stage of erythroid development is brief; however, it is a period of rapid change. Reticulocytes undergo extensive membrane remodeling, volume changes, and eliminate all internal membrane-bound organelles and ribosomes. These transformative processes ensure that critical cellular functions, such as hemoglobin production, oxygen transport, and deformability, are optimized in mature erythrocytes. This review will cover recent advances in our understanding of these processes, and some of the biological implications.
Enucleation has several beneficial effects. Among these, it definitively prevents dedifferentiation and transformation of mature erythroid cells. It also has a potential impact on organismal fitness. The nucleus comprises two-fifths of erythroid cell weight; accordingly, enucleation reduces the circulatory work of the heart by two-fifths . Apart from these theoretical benefits, enucleation plays a direct role in reticulocyte maturation. Enucleation of a late stage erythroblast generates a nascent reticulocyte with abundant cytoplasm and hemoglobin, and a membrane-bound nucleus with a thin rim of cytoplasm, designated the pyrenocyte . Enucleation is accompanied by a sorting process, which directs membrane proteins to either the reticulocyte or the pyrenocyte. Cytoskeletal proteins important for erythrocyte function, such as spectrin, ankyrin, and protein 4.1, are directed to the nascent reticulocyte [7;8]. Integral membrane proteins that are associated with the cytoskeleton, such as glycophorin A , are also directed to the reticulocyte. By contrast, proteins that mediate intercellular attachments, such as erythroblast macrophage protein and β1 integrin are directed to the pyrenocyte. The mechanism is not known, but is thought to involve connectivity to the underlying membrane cytoskeleton. Consistent with this interpretation, mutations of cytoskeletal proteins, such as those seen in hereditary spherocytosis and elliptocytosis, are associated with misorting and deficiencies of nonmutant proteins .
Nuclear condensation, which occurs during terminal erythroid differentiation, is a prerequisite for efficient enucleation. In mammalian erythroid cells, nuclear condensation is associated with histone H3K9 dimethylation and H4K12 deactylation ; however, In contrast to avian species, it is not associated with changes in nucleosome structure or the accumulation of special structural proteins. Histone deactylases (HDAC) 2 and 5 are upregulated during erythroid differentiation, and a loss-of-function analysis implicates HDAC2 in nuclear condensation [11;12]. Further, mIR-191 regulates enculeation through its effect on Riok3, Mxi1, and the histone acetyltransferase Gcn5 . These studies support the notion that histone deactylation plays an important role in nuclear condensation and elimination. However, it does not appear to be sufficient. For example, Retinoblastoma 1 (Rb1) regulates cell cycle and differentiation. Rb1-deficient erythroblasts exhibit defective nuclear condensation and enucleation , which may reflect a primary role of Rb in chromatin condensation . Downregulation of vimentin intermediate filaments releases the nucleus from its moorings, setting the stage for enculeation . Regarding enucleation, several cell autonomous mechanisms have been proposed. Caspases are involved in enucleation in keratinocytes and lens epithelial cells; however, there is no evidence for broad caspase activation in terminally differentiated erythroblasts . Microtubules and microfilaments have also been implicated [7;18], and during enucleation a constrictive actin ring forms between the nucleus and the nascent reticulocyte. Formation of this ring, and enucleation, requires Rac GTPases and the downstream effector mDia2 . Also, Sox6 appears to have a role in this process. Recently, Keerthivasan et al. provided evidence that enucleation is driven by the clatharin-dependent generation and coalescence of vacuoles at the nuclear-cytoplasmic junction . This study suggests that vesicular trafficking has an under appreciated role in enucleation. Vesicle recruitment by the multisubunit tethering eoxcyst complex is also required for asymmetrical abscission at the midbody ring in cytokinesis . Given that enucleation may represent an asymmetrical form of cytokinesis, perhaps the exocyst complex is also involved in erythroid cell enucleation.
Nascent reticulocytes are motile, multilobulated cells . Initially, the membranes of these cells are rigid and unstable. As they mature, reticulocytes lose volume and surface area, assume the shape of a biconcave disk, and acquire stability and deformability [24;25]. To a large extent, these functional and morphological changes can be attributed to maturational changes in the membrane cytoskeleton, as discussed elsewhere in this issue. However, there are also changes in the expression of membrane proteins during reticulocyte maturation; prominent among these is downregulation of the transferrin receptor. The transferrin receptor-1 (TfR1) participates in acycle that transports extracellular iron into erythroid cells (reviewed by Hentze et al. ). In this cycle, iron-loaded transferrin binds to its receptor, and is imported via clatharin-mediated endocytosis into the early endosomal compartment. Internalization is followed by endosomal acidification, release of iron from transferrin, iron reduction, and iron transport into the cytoplasm. Finally, TfR1 returns to the plasma membrane and releases apo-transferrin, completing the cycle. Some TfR1 is recycled rapidly to the membrane, whereas the rest is slowly recycled after passing first through the endosomal recycling compartment. Notably, a naturally-occuring mutation of Sec15l1, a homolog of a yeast exocyst component, is associated with decreased rapid recycling of the TfR, an iron utilization defect, and microcytic anemia [27–30].
Once hemoglobin synthesis is complete, reticulocyte Tfr1 is rapidly downregulated to prevent continued iron importation, and iron-mediated toxicity. This is accomplished by a switch in TfR1 trafficking from the recycling pathways to the multivesicular endosomal (MVE) pathway . MVE are formed by membrane invagination in late endosomes, which is mechanistically related to the process of viral budding. Budding in MVE is initiated by ESCRT complexes (reviewed by Saksena et al. ), although ESCRT-independent mechanisms have been described that are mediated by lipid rafts or lectin-induced aggregation[34;35]. ESCRT complexes bind ubiquitylated proteins, providing one mechanism for protein sorting into MVE. Regarding TfR1, an ESCRT-associated protein, Alix, may play a role in its sorting into exosomes . MVE undergo fusion with lysosomes, followed by degradation, or fusion with the plasma membrane, which leads to the release of intraluminal vesicles into the extracellular space. The release of such vesicles, known as exosomes, was initially described in the context of TfR1 downregulation[37;38], but was subsequently shown to be involved in other biological processes, such as antigen presentation and the transmission of infectious agents[39–41]. Exosome secretion provides a means of rapid TfR1 downregulation. Besides this, it is involved in plasma membrane elimination from reticulocytes[42;43], and volume regulation through downregulation of Na/K-ATPase and aquaporin-1 [38;44]. Secretion of aquaporin-1 is suppressed by a proteasome inhibitor, suggesting that its sorting in MVE may be regulated by ubiquitylation. Other membrane proteins, such as glucose, nucleoside, and amino acid transporters are also downregulated through this pathway [38;44;45].
Nascent reticulocytes consume oxygen, and have a functioning tricarboxylic acid cycle, which is downregulated during maturation[46;47]. Since one of the main functions of mature erythrocytes is oxygen transport, and mitochondria consume oxygen, it is not surprising that mitochondria are targeted for inactivation during reticulocyte maturation. Indeed, mitochondria are eliminated from reticulocytes as they develop, and are absent in mature erythrocytes. Early ultrastructural studies implicated two mechanisms of elimination, structural deterioration by a degradative process , and exocytosis [49–51]. Ultrastructurally, mitochondrial clearance is initiated at the same time as enculeation and continues as the reticulocyte matures. Mitochondria undergoing clearance are surrounded by double-membraned structures, similar in appearance to autophagosomes, and subsequently undergo degradation and elimination by exocytosis. Mitochondria free in the cytoplasm also show signs of deterioration. In this regard, the enzyme 15-lipoxygenase inserts into mitochondrial membranes in reticulocytes and allows access of lytic enzymes to mitochondria[52;53]. However, as will be discussed, studies from our laboratory suggest that mitochondrial degradation and clearance is primarily mediated through vacuolar structures.
NIX is an atypical member of the BH3-only subgroup of BCL2-related proteins (reviewed by Zhang and Ney ). NIX is upregulated during terminal erythroid differentiation . To determine the role of NIX in erythroid development several groups generated mice with targeted disruption of Nix. Consistent with its documented proapoptotic role, Diwan et al. found that NIX-deficient erythroblasts are resistant to cytokine deprivation-induced cell death. Two other groups found that NIX-deficient reticulocytes exhibit a striking defect in mitochondrial clearance [57;58]. Mitochondria in NIX-deficient reticulocytes are not eliminated, but remain in the cytoplasm in association with isolation membranes and the cytoplasmic face of autophagosomes. Ultrastructurally, autophagy membranes are not properly targeted to mitochondria in the absence of NIX; consequently, mitochondria are not incorporated into autophagosomes, and they are not eliminated. NIX-deficient mice are anemic, which is likely related to an increase in caspase activity and the shortened life-span of NIX-deficient erythrocytes. Another likely consequence of the mitochondrial clearance defect, which may be of functional relevance, is that blood from NIX-deficient mice undergoes accelerated deoxygenation ex vivo (Ney PA, unpublished data).
There are several models of the mechanism NIX-mediated mitochondrial clearance. Recent studies implicate defective mitochondrial clearance in the pathogenesis of Parkinson’s disease. The genes Parkin and Pink1 are frequently mutated in autosomal recessive Parkinson’s disease[59;60]. PINK1 is a mitochondrial integral membrane protein that is stabilized by mitochondrial depolarization. PINK1 recruits Parkin, an E3 ligase [61;62]. Ubiquitylation of mitochondrial proteins by Parkin further leads to the recruitment of adaptors and the autophagy machinery[63;64]. Thus, one proposal is NIX acting in its capacity as a proapoptotic protein causes mitochondrial depolarization and consequent elimination by autophagy . Against this possibility, mitochondrial clearance in reticulocytes proceeds in the absence of the proapoptotic proteins BAX and BAK, and independent of the mitochondrial permeability transition pore . Furthermore, the rate of mitochondrial depolarization is greatly diminished when autophagosome formation is impaired, indicating that mitochondrial depolarization in reticulocytes is not the event that initiates mitochondrial clearance. A second model is that NIX directly recruits autophagy proteins to mitochondria. Consistent with this model, NIX has a microtubule-associated protein-1, light chain-3 (LC3)-interaction region near its amino terminus, and a point mutation of this motif diminishes, but does not eliminate NIX-dependent mitochondrial clearance in reticulocytes [66;67]. A third possibility is that NIX promotes autophagy by competing with Beclin-1 for binding to other BCL2-related proteins ; however, at present there is no evidence to suggest that this is the mechanism of mitochondrial clearance in reticulocytes. At present, none of these models can fully account for the activity of NIX in mitochondrial clearance.
Autophagy, or “self-digestion”, is a process for recycling cellular components. In contrast to the other major proteolytic pathway, proteasome-dependent degradation, autophagy can recycle complete subcellular structures, such as cytoplasm, protein aggregates, and organelles. This is accomplished by packaging these structures in double-membranes for delivery to lysosomes. Autophagy was originally described as an ultrastructural phenomenon, in mammalian cells [69;70]. However, in the past decade there has been considerable progress defining the pathways that regulate autophagy in yeast (reviewed by Xie and Klionsky ). Starvation induces nonselective autophagy (macroautophagy, or simply autophagy); however, metabolic changes can also cause selective degradation of organelles, such as peroxisomes and mitochondria. In this regard, Atg32, an integral mitochondrial outer membrane protein (like NIX), mediates mitochondrial autophagy in yeast[72;73].
Most autophagy proteins identified in yeast have one or more mammalian homologs. Atg1 is a serine-threonine kinase, itself a target of Tor kinase, and a critical regulator of autophagy. The mammalian homologs of Atg1 are ULK1 and ULK2; however only ULK1 is expressed at high levels in erythroid cells. ULK1-deficient reticulocytes exhibit a moderate defect in mitochondrial clearance, less severe than that caused by deficiency of NIX, and a mild defect in ribosomal clearance; otherwise, reticulocyte maturation is not affected. Atg7 is an E1-like ubiquitin ligase, and key autophagy enzyme in two parallel ubiquitin-like conjugation pathways. Atg7-deficient reticulocytes exhibit a mild-moderate defect in mitochondrial clearance[65;75], less severe than that caused by deficiency of ULK1, and have no other apparent defect in maturation. Together, these studies demonstrate that autophagy has a role in reticulocyte maturation; however, the absence of a more severe defect suggests that functionally redundant pathways exist.
To create an optimal cell for hemoglobin transportation, reticulocytes jettison all their unnecessary components. Besides the nucleus and mitochondria, this includes the smooth and rough ER, golgi appartus remnants, ferritin particles, and ribosomes. Compared with the nucleus and mitochondria, less is known about the elimination of other structures and organelles, but similar mechanisms appear to be involved. Lamp-2, a marker of the endolysosomal compartment, is present in secreted exosomes, suggesting that this structure is eliminated in part through MVE. Ferritin is observed in cytoplasmic aggregates, in vacuoles, and in MVE-like structures [76;77], although its mechanism of elimination is not yet known. Regarding ribosomes, their degradation by selective autophagy is regulated by a deubiquitinating enzyme in yeast . The mechanism in higher eukaryotes remains to be elucidated. In reticulocytes, ribosomal RNA is degraded by ribonucleases into nucleotides, which in turn are broken down into nucleosides by erythroid pyrimidine 5′-nucleotidase . Deficiency of this enzyme is associated with basophilic stippling, and inherited hemolytic anemia.
The creation of an erythrocyte, a highly specialized cell, requires the upregulation of dedicated proteins, like hemoglobin, and the elimination of all detrimental or simply unnecessary proteins and structures (Figure 1). There is a balance, but both types of processes are required. Because the changes are dramatic and easily studied, the reticulocyte provides an excellent model for the study of cellular remodeling. As discussed here, cellular remodeling in the reticulocyte itself appears to occur through two central processes: vesicular trafficking and autophagy. Although they are considered distinct processes, they may overlap at the cellular and molecular levels. At the cellular level, they can both terminate with exocytosis, suggesting a common mechanism. Further, autophagosomes can directly fuse with MVE, leading to hybrid organelles . At the molecular level, there is recent evidence of shared components between the vesicular trafficking and autophagy pathways [81;82]. In this regard, in higher eukaryotes, inactivation of the ubiqutin-like conjugation pathways leads to only a partial loss of autophagy activity [65;81]. Whether a component of the vesicular trafficking machinery supplies this redundant activity remains to be determined.
Funding sources: National Institutes of Health (R21 DK074519), and the American, Lebanese, and Syrian Associated Charities (ALSAC)
The author acknowledges the contributions of Ji Zhang, Melanie Loyd, and Mindy Randall. This work was supported by the National Institutes of Health (R21 DK074519), and by the American, Lebanese, and Syrian Associated Charities (ALSAC).