Edited by: Lars Kaestner, Saarland University, Germany
Reviewed by: Pablo Martín-Vasallo, Universidad de La Laguna, Spain; Rodrigo F. M. De Almeida, Universidade de Lisboa, Portugal
*Correspondence: Mariano A. Ostuni
This article was submitted to Membrane Physiology and Membrane Biophysics, a section of the journal Frontiers in Physiology
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Erythropoiesis occurs mostly in bone marrow and ends in blood stream. Mature red blood cells are generated from multipotent hematopoietic stem cells, through a complex maturation process involving several morphological changes to produce a highly functional specialized cells. In mammals, terminal steps involved expulsion of the nucleus from erythroblasts that leads to the formation of reticulocytes. In order to produce mature biconcave red blood cells, organelles and ribosomes are selectively eliminated from reticulocytes as well as the plasma membrane undergoes remodeling. The mechanisms involved in these last maturation steps are still under investigation. Enucleation involves dramatic chromatin condensation and establishment of the nuclear polarity, which is driven by a rearrangement of actin cytoskeleton and the clathrin-dependent generation of vacuoles at the nuclear-cytoplasmic junction. This process is favored by interaction between the erythroblasts and macrophages at the erythroblastic island. Mitochondria are eliminated by mitophagy. This is a macroautophagy pathway consisting in the engulfment of mitochondria into a double-membrane structure called autophagosome before degradation. Several mice knock-out models were developed to identify mitophagy-involved proteins during erythropoiesis, but whole mechanisms are not completely determined. Less is known concerning the clearance of other organelles, such as smooth and rough ER, Golgi apparatus and ribosomes. Understanding the modulators of organelles clearance in erythropoiesis may elucidate the pathogenesis of different dyserythropoietic diseases such as myelodysplastic syndrome, leukemia and anemia.
Mature red blood cells (RBCs) result from a finely regulated process called erythropoiesis that produces 2 million RBCs every second in healthy human adults (Palis,
Maturation from erythroid-committed precursors is called terminal erythropoiesis and occurs in the BM within erythroblastic islands, which consist of a central macrophage surrounded by erythroblasts, and ends in the blood stream where reticulocytes complete their maturation within 1–2 days. During this phase, proerythroblasts (Pro-E) undergo morphological changes, such as cell size reduction and chromatin condensation, produce specific proteins, such as hemoglobin, and exhibit a reduced proliferative capacity to give rise to basophilic (Baso-E), polychromatophilic (Poly-E) and orthochromatophilic (Ortho-E) erythroblasts, successively. Even though several growth factors are known to regulate erythropoiesis, Epo is the main regulator of erythropoiesis driving RBC precursor proliferation and differentiation, preventing erythroblast apoptosis (Koury and Bondurant,
At the end of the terminal maturation, mammalian erythroblasts expel their nuclei and lose all their organelles, such as the Golgi apparatus, endoplasmic reticulum (ER), mitochondria and ribosomes. After expelling its nucleus, the reticulocyte maturation continues, losing 20–30% of the cell surface (Waugh et al.,
While extensive literature is done concerning the general mechanisms of erythropoiesis (Palis,
Terminal maturation of erythroblasts.
The most spectacular aspect of mammalian erythropoiesis is the generation of enucleated cells. Enucleation occurs in orthochromatic erythroblasts producing two kinds of cells, the reticulocyte and the pyrenocyte [the nucleus surrounded by a tiny layer of cytoplasm and the plasma membrane (PM)]. Pyrenocytes are rapidly eliminated by the macrophages of the erythroblastic island, where phosphatidylserine exposure acts as an “eat me” signal (Yoshida et al.,
Among the changes occurring during terminal differentiation, cell cycle arrest, chromatin and nuclear condensation and nuclear polarization are important for enucleation. In addition, nucleus expulsion is believed to be dependent on adhesion protein reorganization across the PM and macrophage interactions (Lee et al.,
Nuclear and chromatin condensation is essential for enucleation (Popova et al.,
Many studies demonstrate the cell cycle dependence of enucleation (Gnanapragasam and Bieker,
Cytoskeletal elements play an important role in erythroblast enucleation, acting in a similar manner to cytokinesis but in an asymmetric way. Specifically, as observed by electron and immunofluorescence microscopy, actin filaments (F-actin) condensate behind the extruding nucleus to form the CAR. The use of cytochalasin D, an F-actin inhibitor, causes the complete blockage of enucleation (Koury et al.,
Regarding other cytoskeleton elements, the pharmacological inhibition of vimentin does not affect enucleation, which is in agreement with its decrease during human erythropoiesis (Dellagi et al.,
In 2010, Crispino's group observed, by electron microscopy, the formation of vesicles close to the nuclear extrusion site in both primary murine and human erythroblasts, suggesting that another mechanism contributes to enucleation. Additionally, as shown by genetic invalidation, clathrin is needed for the vesicle formation (Keerthivasan et al.,
Clearly, we are still at the beginning of unraveling the molecular players involved in the enucleation process. Moreover, as shown in Table
Comparison between studies in human or mice erythroid cells or in other cell models.
PS exposition in pyrenocytes | Yoshida et al., |
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Role of KFL1 | Magor et al., |
Parkins et al., |
|
Role of Gcn5 | Jayapal et al., |
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Formation of CAR | Ji et al., |
||
Role of F-actin | Koury et al., |
||
Role of Rac1 and mDia2 | Ji et al., |
||
Role of E2F-2 | Swartz et al., |
||
Role of dynein | Kobayashi et al., |
||
Role of PI3K | Wang et al., |
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Vesicular trafficking | Keerthivasan et al., |
Keerthivasan et al., |
|
Role of survivin/EPS15/clathrin | Keerthivasan et al., |
Keerthivasan et al., |
|
Apoptotic involvement | Krauss, |
Weil et al., |
|
PINK1 accumulation | Narendra et al., |
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Role of Parkin | Kim et al., |
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LC3 Cleavage | Betin et al., |
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Lc3B binding through p62 | Pankiv et al., |
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Engulfment inside the autophagosome | Koury, |
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Role of NIX | Aerbajinai et al., |
Zhang et al., |
Yuan et al., |
Atg7 independent pathway | Honda et al., |
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Role of FUNDC1 | Chen et al., |
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Role of Bcl2-L-13 | Murakawa et al., |
||
Role of optineurin | Wong and Holzbaur, |
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Role of prohibitin 2 | Wei et al., |
||
KRAB/KAP1-miRNA regulatory cascade | Barde et al., |
Barde et al., |
|
Role of 15-LOX | Kühn et al., |
||
Role of Rab | Wang et al., |
Hammerling et al., |
|
Role of hemin regulation | Fader et al., |
||
Role of NF-E2 | Gothwal et al., |
Gothwal et al., |
|
Role of Ulk1 | Kundu et al., |
||
Atg7-Independent pathway | Mortensen et al., |
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Role of macroautophagy | Iwata, |
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Role of 15-LOX | Yokota et al., |
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Role of Lon proteases | Yokota et al., |
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Role of p62 | Hung et al., |
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Role of pyrimidin nucleotidase | Valentine et al., |
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TfR removing | Johnstone et al., |
||
AQP removing | Blanc et al., |
||
α4β1 integrin removing | Rieu et al., |
||
GLUT and AChE removing | Johnstone et al., |
||
GPA+ exosomes | Griffiths et al., |
The main mechanism for mitochondrial clearance is mitophagy, a selective type of autophagy that allows the degradation of damaged mitochondria. The importance of this process is highlighted by knowing that an impairment in mitochondrial function triggers an increase in reactive oxygen species production, which can in turn cause damage to cellular components (proteins, nucleic acid, and lipids) and trigger cell death (Lee et al.,
During regular autophagy processes, stress or nutrient deprivation activates APM-activated protein kinase (AMPK), triggering two ubiquitin-dependent pathways (Figure
Upon mitochondria damage or depolarization, the mitochondrial membrane proteins are exposed and act as a beacon to recruit the phagophore membranes (Liu et al.,
Recently, other mitochondrial receptors were found to participate in mitophagy, such as FUNDC1, induced by MARCH5, an E3 ubiquitin ligase acting in hypoxic condition (Chen et al.,
Canonical Atg proteins also participate in terminal maturation. In human erythropoiesis, LC3 cleavage is under the control of the endopeptidase Atg4 and is needed for autophagosome maturation (Betin et al.,
Some studies suggest that the Atg5/7-independent degradation of mitochondria involves endosomal trafficking regulatory Rab proteins. Autophagosomes, formed in a Ulk1-dependent pathway, fuse with Golgi-derived vesicles and late endosomes in a Rab9a-dependent manner before they are targeted to the lysosomes (Wang et al.,
Mitophagy also appears to be transcriptionally regulated. Indeed, hemin-dependent differentiation of an erythroid cell line shows features of mitophagy (Fader et al.,
In parallel to the autophagic pathway, cytosolic degradation seems to occur during reticulocyte maturation. 15-lipoxygenase (15-LOX), an enzyme that catalyzes the dioxygenation of polyunsaturated fatty acids, is translationally inhibited until the reticulocyte stage and acts to permeabilize organelle membranes, allowing proteasome access and degradation. Interestingly, only mitochondria elimination is affected, while ribosome clearance remains efficient when using a lipoxygenase inhibitor (Grüllich et al.,
In general, autophagy plays an essential role in the elimination of other organelles, such as lysosomes, peroxisomes and ER. However, the literature presents only very few studies in erythroid cells (Table
While Nix is required for mitochondria removal, Ulk1 is involved in ribosome and mitochondria degradation (Schweers et al.,
In non-erythroid cells from mammals, it was proposed that peroxisomes are eliminated by three different pathways: macroautophagy (Iwata,
After enucleation, reticulocytes mature in the bone marrow (R1) and then exit in the blood stream (R2) to complete the process. While the degradation of organelles starts at the time of enucleation, the elimination of mRNA occurs in the blood stream and is mediated by ribonucleases, generating nucleotides that are degraded by the erythroid pyrimidine nucleotidase. This elimination is crucial, as the deficiency in this enzyme causes hemolytic anemia (Valentine et al.,
Exosomes are small vesicles that are secreted into the extracellular medium from various kind of cells. PM invaginations form early endosomes that engulf various targets forming multivesicular bodies (MVB, late endosomes) that eventually fuse with the PM and release exosomes. In reticulocytes, this pathway is thought to be involved in cell volume and membrane remodeling to reduce volume and remove unwanted membrane proteins. This was first discovered in sheep reticulocytes where transferrin receptor (TfR) is first internalized into small vesicles of 100–200 nm before being engulfed into the MVBs (Pan et al.,
While plenty of evidence notes the role of autophagy in removing organelles during terminal maturation, the degradation step itself shows discrepancies with canonical proteolysis involving lysosomal proteins because of the disappearance of the lysosomal compartment during the maturation and removal of LAMP2 by exocytosis (Barres et al.,
It should be pointed out the importance of lipids domain such as cholesterol and sphingomyelin-enriched domains in the PM remodeling, as they were find both in membrane vesiculation specific sites (Leonard et al.,
Even if all the animal models used to identify the molecular players involved during terminal differentiation exhibit maturation defects and anemia, links between organelle clearance and human hematological diseases are still mostly unknown. Erythroid disorders, such as β-thalassemia and myelodysplastic syndrome (MDS), are characterized by ineffective hematopoiesis, anemia, dissociation between proliferation and differentiation of progenitor cells and the inefficient elimination of aggregated protein (Arber et al.,
Unraveling the molecular mechanisms and interplays ruling erythroblast terminal maturation would be priceless in hematological disease therapy. However, much of our knowledge regarding human erythropoiesis is based on animal models and/or
All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
MM is funded by the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 665850. We thank I. Marginedas-Freixa and C. Hattab for helpful discussions.