Clearance Of Organelles Research Articles

Developmentally controlled subcellular remodeling and VND-initiated vacuole-executed PCD module shape xylem-like cells in peat moss

Peat moss (Sphagnum) is a non-vascular higher plant with unique xylem-like hyaline (H) cells that are accompanied by photosynthetic chlorophyllous cells. These cellular structures play crucial roles in water storage and carbon sequestration. However, it is largely unknown how peat moss develops the H cells. This study systematically explored the Sphagnum Developmental Cell Atlas and Lineage and classified leaf cell development into two lineages with six stages (S0-S5) based on changes in key cellular traits, including the formation of spiral secondary cell walls (S4) and the presence of water pores (S5). Cell lineage-specific subcellular remodeling was transcriptionally regulated during leaf development, and vacuole-mediated clearance of organelles and cell death led to mature dead H cells. Interestingly, expression of land plant conserved Vascular-related NAC Domain (VND) genes correlated with H cell formation. Overall, these results suggest that the origination of xylem-like H cells is related to VND, likely through the neofunctionalization of vacuole-mediated cell death to attempt xylem formation in peat moss, suggesting potential uncoupling of xylem and phloem cell origins. This study positions peat moss as a potential model organism for studying integrative evolutionary cell biology.

Autophagy, aging, and age-related neurodegeneration.

Autophagy is a conserved mechanism that degrades damaged or superfluous cellular contents and enables nutrient recycling under starvation conditions. Many neurodegeneration-associated proteins are autophagy substrates, and autophagy upregulation ameliorates disease in many animal models of neurodegeneration by enhancing the clearance of toxic proteins, proinflammatory molecules, and dysfunctional organelles. Autophagy inhibition also induces neuronal and glial senescence, a phenomenon that occurs with increasing age in non-diseased brains as well as in response to neurodegeneration-associated stresses. However, aging and many neurodegeneration-associated proteins and mutations impair autophagy. This creates a potentially detrimental feedback loop whereby the accumulation of these disease-associated proteins impairs their autophagic clearance, facilitating their further accumulation and aggregation. Thus, understanding how autophagy interacts with aging, senescence, and neurodegenerative diseases in a temporal, cellular, and genetic context is important for the future clinical application of autophagy-modulating therapies in aging and neurodegeneration.

Enhancing terminal erythroid differentiation in human embryonic stem cells through TRIB3 overexpression

Tribbles pseudokinase 3 (TRIB3) expression significantly increases during terminal erythropoiesis in vivo. However, we found that TRIB3 expression remained relatively low during human embryonic stem cell (hESC) erythropoiesis, particularly in the late stage, where it is typically active. TRIB3 was expressed in megakaryocyte-erythrocyte progenitor cells and its low expression was necessary for megakaryocyte differentiation. Thus, we proposed that the high expression during late stage of erythropoiesis could be the clue for promotion of maturation of hESC-derived erythroid cells. To our knowledge, the role of TRIB3 in the late stage of erythropoiesis remains ambiguous. To address this, we generated inducible TRIB3 overexpression hESCs, named TRIB3tet-on OE H9, based on a Tet-On system. Then, we analyzed hemoglobin expression, condensed chromosomes, organelle clearance, and enucleation with or without doxycycline treatment. TRIB3tet-on OE H9 cells generated erythrocytes with a high proportion of orthochromatic erythroblast in flow cytometry, enhanced hemoglobin and related protein expression in Western blot, decreased nuclear area size, promoted enucleation rate, decreased lysosome and mitochondria number, more colocalization of LC3 with LAMP1 (lysosome marker) and TOM20 (mitochondria marker) and up-regulated mitophagy-related protein expression after treatment with 2 μg/mL doxycycline. Our results showed that TRIB3 overexpression during terminal erythropoiesis may promote the maturation of erythroid cells. Therefore, our study delineates the role of TRIB3 in terminal erythropoiesis, and reveals TRIB3 as a key regulator of UPS and downstream mitophagy by ensuring appropriate mitochondrial clearance during the compaction of chromatin.

Mitochondrial regulation of erythropoiesis in homeostasis and disease.

Erythroid cells undergo a highly complex maturation process, resulting in dynamic changes that generate red blood cells (RBCs) highly rich in haemoglobin. The end stages of the erythroid cell maturation process primarily include chromatin condensation and nuclear polarization, followed by nuclear expulsion called enucleation and clearance of mitochondria and other organelles to finally generate mature RBCs. While healthy RBCs are devoid of mitochondria, recent evidence suggests that mitochondria are actively implicated in the processes of erythroid cell maturation, erythroblast enucleation and RBC production. However, the extent of mitochondrial participation that occurs during these ultimate steps is not completely understood. This is specifically important since abnormal RBC retention of mitochondria or mitochondrial DNA contributes to the pathophysiology of sickle cell and other disorders. Here we review some of the key findings so far that elucidate the importance of this process in various aspects of erythroid maturation and RBC production under homeostasis and disease conditions.

ROS-responsive nano-medicine for navigating autophagy to enhance targeted therapy of inflammatory bowel disease

Inflammatory bowel disease (IBD) is a chronic gastrointestinal disorder characterized by immune dysregulation and intestinal inflammation. Rapamycin (Ra), an mTORC1 pathway inhibitor, has shown promise for autophagy induction in IBD therapy but is associated with off-target effects and toxicity. To address these issues, we developed an oral liposome responsive to reactive oxygen species (ROS) using lipids and amphiphilic materials. We combined ketone thiol (TK) for ROS responsive and hyaluronic acid (HA) with high affinity for CD44 receptors to prepare rapamycin-loaded nanoparticle (Ra@TH). Owing to its ROS responsive characteristic, Ra@TH can achieve inflammatory colonic targeting. Additionally, Ra@TH can induce autophagy by inhibiting the mTORC1 pathway, leading to the clearance of damaged organelles, pathogenic microorganisms and oxidative stress products. Simultaneously, it also collaboratively inhibits the NF-κB pathway suppressed by the removal of ROS resulting from TK cleavage, thereby mediating the expression of inflammatory factors. Furthermore, Ra@TH enhances the expression of typical tight junction proteins, synergistically restoring intestinal barrier function. Our research not only expands the understanding of autophagy in IBD treatment but also introduces a promising therapeutic approach for IBD patients.

The non-canonical poly(A) polymerase FAM46C promotes erythropoiesis

The post-transcriptional regulation of mRNA is a crucial component of gene expression. The disruption of this process has detrimental effects on the normal development and gives rise to various diseases. Searching for novel post-transcriptional regulators and exploring their roles are essential for understanding development and disease. Through a multimodal analysis of red blood cell trait genome-wide association studies (GWAS) and transcriptomes of erythropoiesis, we identify FAM46C, a non-canonical RNA poly(A) polymerase, as a necessary factor for proper red blood cell development. FAM46C is highly expressed in the late stages of the erythroid lineage, and its developmental upregulation is controlled by an erythroid-specific enhancer. We demonstrate that FAM46C stabilizes mRNA and regulates erythroid differentiation in a polymerase activity-dependent manner. Furthermore, we identify transcripts of lysosome and mitochondria components as highly confident in vivo targets of FAM46C, which aligns with the need of maturing red blood cells for substantial clearance of organelles and maintenance of cellular redox homeostasis. In conclusion, our study unveils a unique role of FAM46C in positively regulating lysosome and mitochondria components, thereby promoting erythropoiesis.

Intra-erythrocytic vacuoles in asplenic patients: elusive genesis and original clearance of unique organelles.

The spleen plays a dual role of immune response and the filtration of red blood cells (RBC), the latter function being performed within the unique microcirculatory architecture of the red pulp. The red pulp filters and eliminates senescent and pathological RBC and can expell intra-erythrocytic rigid bodies through the so-called pitting mechanism. The loss of splenic function increases the risk of infections, thromboembolism, and hematological malignancies. However, current diagnostic tests such as quantification of Howell-Jolly Bodies and splenic scintigraphy lack sensitivity or are logistically demanding. Although not widely available in medical practice, the quantification of RBC containing vacuoles, i.e., pocked RBC, is a highly sensitive and specific marker for hyposplenism. The peripheral blood of hypo/asplenic individuals contains up to 80% RBC with vacuoles, whereas these pocked RBC account for less than 4% of RBC in healthy subjects. Despite their value as a spleen function test, intraerythrocytic vacuoles have received relatively limited attention so far, and little is known about their origin, content, and clearance. We provide an overview of the current knowledge regarding possible origins and mechanisms of elimination, as well as the potential function of these unique and original organelles observed in otherwise "empty" mature RBC. We highlight the need for further research on pocked RBC, particularly regarding their potential function and specific markers for easy counting and sorting, which are prerequisites for functional studies and wider application in medical practice.

PINK1, Keap1, and Rtnl1 regulate selective clearance of endoplasmic reticulum during development

Selective clearance of organelles, including endoplasmic reticulum (ER) and mitochondria, by autophagy plays an important role in cell health. Here, we describe a developmentally programmed selective ER clearance by autophagy. We show that Parkinson's disease-associated PINK1, as well as Atl, Rtnl1, and Trp1 receptors, regulate ER clearance by autophagy. The E3 ubiquitin ligase Parkin functions downstream of PINK1 and is required for mitochondrial clearance while having the opposite function in ER clearance. By contrast, Keap1 and the E3 ubiquitin ligase Cullin3 function downstream of PINK1 to regulate ER clearance by influencing Rtnl1 and Atl. PINK1 regulates a change in Keap1 localization and Keap1-dependent ubiquitylation of the ER-phagy receptor Rtnl1 to facilitate ER clearance. Thus, PINK1 regulates the selective clearance of ER and mitochondria by influencing the balance of Keap1- and Parkin-dependent ubiquitylation of substrates that determine which organelle is removed by autophagy.

Activating transcription factor 4 regulates mitochondrial content, morphology, and function in differentiating skeletal muscle myotubes.

Mitochondrial function is widely recognized as a major determinant of health, emphasizing the importance of understanding the mechanisms promoting mitochondrial quality in various tissues. Recently, the mitochondrial unfolded protein response (UPRmt) has come into focus as a modulator of mitochondrial homeostasis, particularly in stress conditions. In muscle, the necessity for activating transcription factor 4 (ATF4) and its role in regulating mitochondrial quality control (MQC) have yet to be determined. We overexpressed (OE) and knocked down ATF4 in C2C12 myoblasts, differentiated them to myotubes for 5 days, and subjected them to acute (ACA) or chronic (CCA) contractile activity. ATF4 mediated myotube formation through the regulated expression of myogenic factors, mainly Myc and myoblast determination protein 1 (MyoD), and suppressed mitochondrial biogenesis basally through peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1α). However, our data also show that ATF4 expression levels are directly related to mitochondrial fusion and dynamics, UPRmt activation, as well as lysosomal biogenesis and autophagy. Thus, ATF4 promoted enhanced mitochondrial networking, protein handling, and the capacity for clearance of dysfunctional organelles under stress conditions, despite lower levels of mitophagy flux with OE. Indeed, we found that ATF4 promoted the formation of a smaller pool of high-functioning mitochondria that are more responsive to contractile activity and have higher oxygen consumption rates and lower reactive oxygen species levels. These data provide evidence that ATF4 is both necessary and sufficient for mitochondrial quality control and adaptation during both differentiation and contractile activity, thus advancing the current understanding of ATF4 beyond its canonical functions to include the regulation of mitochondrial morphology, lysosomal biogenesis, and mitophagy in muscle cells.

HSF4 Transcriptionally Activates Autophagy by Regulating ATG9a During Lens Terminal Differentiation.

HSF4 mutations are responsible for congenital cataract formation. Dysfunction of HSF4 leads to defects in lens terminal differentiation. We aimed to study the mechanism of how HSF4 promotes organelle degradation during lens differentiation. HSF4del42 mutant mice that developed congenital cataracts were employed. The organelle degradation and autophagic function in lens fibers were detected by immunofluorescence and Immunoblotting. Transcriptome analysis was performed to investigate the differentially expressed genes in HSF4del42 lenses, whereas luciferase report assay and ChIP assay were used to confirm the directly transcriptional regulation of ATG9a by HSF4. HSF4del42 mice displayed delayed organelle clearance and impaired autophagic degradation function in lens fibers. Activation of autophagy by rapamycin ameliorated the defects in organelle clearance in HSF4del42 lenses ex vivo and in vivo. Depletion of HSF4 attenuated autophagic flux by disrupting autophagosome biogenesis and maturation in lens epithelial cells. HSF4 directly transcriptionally activated the core autophagy protein ATG9a. Instead of the canonical ATG9a isoform, the ATG9a-X2 isoform was predominantly expressed in the lens and alleviated autophagic defects in HSF4 KO lens epithelial cells. The ATG9a-X2 protein displayed a short half-life, and rapamycin treatment restored its levels in HSF4 KO lens epithelial cells and HSF4del42 lenses. Our findings demonstrate that HSF4 facilitates organelle degradation probably by transcriptionally activating autophagy during lens terminal differentiation. We first report the involvement of HSF4 in autophagy and the tissue specific splicing of ATG9a. Our study indicates that autophagy activation is a possible therapeutic strategy for HSF4-related congenital cataracts.

Proteasomes of Autophagy-Deficient Cells Exhibit Alterations in Regulatory Proteins and a Marked Reduction in Activity.

Autophagy and the ubiquitin proteasome system are the two major processes for the clearance and recycling of proteins and organelles in eukaryotic cells. Evidence is accumulating that there is extensive crosstalk between the two pathways, but the underlying mechanisms are still unclear. We previously found that autophagy 9 (ATG9) and 16 (ATG16) proteins are crucial for full proteasomal activity in the unicellular amoeba Dictyostelium discoideum. In comparison to AX2 wild-type cells, ATG9-and ATG16- cells displayed a 60%, and ATG9-/16- cells a 90%, decrease in proteasomal activity. Mutant cells also showed a significant increase in poly-ubiquitinated proteins and contained large ubiquitin-positive protein aggregates. Here, we focus on possible reasons for these results. Reanalysis of published tandem mass tag-based quantitative proteomic results of AX2, ATG9-, ATG16-, and ATG9-/16- cells revealed no change in the abundance of proteasomal subunits. To identify possible differences in proteasome-associated proteins, we generated AX2 wild-type and ATG16- cells expressing the 20S proteasomal subunit PSMA4 as GFP-tagged fusion protein, and performed co-immunoprecipitation experiments followed by mass spectrometric analysis. The results revealed no significant differences in the abundance of proteasomes between the two strains. However, we found enrichment as well as depletion of proteasomal regulators and differences in the ubiquitination of associated proteins for ATG16-, as compared to AX2 cells. Recently, proteaphagy has been described as a means to replace non-functional proteasomes. We propose that autophagy-deficient D. discoideum mutants suffer from inefficient proteaphagy, which results in the accumulation of modified, less-active, and also of inactive, proteasomes. As a consequence, these cells exhibit a dramatic decrease in proteasomal activity and deranged protein homeostasis.

A single dose of exercise stimulates skeletal muscle mitochondrial plasticity in myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is the second most common muscular dystrophy after Duchenne and is the most prevalent muscular dystrophy in adults. DM1 patients that participate in aerobic exercise training experience several physiological benefits concomitant with improved muscle mitochondrial function without alterations in typical DM1-specific disease mechanisms, which suggests that correcting organelle health is key to ameliorate the DM1 pathology. However, our understanding of the molecular mechanisms of mitochondrial turnover and dynamics in DM1 skeletal muscle is lacking. Skeletal muscle tissue was sampled from healthy and DM1 mice under sedentary conditions and at several recovery time points following an exhaustive treadmill run. We demonstrate that DM1 patients exhibit an imbalance in the transcriptional apparatus for mitochondrial turnover and dynamics in skeletal muscle. Additionally, DM1 mice displayed elevated expression of autophagy and mitophagy regulators. A single dose of exercise successfully enhanced canonical exercise molecular pathways and skeletal muscle mitochondrial biogenesis despite failing to alter the cellular pathology in DM1 mice. However, treadmill running stimulated coordinated organelle fusion and fission signaling, as well as improved alternative splicing of Optic atrophy 1. Exercise also evoked autophagy and mitophagy pathways in DM1 skeletal muscle resulting in the normalized expression of autophagy- and lysosome-related machinery responsible for the clearance of dysfunctional organelles. Collectively, our data indicate that mitochondrial dynamics and turnover processes in DM1 skeletal muscle are initiated with a single dose of exercise, which may underlie the adaptive benefits previously documented in DM1 mice and patients.

Insufficient or excessive dietary carbohydrates affect gut health through change in gut microbiota and regulation of gene expression of gut epithelial cells in grass carp (Ctenopharyngodon idella)

Dietary carbohydrate levels can affect gut health, but the roles played by gut microbiota and gut epithelial cells, and their interactions remain unclear. In this experiment, we investigated gut health, gut microbiota, and the gene expression profiles of gut epithelial cells in grass carp consuming diets with different carbohydrate levels. Compared to the moderate-carbohydrate diet, low-carbohydrate diet significantly increased the relative abundance of pathogenic bacteria (Ralstonia and Elizabethkingia) and decreased the abundance of metabolism in cofactors and vitamins, implying a dysregulated gut microbiota and compromised metabolic function. Moreover, low-carbohydrate diet inhibited the expression levels of key genes in autophagy-related pathways in gut epithelial cells, which might directly lead to reduced clearance of defective organelles and pathogenic microorganisms. These aforementioned factors may be responsible for the imperfect organization of the intestinal tract. High-carbohydrate diet also significantly increased the abundance of pathogenic bacteria (Flavobacterium), which directly contributed to a decrease in the abundance of immune system of the microbiota. Furthermore, the active pathways of staphylococcus aureus infection and complement and coagulation cascades, as well as the inhibition of the glutathione metabolism pathway were observed. Above results implied that high-carbohydrate diet might ultimately cause severe gut damage by affecting immune function of microbiota, mentioned immune-related pathways, and the antioxidant capacity. Finally, the correlation network diagram revealed strong correlations of the differentially immune-related gene major histocompatibility complex class I antigen (MR1) with Enhydrobacter and Ruminococcus_gnavus_group in low-carbohydrate diet group, and Arenimonas in high-carbohydrate diet group, respectively, suggesting that MR1 might be a central target for immune responses in gut epithelial cells induced by gut microbiota at different levels of dietary carbohydrate. All these results provided insight in the development of antagonistic probiotics and target genes to improve the utilization of carbohydrate.

Ube2c-inhibition alleviated amyloid pathology and memory deficits in APP/PS1 mice model of AD.

Autophagy is a major intracellular degradation pathway for the clearance of damaged organelles and misfolded peptides. Previous studies have indicated that autophagy is involved in the pathogenesis of neurodegenerative disease including Alzheimer's disease (AD). Defective autophagy and highly expressed ubiquitin-conjugating enzyme 2C (Ube2c) have been found in AD patients and mouse. However, little is known about the regulation of autophagy in AD. The association of Ube2c with autophagy, amyloid pathology and cognitive deficits in AD remains unclear. In the present study, we characterized over expression of Ube2c and declined autophagy in amyloid β (Aβ)-treated microglia and demonstrated the protective effects of agomelatine (AGO) in APP/PS1 mice. We found that knockdown of Ube2c with AAV2 encoding shUbe2c resulted in an obvious enhancement of autophagy in BV2 microglia cells, and an alleviation of Aβ pathology and memory deficits in APP/PS1 mice. Further, pharmacological inhibition of Ube2c by AGO significantly reduced Aβ plaques, improved synaptic plasticity and cognitive behaviors in APP/PS1 mice, as well as promoted autophagy in microglia. Our findings uncover a potent role of Ube2c over-expression and autophagy decline in the pathogenesis of AD, and suggest that regulation of Ube2c and autophagy may provide an important clue and a potential target for the novel therapeutics of AD.

The presence of MTDNA mutations in THP1-based cybrid cells affects the efficiently of FCCP-induced mitophagy

Background and Aims : Mitophagy is an effective way to clearance of dysfunctional mitochondria. A disturbance in the clearance of dysfunctional organelles and their accumulation may indicate that mitophagy is defective. We hypothesize that mtDNA mutations may cause defective mitophagy.

JIP3 interacts with dynein and kinesin-1 to regulate bidirectional organelle transport.

The MAP kinase and motor scaffold JIP3 prevents excess lysosome accumulation in axons of vertebrates and invertebrates. How JIP3's interaction with dynein and kinesin-1 contributes to organelle clearance is unclear. We show that human dynein light intermediate chain (DLIC) binds the N-terminal RH1 domain of JIP3, its paralog JIP4, and the lysosomal adaptor RILP. A point mutation in RH1 abrogates DLIC binding without perturbing the interaction between JIP3's RH1 domain and kinesin heavy chain. Characterization of this separation-of-function mutation in Caenorhabditis elegans shows that JIP3-bound dynein is required for organelle clearance in the anterior process of touch receptor neurons. Unlike JIP3 null mutants, JIP3 that cannot bind DLIC causes prominent accumulation of endo-lysosomal organelles at the neurite tip, which is rescued by a disease-associated point mutation in JIP3's leucine zipper that abrogates kinesin light chain binding. These results highlight that RH1 domains are interaction hubs for cytoskeletal motors and suggest that JIP3-bound dynein and kinesin-1 participate in bidirectional organelle transport.

Autophagy reprogramming stem cell pluripotency and multiple-lineage differentiation.

The cellular process responsible for the degradation of cytosolic proteins and subcellular organelles in lysosomes was termed "autophagy." This process occurs at a basal level in most tissues as part of tissue homeostasis that redounds to the regular turnover of components inside cytoplasm. The breakthrough in the autophagy field is the identification of key players in the autophagy pathway, compounded under the name "autophagy-related genes" (ATG) encoding for autophagy effector proteins. Generally, the function of autophagy can be classified into two divisions: intracellular clearance of defective macromolecules and organelles and generation of degradation products. Therapeutic strategies using stem cell-based approach come as a promising therapy and develop rapidly recently as stem cells have high self-renewability and differentiation capability as known as mesenchymal stem cells (MSCs). They are defined as adherent fibroblast-like population with the abilities to self-renew and multi-lineage differentiate into osteogenic, adipogenic, and chondrogenic lineage cells. To date, they are the most extensively applied adult stem cells in clinical trials. The properties of MSCs, such as immunomodulation, neuroprotection, and tissue repair pertaining to cell differentiation, processes to replace lost, or damaged cells, for aiding cell repair and revival. Autophagy has been viewed as a remarkable mechanism for maintaining homeostasis, ensuring the adequate function and survival of long-lived stem cells. In addition, authophagy also plays a remarkable role in protecting stem cells against cellular stress when the stem cell regenerative capacity is harmed in aging and cellular degeneration. Understanding the under-explored mechanisms of MSC actions and expanding the spectrum of their clinical applications may improve the utility of the MSC-based therapeutic approach in the future.

Lysosomal inhibition sensitizes TMEM16A-expressing cancer cells to chemotherapy

Squamous cell carcinoma of the head and neck (SCCHN) is a devastating disease that continues to have low cure rates despite the recent advances in therapies. Cisplatin is the most used chemotherapy agent, and treatment failure is largely driven by resistance to this drug. Amplification of chromosomal band 11q13 occurs in ∼30% of SCCHN tumors. This region harbors the ANO1 gene that encodes the TMEM16A ion channel, which is responsible for calcium-activated chloride transport in epithelial tissues. TMEM16A overexpression is associated with cisplatin resistance, and high TMEM16A levels correlate with decreased survival. However, the mechanistic underpinning of this effect remains unknown. Lysosomal biogenesis and exocytosis have been implicated in cancer because of their roles in the clearance of damaged organelles and exocytosis of chemotherapeutic drugs and toxins. Here, we show that TMEM16A overexpression promotes lysosomal biogenesis and exocytosis, which is consistent with the expulsion of intracellular cisplatin. Using a combination of genetic and pharmacologic approaches, we find that TMEM16A promotes lysosomal flux in a manner that requires reactive oxygen species, TRPML1, and the activation of the β-catenin–melanocyte-inducing transcription factor pathway. The lysosomal inhibitor hydroxychloroquine (HCQ) synergizes with cisplatin in killing SCCHN cells in vitro. Using a murine model of SCCHN, we show that HCQ and cisplatin retard the growth of cisplatin-resistant patient-derived xenografts in vivo. We propose that TMEM16A enables cell survival by the up-regulation of lysosomal sequestration and exocytosis of the cytotoxic drugs. These results uncover a model of treatment for resistance in cancer, its reversal, and a role for TMEM16A.

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.

Proteome remodeling and organelle clearance in mammalian terminal erythropoiesis.

The differentiation from colony forming unit-erythroid (CFU-E) cells to mature enucleated red blood cells is named terminal erythropoiesis in mammals. Apart from enucleation, several unique features during these developmental stages include proteome remodeling and organelle clearance that are important to achieve hemoglobin enrichment. Here, we review the recent advances in the understanding of novel regulatory mechanisms in these processes, focusing on the master regulators that link these major events during terminal erythropoiesis. Comprehensive proteomic studies revealed a mismatch of protein abundance to their corresponding transcript abundance, which indicates that the proteome remodeling is regulated in a complex way from transcriptional control to posttranslational modifications. Key regulators in organelle clearance were also found to play critical roles in proteome remodeling. These studies demonstrate that the complexity of terminal erythropoiesis is beyond the conventional transcriptomic centric perspective. Posttranslational modifications such as ubiquitination are critical in terminal erythroid proteome remodeling that is also closely coupled with organelle clearance.