Erythroid Cell Mitochondria Receive Endosomal Iron by a “Kiss-And-Run” Mechanism

Biochimica et Biophysica Acta 1863 (2016) 2859–2867

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Erythroid cell mitochondria receive endosomal iron by a “kiss-and-run” mechanism

Amel Hamdi a,b,1, Tariq M. Roshan a,1, Tanya M. Kahawita a,b,AnneB.Masonc,AlexD.Shefteld,e,PremPonkaa,b,⁎ a Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Quebec, Canada b Department of Physiology, McGill University, Montreal, Quebec, Canada c Department of Biochemistry, University of Vermont, Burlington, VT, USA d Spartan Bioscience Inc., Ottawa, Ontario, Canada e High Impact Editing, Ottawa, Ontario, Canada article info abstract

Article history: In erythroid cells, more than 90% of transferrin-derived iron enters mitochondria where ferrochelatase inserts Fe2+ Received 7 July 2016 into protoporphyrin IX. However, the path of iron from endosomes to mitochondrial ferrochelatase remains elu- Received in revised form 24 August 2016 sive. The prevailing opinion is that, after its export from endosomes, the redox-active metal spreads into the cytosol Accepted 8 September 2016 and mysteriously finds its way into mitochondria through passive diffusion. In contrast, this study supports the hy- Available online 11 September 2016 pothesis that the highly efficient transport of iron toward ferrochelatase in erythroid cells requires a direct inter- “ ” Keywords: action between transferrin-endosomes and mitochondria (the kiss-and-run hypothesis). Using a novel fl Iron method ( ow sub-cytometry), we analyze lysates of reticulocytes after labeling these organelles with different Transferrin fluorophores. We have identified a double-labeled population definitively representing endosomes interacting Endosomes with mitochondria, as demonstrated by confocal microscopy. Moreover, we conclude that this endosome- Mitochondria mitochondrion association is reversible, since a “chase” with unlabeled holotransferrin causes a time-dependent Erythroid cells decrease in the size of the double-labeled population. Importantly, the dissociation of endosomes from mitochondria does not occur in the absence of holotransferrin. Additionally, mutated recombinant holotransferrin, that cannot release iron, significantly decreases the uptake of 59Fe by reticulocytes and diminishes 59Fe incorporation into heme. This suggests that endosomes, which are unable to provide iron to mitochondria, cause a “trafficjam” lead- ing to decreased endocytosis of holotransferrin. Altogether, our results suggest that a molecular mechanism exists to coordinate the iron status of endosomal transferrin with its trafficking. Besides its contribution to the field of iron metabolism, this study provides evidence for a new intracellular trafficking pathway of organelles. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Developing erythroid cells, which have a capacity to obtain and process iron with astonishing efficacy [8], are capable of concentrating iron Iron is a transition metal whose properties make it useful to vital bi- (in the form of heme in the red cell) to approximately 7000-fold what is ologic processes in virtually all living organisms. Essential cellular func- present in the plasma (as diferric transferrin [Fe2-Tf]). Additionally, the tions include use of iron for oxygen transport, electron transfer, DNA delivery of iron into hemoglobin occurs extremely efficiently, since ma- synthesis and innumerable other purposes [1–4]. However, under phys- ture erythrocytes contain about 45,000-fold more heme iron than non- iological conditions iron is virtually insoluble and can be extremely toxic heme iron [9]. These facts suggest that in erythroid cells the iron trans- when not properly shielded [5–7].Hence,ironplaysaroleintheforma- port and heme biosynthetic machineries are fully integrated and are tion of toxic oxygen radicals that damage various cell structures. There- part of the same metabolic pathway that leads to a remarkably efficient fore, it is critical that we gain a better understanding of the mechanisms production of heme. Delivery of iron to these cells occurs following the involved in normal and abnormal intracellular iron trafficking. binding of Fe2-Tf to its cognate receptors (TfR) on the cell membrane. The Fe2-Tf-TfR complexes are then internalized via endocytosis, and iron is released by a process which includes both acidification and the participation of the TfR [1,9–12]. Iron, following its reduction to Fe2+ ⁎ Corresponding author at: Jewish General Hospital, Lady Davis Institute for Medical by Steap3 (six-transmembrane epithelial antigen of the prostate [13]), Research, and the Department of Physiology, McGill University, 3755 Chemin Côte-Ste- likely together with ascorbate [14] is then transported across Catherine, Montréal, QC H3T 1E2, Canada. E-mail address: [email protected] (P. Ponka). the endosomal membrane by the divalent metal transporter 1, DMT1/ 1 Co-first author. Nramp2 [15].

Following its egress from endosomes, iron is transported to intracel- the “chaperone”-like function of endosomes may be one of the mecha- lular sites of use and/or storage in ferritin, but this aspect of iron metab- nisms that keeps the concentrations of reactive Fe2+ at extremely low olism remains elusive or is at best controversial. It has been commonly levels in the oxygen-rich cytosol of erythroblasts and reticulocytes, believed that a low molecular weight intermediate chaperones iron preventing ferrous iron's participation in a dangerous Fenton reaction. from endosomes to mitochondria and other sites of utilization [16]. There is accumulating evidence for transfer of metabolites directly be- However, this long sought iron binding intermediate, that would consti- tween interacting organelles [33–36].However,theroleofendosomesin tute the labile iron pool, has never been identified. In fact, earlier work distributing intracellular iron is accepted without enthusiasm [20], from this laboratory demonstrated [17] that there was virtually no misinterpreted [16] or simply ignored [37]. Hence, we deemed it essential low molecular weight iron pool in hemoglobin-synthesizing cells and to seek further evidence for our “kiss-and-run” hypothesis. that iron present in this pool does not behave as an intermediate, thus In the current study, we used 3D live confocal imaging of reticulo- corresponding to an end product. Although it has recently been pro- cytes following their incubation with MitoTracker Deep Red (MTDR) posed that the ferrous iron in this pool is associated with glutathione and Alexa Green Transferrin (AGTf) and demonstrated transient inter- [18], this conclusion was only based on chemical interactions. Hence, actions of endosomes with mitochondria. We also demonstrate these the relevance of this study to the physiological mechanisms involved interactions by a novel method exploiting flow sub-cytometry to ana- in the intracellular iron transport remains in doubt. Additionally, it has lyze reticulocyte lysates labeled with MTDR and AGTf. This strategy been proposed that human poly(rC)-binding protein 1 (PCBP1) “chap- identified a population double-labeled with both fluorescent markers erones” iron into ferritin and non-heme iron proteins [19,20]. On the representing endosomes interacting with mitochondria. FACS sorting other hand, more recent reports [21,22] have proposed PCBP2, rather followed by 2D confocal microscopy confirmed the association of both than PCBP1, to play an “iron-chaperone” role. This diversity is probably organelles in the double-labeled population. The results of these exper- due to different cell types and/or experimental strategies used. Howev- iments as well as those exploiting a mutated recombinant Fe2-Tf unable er, no direct evidence has been provided that PCBPs acquire iron from to release iron efficiently further support the hypothesis that the effi- transferrin-endosomes, carry it in cytosol and, more crucially, deliver cient iron transfer to PPIX requires a transient interaction between the it to mitochondria. endosome and the mitochondrion. In erythroid cells, more than 90% of iron enters mitochondria where ferrochelatase, the enzyme that inserts Fe2+ into protoporphyrin IX 2. Results (PPIX), resides in the matrix side of the inner mitochondrial membrane. Importantly, in these cells, strong evidence exists for specifictargeting 2.1. Three-dimensional confocal microscopy of live cells reveals of iron toward mitochondria. This mitochondria-directed transport is endosome-mitochondria interactions demonstrated in hemoglobin-synthesizing cells, in which iron acquired from Tf continues to flow into mitochondria even when the synthesis of Using two-dimensional (2D) confocal microscopy, we have previ- PPIX is suppressed either experimentally [17,23–25] or in hereditary ously shown that endosomes come in contact with mitochondria [31]. sideroblastic anemia caused by defects in heme synthesis [26–28].Of To avoid any pseudo-interactions of these two organelles, i.e., the endo- note, a compelling candidate for the transport of iron through the mito- some hovering over the mitochondria, we have used a Quorum WaveFx chondria towards ferrochelatase, mitoferrin, has been identified [29]. spinning disc confocal system to capture three-dimensional (3D) live Based on the above considerations, we have formulated a hypothesis cell imaging of reticulocytes with fluorescently labeled mitochondria that in erythroid cells a transient mitochondrion-endosome interaction and endosomes. An individual cell at different time intervals is present- is involved in iron translocation to its final destination [17,30].Wehave ed in three different orientations (Fig. 1,A–C) and shows the proximity collected the following experimental evidence to support this hypothe- of endosomes and mitochondria at different angles (Link for Video; sis: 1) iron, delivered to mitochondria via the Tf-TfR pathway, is un- please see Supplementary Material). available to cytoplasmic chelators [31]; 2) Tf-containing endosomes move to and contact mitochondria [31] in erythroid cells; 3) endosomal 2.2. Development of the flow sub-cytometry method to study movement is required for iron delivery to mitochondria [31,32].We endosome-mitochondria interactions have also demonstrated that “free” cytoplasmic iron is not efficiently used for heme biosynthesis and that the endosome-mitochondrion in- We developed a new method, “flow sub-cytometry”, based on the teraction increases chelatable mitochondrial iron [31]. standard flow cytometry method, to analyze lysates obtained from re- As already mentioned, the substrate for the endosomal transporter, ticulocytes with fluorescently labeled mitochondria (MTDR) and DMT1, is Fe2+, the redox form of iron which is also the substrate for endosomes (AGTf). Employing this strategy, three distinct populations: ferrochelatase. These facts make our hypothesis quite attractive, since endosomes, mitochondria, and a population double labeled with both

Fig. 1. 3D confocal microscopy demonstrates the association of transferrin-endosomes (green) with mitochondria (red). At one time interval (“3”, marked by white arrows), endosome- mitochondria pairs are shown in three different orientations. A. Hamdi et al. / Biochimica et Biophysica Acta 1863 (2016) 2859–2867 2861

fluorescent markers have been identified (Fig. 2A). To confirm the inter- 2.3. Holotransferrin causes the detachment of endosomes from action between endosomes and mitochondria, we used FACS-sorting to mitochondria isolate the population double-labeled with both fluorescent markers as well as MTDR-labeled population (as a negative control), followed by To investigate whether the association of endosomes with mito- 2D examination using a confocal fluorescence microscope. A multichan- chondria is reversible, we incubated reticulocytes with both fluorescent nel protocol was used to scan the samples for AGTf and MTDR labeled markers for 20 min, washed the cells and reincubated with or without endosomes and mitochondria, respectively. The colocalization of the holotransferrin (Fe2-Tf) for 60 min. The presence of non-fluorescent two organelles was demonstrated by overlapping the green and the Fe2-Tf caused a time-dependent decrease in the size of the double- red images of endosomes and mitochondria, where the merged images labeled population (Fig. 4A and B). Hence, AGTf-endosomes associated represent both organelles together (Fig. 2B). This experiment has un- with mitochondria are replaced by incoming non-fluorescent Fe2-Tf- equivocally demonstrated the presence of the Tf-endosome interacting endosomes, indicating the reversibility of the interaction. AGTf- with the mitochondrion in the double-labeled population (UR). Impor- endosomes leaving mitochondria cannot be detected by FACS because tantly, the LR population contains exclusively MTDR-labeled mitochon- of their small size. Importantly, this finding, together with the fact that dria and the LL population is devoid of any fluorescence. Moreover, we endosomes remain bound to mitochondria in the absence of Fe2-Tf, sug- have shown that the size of the double-labeled population representing gests that the Tf-endosome drives the detachment. endosomes associated with mitochondria (UR quadrant) increases with incubation time with AGTf and reaches a plateau in ~20 min (Fig. 3). 2.4. Intra-endosomal transferrin saturation governs interorganellar association

We investigated whether the degree of endosomal Tf saturation with iron affects the dynamics of the endosome-mitochondrion associ- A ation. When bafilomycin A1, a specific V-ATPase inhibitor known to de- E EM crease acidification of endosomes [38], is added to reticulocytes, the size of the double-labeled population dramatically decreased (Fig. 5A). Bafilomycin A1 has been shown to block the incorporation of 59Fe into heme from 59Fe-Tf-endosomes [32]. Additionally, hemin treatment of reticulocytes also considerably decreased the size of the double- labeled population (Fig. 5B). Heme has been shown to feedback inhibit the release of iron from Tf within reticulocyte endosomes [39] and, sim- ilarly as bafilomycin A1, can be expected to increase endosomal Tf saturation. M The most likely explanation for these observations is that bafilomycin A1 as well as hemin block Fe2+ export from endosomes already associated with mitochondria, extending their residence time on these organelles. This causes a “trafficjam” that decelerates the movement of endosomes containing fluorescent Tf towards mitochondria. Furthermore, the presence of heme during the reincubation of double-

labeled reticulocytes with unlabeled Fe2-Tf slowed down the disappear- ance of the double population (Fig. 5C), suggesting that the efﬁcient dis- MTDR AGTf Merge B sociation of endosomes requires them to be iron-free. UR(EM) 2.5. Calmodulin antagonist (W-7) decreases the size of double-labeled population

N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7), a calmodulin antagonist, is known to inhibit the microﬁlament motor, myosin, which is involved in intracellular movement of endosomes MTDR AGTf Merge and causes signiﬁcant inhibition of 59Fe incorporation from 59Fe- LR(M) endosomes into heme [32]. A positive experiment presented in Fig. S2 shows that W-7, as expected, inhibits dramatically the association of endosomes with mitochondria.

2.6. Transferrin mutant unable to release iron (Holo-rTf) impairs the uptake 59 59 of Fe from Fe2-Tf

Fig. 2. Flow sub-cytometry method. (A) Using FACS analysis, four different populations in To assess whether endosomes unable to release iron affect intracel- lysates of reticulocytes, previously labeled with MTDR and AGTf for 20 min, were lular iron trafficking, reticulocytes were preincubated with Holo-rTf at identified: The lower left (LL) quadrant displays events devoid of all fluorescence (cell 37 °C for 30 min, washed with cold PBS and incubated with 59Fe-Tf for debris). The upper left (UL) quadrant displays events labeled only with AGTf and, the indicated time intervals. After washing with cold PBS, total cell therefore, represents the endosomal population. The lower right (LR) quadrant displays 59 events labeled only with MTDR and, therefore, corresponds to a mitochondrial population and heme Fe-radioactivities were measured. The presence of Holo- 59 (M). The upper right (UR) quadrant, labeled with both AGTf and MTDR, is referred to as rTf during incubation significantly inhibited Fe uptake by reticulocytes the double-labeled population representing endosomes interacting with mitochondria from wild-type 59Fe-Tf and also diminished 59Fe incorporation into (EM). (B) Populations present in UR, LR and LL quadrants were isolated by FACS-sorting heme (Fig. 6). This result suggests that the failure to release iron from and then examined by 2D confocal microscopy. UR is composed only of AGTf-labeled Holo-rTf interferes with the process of endocytosis or endosomal traf- endosomes that colocalize with MTDR-labeled mitochondria. LR contains exclusively fi 59 MTDR-labeled mitochondria. The confocal images show four separate structures. Scale cking, causing a decrease in the total Fe uptake by the reticulocytes bar is 5 μm. as compared to controls. 2862 A. Hamdi et al. / Biochimica et Biophysica Acta 1863 (2016) 2859–2867

A Time (min) 0 1 2 5

0.31% 10.01% 16.10% 23.31% ) AGTf

10 20 30 27.75% 29.68% 31.47% Endosomes (

Mitochondria (MTDR) B 35

Percentage of total population 0 0 5 10 15 20 25 30 Time (min)

Fig. 3. Flow sub-cytometry analysis of the endosome-mitochondria interaction. (A) Percentage of mitochondria associated with endosomes increases with time. (B) Graphical presentation of results shown in (A). Error bars were determined from triplicate values.

2.7. Endosomes interact with mitochondria also in erythroid progenitors basis, the rate of heme synthesis in developing erythroid cells is at least an order of magnitude higher than in the liver, the second highest heme In this experiment, we used murine fetal liver (FL) cells, primary producer in the body. mouse erythroid cells that closely recapitulate several aspects of termi- As described in the Introduction, most mammalian cells take up iron nal maturation in vivo, in particular very active hemoglobinization [8] via receptor-mediated endocytosis by the TfR. The prevailing opinion is We deemed it important to ascertain whether endosomes interact that after its escape from endosomes, iron spreads into the cytosol [40] with mitochondria only in differentiating erythroid cells or if this phe- and mysteriously finds its way into mitochondria. Based on the fact that nomenon operates also before the onset of hemoglobin synthesis. As Tf-bound iron is extremely efficiently used for hemoglobin synthesis, predicted, subjecting these cells to flow sub-cytometry as performed targeted into erythroid mitochondria, and that no cytoplasmic iron above with reticulocytes, revealed a large pool of the double-labeled transport intermediate has ever been identified in erythroid cells, we population in FL cells induced to differentiate by erythropoietin. Impor- have suggested a new hypothesis of intracellular iron transport [17, tantly, we also observed that the endosome-mitochondria population, 30]. This model proposes [17] that after iron is released from Tf in Hb- although smaller than in erythropoietin-induced FL cells, is present al- synthesizing cells, it would bypass the cytosol and be directly trans- ready in undifferentiated cells (Fig. S3). ferred from protein to protein (probably in hydrophobic environments) until it reaches ferrochelatase in the mitochondrion. 3. Discussion Previous work from our laboratory has suggested that iron is directly transferred from endosomes to mitochondria [31,32] likely through During differentiation, immature erythroid cells acquire vast amounts interorganellar interaction. However, this model has not been accepted of iron at a “breakneck” rate. Proper coordination of iron delivery and by many [16,20]. In the present study, we used 2D and 3D confocal mi- utilization in heme synthesis is essential; disruption of this process likely croscopy and developed a novel experimental strategy based on flow underlies iron loading disorders such as sideroblastic anemia and cytometry analysis of fluorescently labeled organelles (“flow sub- myelodysplastic syndrome with ringed sideroblasts [28]. On a per-cell cyometry”) to provide evidence for our hypothesis. A. Hamdi et al. / Biochimica et Biophysica Acta 1863 (2016) 2859–2867 2863

Fig. 4. Holotransferrin causes the detachment of endosomes from mitochondria. (A) Flow sub-cytometry analysis of reticulocytes preincubated (20 min) with MTDR and AGTf, followed by their incubation (60 min) with or without Fe2-Tf. (B) Graphical presentation of the results shown in (A) as the percentage decrease from 100% (Time 0, the start of the “chase” experiment) in the presence or absence of holotransferrin as compared to control. ■,Fe2-Tf; ▲, minus Tf. Error bars were determined from triplicate values.

Employing 3D confocal system, we acquired video images, allowing Both bafilomycin A1 and heme interfere with iron release from Tf and us to capture fluorescent Tf-endosomes interacting with labeled mito- cause a significant decrease in the size of the double-labeled population chondria in space, excluding the possibility of “pseudo-interactions” (Fig. 5AandB).TheseagentsobstructFe2+ export from endosomes al- caused by a coincidental overlapping. This technique very clearly re- ready associated with mitochondria, extending the residence time of vealed contacts of endosomes with mitochondria in 3D space (Fig. 1). endosomes on mitochondria, thus blocking the movement of endosomes Using a flow sub-cytometry to analyze lysates obtained from reticulo- containing fluorescent Tf towards mitochondria. Additionally, hemin cytes with fluorescently labeled mitochondria (MTDR) and endosomes slows down the dissociation of labeled endosomes from mitochondria (AGTf), we have identified three distinct populations: endosomes, mito- (Fig. 5C). Hence, our results suggest that a molecular mechanism exists chondria, and a population double-labeled with both fluorescent markers to coordinate the iron status of endosomal Tf with its trafficking. (Fig. 2A). The double-labeled population was sorted by FACS and shown Moreover, the presence of a Tf mutant unable to release iron (Holo- 59 59 by 2D confocal microscopy to be composed of endosomes associated with rTf) impairs the uptake of Fe from Fe2-Tf (Fig. 6), suggesting that ef- mitochondria (Fig. 2B). The size of the double-labeled population in- ficient endocytosis and/or endosomal trafficking requires the transfer of creases with the incubation time and reaches a plateau at approximately iron from endosomes to mitochondria. In other words, the endosomal 20 min (Fig. 3). mitochondrial interface plays a role in regulating the cellular iron up- The reversibility of the endosome-mitochondria association was take, probably by controlling Tf endocytosis. demonstrated by the experiment in which we first preincubated reticu- Our finding of endosomes interacting with mitochondria in undiffer- locytes with both fluorescent markers for 20 min followed by their incu- entiated fetal liver cells (Fig. S3) is of considerable importance. It suggests bation (“chase”) with or without unlabeled holotransferrin (Fig. 4Aand that the endosome-mitochondrion interaction may be a common mech-

B). The presence of unlabeled Fe2-Tf caused a time-dependent decrease anism involved in iron delivery for heme synthesis. However, since FL in the size of the double-labeled population indicating the reversibility progenitors are committed to synthesize hemoglobin, further experi- of the endosome-mitochondria contact. Importantly, in the absence of ments with non-erythroid cells are needed to establish whether the holotransferrin, endosomes remain associated with mitochondria. kiss-and-run mechanism is generally involved in the transport of Tf- Taken together, our ﬁnding indicates that Tf-endosome drives the borne iron to mitochondria. If the so-called kiss-and-run mechanism of detachment. iron delivery to mitochondria proves to be ubiquitous, then mitochondria 2864 A. Hamdi et al. / Biochimica et Biophysica Acta 1863 (2016) 2859–2867

7000 A *** 35 6500 Total Control 6000 30 ** Heme 5500 25 Bafilomycin A1 5000 4500 20 4000 3500 15 3000 10 2500 2000 5 1500 CPM / 500 million reticulocytes Percentage of total population 1000 0 05101520500 0 B Time (min) 0 5 10 20 0 5 10 20 60 lortnoC fTr-oloH Time (min) Control 50 59 59 Hemin Fig. 6. Holo-rTf inhibits Fe incorporation into reticulocytes and heme from Fe2-Tf. Error bars were determined from triplicate values. ⁎⁎ p ≤ 0.01; ⁎⁎⁎ p ≤ 0.001. 40

30 recessive inheritance; some such patients have a defect in the gene encoding the erythroid-specific mitochondrial carrier protein, SLC25A38 20 [41]. It has been suggested that SLC25A38 may translocate glycine into mitochondria, a proposal recently confirmed by Fernández-Murray 10 et al. [42] and Lunetti et al. [43]. Percentage of total population In our opinion, at least four factors are responsible for the pathogen- 0 esis of ring sideroblast formation in congenital sideroblastic anemias: 0 5 10 15 20 25 30 fi C Time (min) (i) As extensively discussed in this report, the ef cient delivery of iron to ferrochelatase in erythroid cells requires the direct interaction of 100 endosomes with mitochondria. (ii) Mitochondrial iron cannot be ade- quately utilized due to the lack of PPIX. (iii) Erythroid cells, but not 80 non-erythroid cells, are equipped with a negative feedback mechanism in which ‘uncommitted’ heme inhibits iron acquisition from Tf [30].Al- though the molecular mechanism of this inhibition is still unknown, the 60 lack of heme plays an important role in mitochondrial iron accumula- tion in erythroid cells. (iv) Iron can leave erythroid mitochondria only 40 after being inserted into PPIX [24]. population As already alluded to, there is no consensus regarding the mecha- Hemin 20 nism of iron transfer from plasma Tf to mitochondria. One of the pro- Control posed intermediates in the intracellular iron path is the iron storage Percentage of double-labeled Percentage of protein, ferritin [44,45]. Nevertheless, a strong argument against this 0 claim was collected and reported [46]. Somewhat surprisingly, it was 0 102030405060 Time (min) suggested in a recent report [47] that the autophagic turnover of ferritin via NCOA4 is a critical process for regulating intracellular iron bioavail-

Fig. 5. Bafilomycin A1 and hemin decrease the association of endosomes with mitochondria. ability. However, Darshan et al. [48] demonstrated that the conditional (A) The percentage of the double-labeled population after treatment with bafilomycin. ■, deletion of the ferritin heavy chain in adult mice did not cause any de- Control; ♦,bafilomycin A1 (100 nM). Results presented are representative of 6 replicate crease in hematocrit or hemoglobin levels, strongly indicating that ferri- experiments. (B) The percentage of the double-labeled population after treatment with tin is not involved in hemoglobinization. In this context it is pertinent to hemin. ■, Control; ▲, hemin (5 μM). Results presented are representative of 4 replicate mention that the nuclear receptor coactivator 4 (NCOA4), also known as experiments. (C) Hemin prevents the dissociation of labeled endosomes from fi fi mitochondria. ■, Control; ▲, hemin (5 μM). Error bars were determined from triplicate ARA70, was initially identi ed as a speci c coactivator for the androgen values. receptor in human prostate cells [49]. Additionally, another study [50] has provided evidence that NCOA4 knockout is not lethal and NCOA4- may be the entry site of iron into the cell. In this context it needs to be null mice have only a mild microcytic hypochromic anemia. These find- pointed out that the only known function of internalized Tf is to deliver ings concur with our previous and current studies demonstrating that iron to its intracellular destination [31].Hence,thedescribedendosome the efficient utilization of iron for hemoglobin synthesis requires a di- mitochondria association facilitates interorganellar iron transfer. rect contact of endosomes with mitochondria. Our study is relevant to sideroblastic anemias, a heterogeneous Finally, recent experimental support for the kiss-and-run hypothesis group of disorders, characterized by mitochondrial iron overload in de- came from a study that investigated the interaction between several mi- veloping red blood cells [26–28]. The unifying characteristic of all tochondrial heme biosynthetic enzymes and proteins involved in iron ho- sideroblastic anemias is the ring sideroblast, a pathological erythroid meostasis [51]. This study revealed a partnership between ferrochelatase precursor containing excessive deposits of non-heme iron in mitochon- (a mitochondrial protein) and TfR (an endosome associated protein) that dria with perinuclear distribution creating a ring appearance. maybemediatedbyayettobeidentified bridge protein. It will be worth The majority of congenital sideroblastic anemias are X-linked; most of examining whether one of the PCBPs could serve as such protein [19,21, them are caused by mutations in the gene encoding erythroid-specific5- 52]. Medlock et al. [45] further state, “(their) data…support a model of aminolevulinate synthase [26–28]. However, there is a certain proportion transient protein-protein interactions within a dynamic protein of patients with hereditary sideroblastic anemia who exhibit autosomal complex.” Our model, shown in Fig. 7, suggests a potential outer A. Hamdi et al. / Biochimica et Biophysica Acta 1863 (2016) 2859–2867 2865

Fig. 7. Scheme of iron delivery to mitochondria for heme biosynthesis in erythroid cells. Iron is released from transferrin within endosomes by a combinationofFe3+ reduction by Steap3 (likely when it is still bound to the transferrin receptor) and a decrease in pH (~pH 5.5); following this, Fe2+ is transported through the endosomal membrane by DMT1. Our model proposes that DMT1 conveys Fe2+ to an outer mitochondrial membrane protein. Our preliminary studies suggest that the voltage-dependent anion channel (VDAC) interacts with the intracellular loops of DMT1 (Hamdi A, Roshan TM, Sheftel AD, Ponka P. Interaction of transferrin-endosomes with mitochondria: Implications for iron transport to ferrochelatase in erythroid cells [abstract]. Blood, 2015; 126(23): 407). VDAC, or possibly another protein, then delivers Fe2+ to mitoferrin 1 (Mfrn1) which, in turn, supplies Fe2+ to ferrochelatase (FC) that inserts it into protoporphyrin IX (PPIX). Maroon and red balls stand for ferric and ferrous iron, respectively. ALA, 5-aminolevulinic acid; ALA-S2, erythroid-speciﬁc ALA synthase.

mitochondrial membrane protein(s) that may interact with the intracel- 4.2. Animals lular loop of DMT1 for the delivery of iron to ferrochelatase. In summary, our study provides strong evidence that the highly effi- All the animal studies performed in this work were approved by the cient delivery of iron to heme in hemoglobin-synthesizing cells requires Lady Davis Institute animal care committee following the guidelines of the direct interaction of transferrin-endosomes with mitochondria. To the Canadian Council on Animal Care. the best of our knowledge, this is the first evidence of the interorganellar CD1 and C57BL/6 mice (females; 6 months old) retired breeders interaction having the functional consequence of facilitating the transport were obtained from Charles River Laboratories (Wilmington, MA) and of a micronutrient. A full understanding of the molecular mechanisms in- were housed in microisolator cages. volved in the endosome-mitochondria system will require the identification of the “signal(s)” that direct iron-carrying endosomes towards 4.3. Isolation of reticulocytes mitochondria, the players involved in the docking of endosomes to mitochondria and the “signal(s)” that determine the detachment of iron-free Adult, female CD1 female mice were injected with neutralized endosomes from mitochondria. We believe that the current work pro- pheynylhydrazine intraperitoneally at the dose of 50 mg/kg/day for vides a strong platform to pursue such studies. three consecutive days. Mice were then anesthetized after 2–3daysfol- lowing the last injection by using 1 mL of 2.5% avertin. Blood was collected via direct cardiac puncture using heparinized syringe and 4. Materials and methods resuspended in cold PBS. Cells were then washed with ice-cold PBS three times. By using this technique we obtained 30–45% reticulocytes 4.1. Materials as verified by thiozole orange staining. MitoTracker Deep Red 633 (MTDR) and Alexa Green Transferrin 488 (AGTf) were purchased from Molecular Probes (Invitrogen, Carlsbad, 4.4. Cell culture of primary mouse fetal liver cells CA). Protease inhibitor was purchased from Roche (Penzberg, Germany). Pronase, heparin and bafilomycin A1 were purchased from Primary erythroid cells were cultured as described [8].Briefly, cells

Bioshop (Burlington, Ontario). Holotransferrin (Fe2-transferrin), were grown from FL cells that were obtained from embryonic day avertin, phenylhydrazine, thiozole orange and N-(6-Aminohexyl)-5- 12.5 embryos of wild type C57BL/6 mice. FL cells were grown in chroro-1-napthalenesulfonamide hydrochloride (W7) were purchased serum-free StemPro-34 medium plus Nutrient Supplement (Invitrogen, from Sigma-Aldrich (St. Louis, MO). Fluorescent mounting media were Carlsbad, CA) and 2 mM L-glutamine, with 100 ng/mL murine recombi- purchased from Dako (Glostrup, Denmark). Microslides and micro cov- nant stem cell factor (SCF), 10−6 M synthetic glucocorticoid dexameth- erslips were purchased from VWR (West Chester, PA). Minimum Essen- asone (Dex), 40 ng/mL insulin-like growth factor 1 (IGF-1), 0.2% (vol/ tial Media (MEM) were purchased from Invitrogen (Gibco® Life vol) gentamicin, 0.2% amphotericin, and 2 U/mL human recombinant Technologies™, Carlsbad, CA) supplemented with 25 mM HEPES. Mito- erythropoietin (Epo). FL cells were kept either in an undifferentiated chondrial isolation buffer contains 220 mM mannitol, 60 mM sucrose, state or induced to terminal differentiation (48 h) by re-suspending 20 mM HEPES, pH 7.4, 80 mM KCl, 2 mM magnesium acetate, 0.5 mM them in differentiation medium (StemPro-34) containing Nutrient Sup- −4 EGTA and protease inhibitors. The human mutant recombinant Fe2-Tf plement, 10 U/mL Epo, 2 mM L-glutamine, 4 × 10 IU/mL insulin, unable to release iron (K206E/K534A) [53,54] was generated in Dr. 3×10−6 M RU486, and 1 mg/mL iron-saturated human transferrin

Mason's laboratory. (Fe2-Tf; Sigma, St. Louis, MO). 2866 A. Hamdi et al. / Biochimica et Biophysica Acta 1863 (2016) 2859– 2867

4.5. Confocal microscopy Author contributions

Reticulocytes were labeled with MTDR and AGTf and kept at 4 °C A.H. and T.M.R. performed research, analyzed data, and wrote the until their analysis by spinning disc confocal microscopy. The tempera- manuscript; T.M.K. performed research and analyzed data; A.B.M. and ture of the chamber was kept at 37 °C. One-milliliter samples of reticu- A.D.S. analyzed data, and wrote the manuscript; and P.P. conceived locytes suspension were resuspended in 50 μL of medium without the study, analyzed data, and wrote the manuscript. phenol red. Cells were then mounted on a pre-warmed (30 °C) heated stage and were allowed to equilibrate for 15 min before imaging. Retic- Conﬂict of interest ulocytes were not kept for more than 10 min in the chamber after ex- posing to laser. Three-dimensional images were acquired over time. The authors declare no competing or ﬁnancial interests. Optical sections of 0.5 μm were acquired in z-plane through a ±2.5- μm(totalof5.0-μm stack). Single endosome was tracked in a cell in different planes from the start of endocytosis to its interaction with mito- Funding chondria and leaving in a different plane. Microscopy was performed with a Zeiss LSM 5 Pascal confocal inverted microscope. Live cell imag- The research of P.P. was funded by grants from the Canadian Insti- ing of reticulocytes was done with Wave FX Spinning Disc Confocal tutes of Health Research (MOP-14100 and MOP-126064). (SDC) microscopy by Quorum Technologies Inc. (Guelph, Ontario). Ve- locity 3D Image Analysis Software from PerkinElmer was used to ana- Transparency document lyze live cell imaging. Rapid imaging of these instruments makes it possible to determine the 3D trajectory of an intra-cellular object. The Transparency document associated with this article can be found, in online version.

4.6. Flow sub-cytometry Acknowledgments Reticulocytes were washed three times with ice-cold PBS, spun down at 800 g for 5 min and pellet was re-suspended in MEM. Reticulo- The authors are grateful to Dr. Heidi McBride for her valuable advice cytes were then incubated with 500 nM MTDR for 30 min at 37 °C in a and helpful suggestions during this research. The authors also thank shaking water-bath following which they were washed 3-times with Drs. Julie Desbarats, Daniel Garcia-Santos, Matthias Schranzhofer, ice-cold PBS and centrifuged at 800g for 5 min at 4 °C. Cells were re- Marc Mikhael and Volker Blank for their useful comments and Christian suspended in MEM containing 500 nM AGTf and kept on ice for Young and Dr. Lilian Amrein for their technical assistance with FACS 30 min in dark. After incubating with AGTf samples were divided and analyses and confocal microscopy. incubated at 37 °C for the indicated time intervals. 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