Biochimica et Biophysica Acta 1863 (2016) 2859–2867
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Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbamcr
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, Anne B. Mason c, Alex D. Sheftel d,e, Prem Ponka a,b,⁎
a
Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Quebec, Canada
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
b
a r t i c l e
i n f o
Article history:
Received 7 July 2016
Received in revised form 24 August 2016
Accepted 8 September 2016
Available online 11 September 2016
Keywords:
Iron
Transferrin
Endosomes
Mitochondria
Erythroid cells
a b s t r a c t
In erythroid cells, more than 90% of transferrin-derived iron enters mitochondria where ferrochelatase inserts Fe2+
into protoporphyrin IX. However, the path of iron from endosomes to mitochondrial ferrochelatase remains elusive. The prevailing opinion is that, after its export from endosomes, the redox-active metal spreads into the cytosol
and mysteriously finds its way into mitochondria through passive diffusion. In contrast, this study supports the hypothesis that the highly efficient transport of iron toward ferrochelatase in erythroid cells requires a direct interaction between transferrin-endosomes and mitochondria (the “kiss-and-run” hypothesis). Using a novel
method (flow sub-cytometry), we analyze lysates of reticulocytes after labeling these organelles with different
fluorophores. We have identified a double-labeled population definitively representing endosomes interacting
with mitochondria, as demonstrated by confocal microscopy. Moreover, we conclude that this endosomemitochondrion association is reversible, since a “chase” with unlabeled holotransferrin causes a time-dependent
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 “traffic jam” leading 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
Iron is a transition metal whose properties make it useful to vital biologic processes in virtually all living organisms. Essential cellular functions include use of iron for oxygen transport, electron transfer, DNA
synthesis and innumerable other purposes [1–4]. However, under physiological conditions iron is virtually insoluble and can be extremely toxic
when not properly shielded [5–7]. Hence, iron plays a role in the formation of toxic oxygen radicals that damage various cell structures. Therefore, it is critical that we gain a better understanding of the mechanisms
involved in normal and abnormal intracellular iron trafficking.
⁎ Corresponding author at: Jewish General Hospital, Lady Davis Institute for Medical
Research, and the Department of Physiology, McGill University, 3755 Chemin Côte-SteCatherine, Montréal, QC H3T 1E2, Canada.
E-mail address: [email protected] (P. Ponka).
1
Co-first author.
http://dx.doi.org/10.1016/j.bbamcr.2016.09.008
0167-4889/© 2016 Elsevier B.V. All rights reserved.
Developing erythroid cells, which have a capacity to obtain and process iron with astonishing efficacy [8], are capable of concentrating iron
(in the form of heme in the red cell) to approximately 7000-fold what is
present in the plasma (as diferric transferrin [Fe2-Tf]). Additionally, the
delivery of iron into hemoglobin occurs extremely efficiently, since mature erythrocytes contain about 45,000-fold more heme iron than nonheme iron [9]. These facts suggest that in erythroid cells the iron transport and heme biosynthetic machineries are fully integrated and are
part of the same metabolic pathway that leads to a remarkably efficient
production of heme. Delivery of iron to these cells occurs following the
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+
by Steap3 (six-transmembrane epithelial antigen of the prostate [13]),
likely together with ascorbate [14] is then transported across
the endosomal membrane by the divalent metal transporter 1, DMT1/
Nramp2 [15].
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Following its egress from endosomes, iron is transported to intracellular sites of use and/or storage in ferritin, but this aspect of iron metabolism remains elusive or is at best controversial. It has been commonly
believed that a low molecular weight intermediate chaperones iron
from endosomes to mitochondria and other sites of utilization [16].
However, this long sought iron binding intermediate, that would constitute the labile iron pool, has never been identified. In fact, earlier work
from this laboratory demonstrated [17] that there was virtually no
low molecular weight iron pool in hemoglobin-synthesizing cells and
that iron present in this pool does not behave as an intermediate, thus
corresponding to an end product. Although it has recently been proposed that the ferrous iron in this pool is associated with glutathione
[18], this conclusion was only based on chemical interactions. Hence,
the relevance of this study to the physiological mechanisms involved
in the intracellular iron transport remains in doubt. Additionally, it has
been proposed that human poly(rC)-binding protein 1 (PCBP1) “chaperones” iron into ferritin and non-heme iron proteins [19,20]. On the
other hand, more recent reports [21,22] have proposed PCBP2, rather
than PCBP1, to play an “iron-chaperone” role. This diversity is probably
due to different cell types and/or experimental strategies used. However, no direct evidence has been provided that PCBPs acquire iron from
transferrin-endosomes, carry it in cytosol and, more crucially, deliver
it to mitochondria.
In erythroid cells, more than 90% of iron enters mitochondria where
ferrochelatase, the enzyme that inserts Fe2 + into protoporphyrin IX
(PPIX), resides in the matrix side of the inner mitochondrial membrane.
Importantly, in these cells, strong evidence exists for specific targeting
of iron toward mitochondria. This mitochondria-directed transport is
demonstrated in hemoglobin-synthesizing cells, in which iron acquired
from Tf continues to flow into mitochondria even when the synthesis of
PPIX is suppressed either experimentally [17,23–25] or in hereditary
sideroblastic anemia caused by defects in heme synthesis [26–28]. Of
note, a compelling candidate for the transport of iron through the mitochondria towards ferrochelatase, mitoferrin, has been identified [29].
Based on the above considerations, we have formulated a hypothesis
that in erythroid cells a transient mitochondrion-endosome interaction
is involved in iron translocation to its final destination [17,30]. We have
collected the following experimental evidence to support this hypothesis: 1) iron, delivered to mitochondria via the Tf-TfR pathway, is unavailable to cytoplasmic chelators [31]; 2) Tf-containing endosomes
move to and contact mitochondria [31] in erythroid cells; 3) endosomal
movement is required for iron delivery to mitochondria [31,32]. We
have also demonstrated that “free” cytoplasmic iron is not efficiently
used for heme biosynthesis and that the endosome-mitochondrion interaction increases chelatable mitochondrial iron [31].
As already mentioned, the substrate for the endosomal transporter,
DMT1, is Fe2+, the redox form of iron which is also the substrate for
ferrochelatase. These facts make our hypothesis quite attractive, since
the “chaperone”-like function of endosomes may be one of the mechanisms that keeps the concentrations of reactive Fe2+ at extremely low
levels in the oxygen-rich cytosol of erythroblasts and reticulocytes,
preventing ferrous iron's participation in a dangerous Fenton reaction.
There is accumulating evidence for transfer of metabolites directly between interacting organelles [33–36]. However, the role of endosomes in
distributing intracellular iron is accepted without enthusiasm [20],
misinterpreted [16] or simply ignored [37]. Hence, we deemed it essential
to seek further evidence for our “kiss-and-run” hypothesis.
In the current study, we used 3D live confocal imaging of reticulocytes following their incubation with MitoTracker Deep Red (MTDR)
and Alexa Green Transferrin (AGTf) and demonstrated transient interactions of endosomes with mitochondria. We also demonstrate these
interactions by a novel method exploiting flow sub-cytometry to analyze reticulocyte lysates labeled with MTDR and AGTf. This strategy
identified a population double-labeled with both fluorescent markers
representing endosomes interacting with mitochondria. FACS sorting
followed by 2D confocal microscopy confirmed the association of both
organelles in the double-labeled population. The results of these experiments as well as those exploiting a mutated recombinant Fe2-Tf unable
to release iron efficiently further support the hypothesis that the efficient iron transfer to PPIX requires a transient interaction between the
endosome and the mitochondrion.
2. Results
2.1. Three-dimensional confocal microscopy of live cells reveals
endosome-mitochondria interactions
Using two-dimensional (2D) confocal microscopy, we have previously shown that endosomes come in contact with mitochondria [31].
To avoid any pseudo-interactions of these two organelles, i.e., the endosome hovering over the mitochondria, we have used a Quorum WaveFx
spinning disc confocal system to capture three-dimensional (3D) live
cell imaging of reticulocytes with fluorescently labeled mitochondria
and endosomes. An individual cell at different time intervals is presented in three different orientations (Fig. 1, A–C) and shows the proximity
of endosomes and mitochondria at different angles (Link for Video;
please see Supplementary Material).
2.2. Development of the flow sub-cytometry method to study
endosome-mitochondria interactions
We developed a new method, “flow sub-cytometry”, based on the
standard flow cytometry method, to analyze lysates obtained from reticulocytes with fluorescently labeled mitochondria (MTDR) and
endosomes (AGTf). Employing this strategy, three distinct populations:
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), endosomemitochondria pairs are shown in three different orientations.
A. Hamdi et al. / Biochimica et Biophysica Acta 1863 (2016) 2859–2867
fluorescent markers have been identified (Fig. 2A). To confirm the interaction between endosomes and mitochondria, we used FACS-sorting to
isolate the population double-labeled with both fluorescent markers as
well as MTDR-labeled population (as a negative control), followed by
2D examination using a confocal fluorescence microscope. A multichannel protocol was used to scan the samples for AGTf and MTDR labeled
endosomes and mitochondria, respectively. The colocalization of the
two organelles was demonstrated by overlapping the green and the
red images of endosomes and mitochondria, where the merged images
represent both organelles together (Fig. 2B). This experiment has unequivocally demonstrated the presence of the Tf-endosome interacting
with the mitochondrion in the double-labeled population (UR). Importantly, the LR population contains exclusively MTDR-labeled mitochondria and the LL population is devoid of any fluorescence. Moreover, we
have shown that the size of the double-labeled population representing
endosomes associated with mitochondria (UR quadrant) increases with
incubation time with AGTf and reaches a plateau in ~20 min (Fig. 3).
A
E
EM
M
B
MTDR
AGTf
Merge
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2.3. Holotransferrin causes the detachment of endosomes from
mitochondria
To investigate whether the association of endosomes with mitochondria is reversible, we incubated reticulocytes with both fluorescent
markers for 20 min, washed the cells and reincubated with or without
holotransferrin (Fe2-Tf) for 60 min. The presence of non-fluorescent
Fe2-Tf caused a time-dependent decrease in the size of the doublelabeled population (Fig. 4A and B). Hence, AGTf-endosomes associated
with mitochondria are replaced by incoming non-fluorescent Fe2-Tfendosomes, indicating the reversibility of the interaction. AGTfendosomes leaving mitochondria cannot be detected by FACS because
of their small size. Importantly, this finding, together with the fact that
endosomes remain bound to mitochondria in the absence of Fe2-Tf, suggests that the Tf-endosome drives the detachment.
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 association. When bafilomycin A1, a specific V-ATPase inhibitor known to decrease 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 doublelabeled population (Fig. 5B). Heme has been shown to feedback inhibit
the release of iron from Tf within reticulocyte endosomes [39] and, similarly as bafilomycin A1, can be expected to increase endosomal Tf
saturation.
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 “traffic jam” that decelerates the movement of endosomes containing fluorescent Tf towards mitochondria.
Furthermore, the presence of heme during the reincubation of doublelabeled reticulocytes with unlabeled Fe2-Tf slowed down the disappearance of the double population (Fig. 5C), suggesting that the efficient dissociation of endosomes requires them to be iron-free.
UR(EM)
2.5. Calmodulin antagonist (W-7) decreases the size of double-labeled
population
MTDR
AGTf
Merge
LR(M)
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
and causes significant inhibition of 59Fe incorporation from 59Feendosomes 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
of 59Fe from 59Fe2-Tf
Fig. 2. Flow sub-cytometry method. (A) Using FACS analysis, four different populations in
lysates of reticulocytes, previously labeled with MTDR and AGTf for 20 min, were
identified: The lower left (LL) quadrant displays events devoid of all fluorescence (cell
debris). The upper left (UL) quadrant displays events labeled only with AGTf and,
therefore, represents the endosomal population. The lower right (LR) quadrant displays
events labeled only with MTDR and, therefore, corresponds to a mitochondrial population
(M). The upper right (UR) quadrant, labeled with both AGTf and MTDR, is referred to as
the double-labeled population representing endosomes interacting with mitochondria
(EM). (B) Populations present in UR, LR and LL quadrants were isolated by FACS-sorting
and then examined by 2D confocal microscopy. UR is composed only of AGTf-labeled
endosomes that colocalize with MTDR-labeled mitochondria. LR contains exclusively
MTDR-labeled mitochondria. The confocal images show four separate structures. Scale
bar is 5 μm.
To assess whether endosomes unable to release iron affect intracellular iron trafficking, reticulocytes were preincubated with Holo-rTf at
37 °C for 30 min, washed with cold PBS and incubated with 59Fe-Tf for
the indicated time intervals. After washing with cold PBS, total cell
and heme 59Fe-radioactivities were measured. The presence of HolorTf during incubation significantly inhibited 59Fe uptake by reticulocytes
from wild-type 59Fe-Tf and also diminished 59Fe incorporation into
heme (Fig. 6). This result suggests that the failure to release iron from
Holo-rTf interferes with the process of endocytosis or endosomal trafficking, causing a decrease in the total 59Fe uptake by the reticulocytes
as compared to controls.
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A
Time (min)
0
1
Endosomes (AGTf)
2
10
23.31%
30
20
27.75%
5
16.10%
10.01%
0.31%
31.47%
29.68%
Mitochondria (MTDR)
B
Percentage of total population
35
30
25
20
15
10
5
0
0
5
10
15
20
Time (min)
25
30
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
In this experiment, we used murine fetal liver (FL) cells, primary
mouse erythroid cells that closely recapitulate several aspects of terminal maturation in vivo, in particular very active hemoglobinization [8]
We deemed it important to ascertain whether endosomes interact
with mitochondria only in differentiating erythroid cells or if this phenomenon operates also before the onset of hemoglobin synthesis. As
predicted, subjecting these cells to flow sub-cytometry as performed
above with reticulocytes, revealed a large pool of the double-labeled
population in FL cells induced to differentiate by erythropoietin. Importantly, we also observed that the endosome-mitochondria population,
although smaller than in erythropoietin-induced FL cells, is present already in undifferentiated cells (Fig. S3).
3. Discussion
During differentiation, immature erythroid cells acquire vast amounts
of iron at a “breakneck” rate. Proper coordination of iron delivery and
utilization in heme synthesis is essential; disruption of this process likely
underlies iron loading disorders such as sideroblastic anemia and
myelodysplastic syndrome with ringed sideroblasts [28]. On a per-cell
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
producer in the body.
As described in the Introduction, most mammalian cells take up iron
via receptor-mediated endocytosis by the TfR. The prevailing opinion is
that after its escape from endosomes, iron spreads into the cytosol [40]
and mysteriously finds its way into mitochondria. Based on the fact that
Tf-bound iron is extremely efficiently used for hemoglobin synthesis,
targeted into erythroid mitochondria, and that no cytoplasmic iron
transport intermediate has ever been identified in erythroid cells, we
have suggested a new hypothesis of intracellular iron transport [17,
30]. This model proposes [17] that after iron is released from Tf in Hbsynthesizing cells, it would bypass the cytosol and be directly transferred from protein to protein (probably in hydrophobic environments)
until it reaches ferrochelatase in the mitochondrion.
Previous work from our laboratory has suggested that iron is directly
transferred from endosomes to mitochondria [31,32] likely through
interorganellar interaction. However, this model has not been accepted
by many [16,20]. In the present study, we used 2D and 3D confocal microscopy and developed a novel experimental strategy based on flow
cytometry analysis of fluorescently labeled organelles (“flow subcyometry”) to provide evidence for our hypothesis.
A. Hamdi et al. / Biochimica et Biophysica Acta 1863 (2016) 2859–2867
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A
B
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
us to capture fluorescent Tf-endosomes interacting with labeled mitochondria in space, excluding the possibility of “pseudo-interactions”
caused by a coincidental overlapping. This technique very clearly revealed contacts of endosomes with mitochondria in 3D space (Fig. 1).
Using a flow sub-cytometry to analyze lysates obtained from reticulocytes with fluorescently labeled mitochondria (MTDR) and endosomes
(AGTf), we have identified three distinct populations: endosomes, mitochondria, and a population double-labeled with both fluorescent markers
(Fig. 2A). The double-labeled population was sorted by FACS and shown
by 2D confocal microscopy to be composed of endosomes associated with
mitochondria (Fig. 2B). The size of the double-labeled population increases with the incubation time and reaches a plateau at approximately
20 min (Fig. 3).
The reversibility of the endosome-mitochondria association was
demonstrated by the experiment in which we first preincubated reticulocytes with both fluorescent markers for 20 min followed by their incubation (“chase”) with or without unlabeled holotransferrin (Fig. 4A and
B). The presence of unlabeled Fe2-Tf caused a time-dependent decrease
in the size of the double-labeled population indicating the reversibility
of the endosome-mitochondria contact. Importantly, in the absence of
holotransferrin, endosomes remain associated with mitochondria.
Taken together, our finding indicates that Tf-endosome drives the
detachment.
Both bafilomycin A1 and heme interfere with iron release from Tf and
cause a significant decrease in the size of the double-labeled population
(Fig. 5A and B). These agents obstruct Fe2+ export from endosomes already associated with mitochondria, extending the residence time of
endosomes on mitochondria, thus blocking the movement of endosomes
containing fluorescent Tf towards mitochondria. Additionally, hemin
slows down the dissociation of labeled endosomes from mitochondria
(Fig. 5C). Hence, our results suggest that a molecular mechanism exists
to coordinate the iron status of endosomal Tf with its trafficking.
Moreover, the presence of a Tf mutant unable to release iron (HolorTf) impairs the uptake of 59Fe from 59Fe2-Tf (Fig. 6), suggesting that efficient endocytosis and/or endosomal trafficking requires the transfer of
iron from endosomes to mitochondria. In other words, the endosomal
mitochondrial interface plays a role in regulating the cellular iron uptake, probably by controlling Tf endocytosis.
Our finding of endosomes interacting with mitochondria in undifferentiated fetal liver cells (Fig. S3) is of considerable importance. It suggests
that the endosome-mitochondrion interaction may be a common mechanism involved in iron delivery for heme synthesis. However, since FL
progenitors are committed to synthesize hemoglobin, further experiments with non-erythroid cells are needed to establish whether the
kiss-and-run mechanism is generally involved in the transport of Tfborne iron to mitochondria. If the so-called kiss-and-run mechanism of
iron delivery to mitochondria proves to be ubiquitous, then mitochondria
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7000
A
30
Control
25
Bafilomycin A1
20
15
10
5
0
5
10
Time (min)
B
15
20
Percentage of total population
**
5500
5000
4500
4000
3500
3000
2500
2000
1500
500
0
0
5
10
20
0
Control
60
50
40
30
20
10
5
10
15
20
25
30
Time (min)
100
80
60
40
Hemin
20
Control
0
0
10
10
20
Holo-rTf
Fig. 6. Holo-rTf inhibits 59Fe incorporation into reticulocytes and heme from 59Fe2-Tf. Error
bars were determined from triplicate values. ⁎⁎ p ≤ 0.01; ⁎⁎⁎ p ≤ 0.001.
Hemin
0
5
Time (min)
Control
0
Percentage of double-labeled
population
Total
Heme
6000
1000
0
C
***
6500
CPM / 500 million reticulocytes
Percentage of total population
35
20
30
40
50
60
Time (min)
Fig. 5. Bafilomycin A1 and hemin decrease the association of endosomes with mitochondria.
(A) The percentage of the double-labeled population after treatment with bafilomycin. ■,
Control; ♦, bafilomycin A1 (100 nM). Results presented are representative of 6 replicate
experiments. (B) The percentage of the double-labeled population after treatment with
hemin. ■, Control; ▲, hemin (5 μM). Results presented are representative of 4 replicate
experiments. (C) Hemin prevents the dissociation of labeled endosomes from
mitochondria. ■, Control; ▲, hemin (5 μM). Error bars were determined from triplicate
values.
may be the entry site of iron into the cell. In this context it needs to be
pointed out that the only known function of internalized Tf is to deliver
iron to its intracellular destination [31]. Hence, the described endosome
mitochondria association facilitates interorganellar iron transfer.
Our study is relevant to sideroblastic anemias, a heterogeneous
group of disorders, characterized by mitochondrial iron overload in developing red blood cells [26–28]. The unifying characteristic of all
sideroblastic anemias is the ring sideroblast, a pathological erythroid
precursor containing excessive deposits of non-heme iron in mitochondria with perinuclear distribution creating a ring appearance.
The majority of congenital sideroblastic anemias are X-linked; most of
them are caused by mutations in the gene encoding erythroid-specific 5aminolevulinate synthase [26–28]. However, there is a certain proportion
of patients with hereditary sideroblastic anemia who exhibit autosomal
recessive inheritance; some such patients have a defect in the gene
encoding the erythroid-specific mitochondrial carrier protein, SLC25A38
[41]. It has been suggested that SLC25A38 may translocate glycine into
mitochondria, a proposal recently confirmed by Fernández-Murray
et al. [42] and Lunetti et al. [43].
In our opinion, at least four factors are responsible for the pathogenesis of ring sideroblast formation in congenital sideroblastic anemias:
(i) As extensively discussed in this report, the efficient delivery of iron
to ferrochelatase in erythroid cells requires the direct interaction of
endosomes with mitochondria. (ii) Mitochondrial iron cannot be adequately utilized due to the lack of PPIX. (iii) Erythroid cells, but not
non-erythroid cells, are equipped with a negative feedback mechanism
in which ‘uncommitted’ heme inhibits iron acquisition from Tf [30]. Although the molecular mechanism of this inhibition is still unknown, the
lack of heme plays an important role in mitochondrial iron accumulation in erythroid cells. (iv) Iron can leave erythroid mitochondria only
after being inserted into PPIX [24].
As already alluded to, there is no consensus regarding the mechanism of iron transfer from plasma Tf to mitochondria. One of the proposed intermediates in the intracellular iron path is the iron storage
protein, ferritin [44,45]. Nevertheless, a strong argument against this
claim was collected and reported [46]. Somewhat surprisingly, it was
suggested in a recent report [47] that the autophagic turnover of ferritin
via NCOA4 is a critical process for regulating intracellular iron bioavailability. However, Darshan et al. [48] demonstrated that the conditional
deletion of the ferritin heavy chain in adult mice did not cause any decrease in hematocrit or hemoglobin levels, strongly indicating that ferritin is not involved in hemoglobinization. In this context it is pertinent to
mention that the nuclear receptor coactivator 4 (NCOA4), also known as
ARA70, was initially identified as a specific coactivator for the androgen
receptor in human prostate cells [49]. Additionally, another study [50]
has provided evidence that NCOA4 knockout is not lethal and NCOA4null mice have only a mild microcytic hypochromic anemia. These findings concur with our previous and current studies demonstrating that
the efficient utilization of iron for hemoglobin synthesis requires a direct contact of endosomes with mitochondria.
Finally, recent experimental support for the kiss-and-run hypothesis
came from a study that investigated the interaction between several mitochondrial heme biosynthetic enzymes and proteins involved in iron homeostasis [51]. This study revealed a partnership between ferrochelatase
(a mitochondrial protein) and TfR (an endosome associated protein) that
may be mediated by a yet to be identified bridge protein. It will be worth
examining whether one of the PCBPs could serve as such protein [19,21,
52]. Medlock et al. [45] further state, “(their) data…support a model of
transient protein-protein interactions within a dynamic protein
complex.” Our model, shown in Fig. 7, suggests a potential outer
A. Hamdi et al. / Biochimica et Biophysica Acta 1863 (2016) 2859–2867
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Fig. 7. Scheme of iron delivery to mitochondria for heme biosynthesis in erythroid cells. Iron is released from transferrin within endosomes by a combination of Fe3+ 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 intracellular loop of DMT1 for the delivery of iron to ferrochelatase.
In summary, our study provides strong evidence that the highly efficient delivery of iron to heme in hemoglobin-synthesizing cells requires
the direct interaction of transferrin-endosomes with mitochondria. To
the best of our knowledge, this is the first evidence of the interorganellar
interaction having the functional consequence of facilitating the transport
of a micronutrient. A full understanding of the molecular mechanisms involved in the endosome-mitochondria system will require the identification of the “signal(s)” that direct iron-carrying endosomes towards
mitochondria, the players involved in the docking of endosomes to mitochondria and the “signal(s)” that determine the detachment of iron-free
endosomes from mitochondria. We believe that the current work provides a strong platform to pursue such studies.
4. Materials and methods
4.1. Materials
MitoTracker Deep Red 633 (MTDR) and Alexa Green Transferrin 488
(AGTf) were purchased from Molecular Probes (Invitrogen, Carlsbad,
CA). Protease inhibitor was purchased from Roche (Penzberg,
Germany). Pronase, heparin and bafilomycin A1 were purchased from
Bioshop (Burlington, Ontario). Holotransferrin (Fe2-transferrin),
avertin, phenylhydrazine, thiozole orange and N-(6-Aminohexyl)-5chroro-1-napthalenesulfonamide hydrochloride (W7) were purchased
from Sigma-Aldrich (St. Louis, MO). Fluorescent mounting media were
purchased from Dako (Glostrup, Denmark). Microslides and micro coverslips were purchased from VWR (West Chester, PA). Minimum Essential Media (MEM) were purchased from Invitrogen (Gibco® Life
Technologies™, Carlsbad, CA) supplemented with 25 mM HEPES. Mitochondrial isolation buffer contains 220 mM mannitol, 60 mM sucrose,
20 mM HEPES, pH 7.4, 80 mM KCl, 2 mM magnesium acetate, 0.5 mM
EGTA and protease inhibitors. The human mutant recombinant Fe2-Tf
unable to release iron (K206E/K534A) [53,54] was generated in Dr.
Mason's laboratory.
4.2. Animals
All the animal studies performed in this work were approved by the
Lady Davis Institute animal care committee following the guidelines of
the Canadian Council on Animal Care.
CD1 and C57BL/6 mice (females; 6 months old) retired breeders
were obtained from Charles River Laboratories (Wilmington, MA) and
were housed in microisolator cages.
4.3. Isolation of reticulocytes
Adult, female CD1 female mice were injected with neutralized
pheynylhydrazine intraperitoneally at the dose of 50 mg/kg/day for
three consecutive days. Mice were then anesthetized after 2–3 days following the last injection by using 1 mL of 2.5% avertin. Blood was collected via direct cardiac puncture using heparinized syringe and
resuspended in cold PBS. Cells were then washed with ice-cold PBS
three times. By using this technique we obtained 30–45% reticulocytes
as verified by thiozole orange staining.
4.4. Cell culture of primary mouse fetal liver cells
Primary erythroid cells were cultured as described [8]. Briefly, cells
were grown from FL cells that were obtained from embryonic day
12.5 embryos of wild type C57BL/6 mice. FL cells were grown in
serum-free StemPro-34 medium plus Nutrient Supplement (Invitrogen,
Carlsbad, CA) and 2 mM L-glutamine, with 100 ng/mL murine recombinant stem cell factor (SCF), 10−6 M synthetic glucocorticoid dexamethasone (Dex), 40 ng/mL insulin-like growth factor 1 (IGF-1), 0.2% (vol/
vol) gentamicin, 0.2% amphotericin, and 2 U/mL human recombinant
erythropoietin (Epo). FL cells were kept either in an undifferentiated
state or induced to terminal differentiation (48 h) by re-suspending
them in differentiation medium (StemPro-34) containing Nutrient Supplement, 10 U/mL Epo, 2 mM L-glutamine, 4 × 10− 4 IU/mL insulin,
3 × 10− 6 M RU486, and 1 mg/mL iron-saturated human transferrin
(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
until their analysis by spinning disc confocal microscopy. The temperature of the chamber was kept at 37 °C. One-milliliter samples of reticulocytes suspension were resuspended in 50 μL of medium without
phenol red. Cells were then mounted on a pre-warmed (30 °C) heated
stage and were allowed to equilibrate for 15 min before imaging. Reticulocytes were not kept for more than 10 min in the chamber after exposing to laser. Three-dimensional images were acquired over time.
Optical sections of 0.5 μm were acquired in z-plane through a ± 2.5μm (total of 5.0-μm stack). Single endosome was tracked in a cell in different planes from the start of endocytosis to its interaction with mitochondria and leaving in a different plane. Microscopy was performed
with a Zeiss LSM 5 Pascal confocal inverted microscope. Live cell imaging of reticulocytes was done with Wave FX Spinning Disc Confocal
(SDC) microscopy by Quorum Technologies Inc. (Guelph, Ontario). Velocity 3D Image Analysis Software from PerkinElmer was used to analyze live cell imaging. Rapid imaging of these instruments makes it
possible to determine the 3D trajectory of an intra-cellular object.
A.H. and T.M.R. performed research, analyzed data, and wrote the
manuscript; T.M.K. performed research and analyzed data; A.B.M. and
A.D.S. analyzed data, and wrote the manuscript; and P.P. conceived
the study, analyzed data, and wrote the manuscript.
Conflict of interest
The authors declare no competing or financial interests.
Funding
The research of P.P. was funded by grants from the Canadian Institutes of Health Research (MOP-14100 and MOP-126064).
Transparency document
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. Reticulocytes were then incubated with 500 nM MTDR for 30 min at 37 °C in a
shaking water-bath following which they were washed 3-times with
ice-cold PBS and centrifuged at 800g for 5 min at 4 °C. Cells were resuspended in MEM containing 500 nM AGTf and kept on ice for
30 min in dark. After incubating with AGTf samples were divided and
incubated at 37 °C for the indicated time intervals. The samples were
then immediately placed on ice and washed three-times with ice-cold
PBS to stop endocytosis. The samples were treated with pronase
(1 mg/mL) on ice to remove any fluorescent probe bound to TfR present
on the cell surface. The cells were then washed three-times with icecold PBS, re-suspended in 1 mL of mitochondrial isolation buffer and
then subjected to four cycles of rapid freeze (liquid nitrogen) and
thaw. The samples were spun at 800 g for 10 min at 4 °C and supernatants were analyzed by flow cytometer (Fig. S1). Fluorescence signals
were determined using Becton Dickinson FACS Caliber (BD). AGTf 488
and MTDR 633 were determined by using a 530/30 and a 660/20 band
pass filter, respectively. Data was analyzed using FCS Express V3 software. Double labeled population were also sorted on a FACSAria sorter
(BD) followed by 2D examination in confocal microscopy. The effects
of bafilomycin A1, hemin and W7 were examined by their addition to
MTDR pre-labeled reticulocytes followed by the incubation with AGTf.
4.7. Iron uptake and incorporation into heme
59
Fe2-Tf was made from 59FeCl3 (PerkinElmer, Santa Clara, USA;
2 μCi) as described previously [55,56]. The acid precipitation method
was used to measure 59Fe in heme and non-heme fractions as described
previously [23,31,32]. Reticulocytes were collected by centrifugation
(4000g for 30 s), lysed in water and then boiled in 1 mL of 0.2 M HCL.
Samples were the transferred to an ice-bath and 59Fe-heme-containing
proteins were precipitated with ice-cold 7% trichoroacetic acid (TCA;
Bioshop Canada Inc., Burlington, Canada) solution, for 2 h and collected
by centrifugation (2800g for 5 min at 4 °C). Precipitated proteins (containing 59Fe in heme) and supernatants (containing non-heme 59Fe)
were separated in different tubes and radioactivity in heme and nonheme fraction was measured in a Packard Cobra gamma counter
(PerkinElmer, Santa Clara, USA.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.bbamcr.2016.09.008.
The authors are grateful to Dr. Heidi McBride for her valuable advice
and helpful suggestions during this research. The authors also thank
Drs. Julie Desbarats, Daniel Garcia-Santos, Matthias Schranzhofer,
Marc Mikhael and Volker Blank for their useful comments and Christian
Young and Dr. Lilian Amrein for their technical assistance with FACS
analyses and confocal microscopy.
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