Autophagy 8:7, 1150–1151; July 2012;
G
2012 Landes Bioscience
The ins and outs of human reticulocyte maturation
Rebecca E. Griffiths,2,3 Sabine Kupzig,2,3 Nicola Cogan,2,3 Tosti J. Mankelow,2,3 Virginie M.S. Betin,1 Kongtana Trakarnsanga,2
Edwin J. Massey,3 Stephen F. Parsons,2,3 David J. Anstee2,3,* and Jon D. Lane1,*
1
Cell Biology Laboratories; School of Biochemistry; University of Bristol; University Walk; Bristol UK; 2Bristol Institute for Transfusion Sciences; Bristol, Avon UK;
NHS Blood and Transplant; North Bristol Park; Filton, Bristol UK
3
T
Keywords: erythropoiesis, reticulocyte
maturation, autophagosome, endosome,
exosome
Submitted: 04/24/12
Revised: 05/04/12
Accepted: 05/04/12
http://dx.doi.org/10.4161/auto.20648
*Correspondence to: David J. Anstee or Jon D. Lane;
Email: [email protected]
or [email protected]
Punctum to: Griffiths RE, Kupzig S, Cogan N,
Mankelow TJ, Betin VM, Trakarnsanga K, et al.
Maturing reticulocytes internalise plasma membrane in glycophorin A-containing vesicles which
fuse with autophagosomes prior to exocytosis.
Blood 2012; 119:6296–306; PMID:22490681;
http://dx.doi.org/10.1182/blood-2011-09-376475
1150
he maturation of reticulocytes into
functional erythrocytes is a complex process requiring extensive cytoplasmic and plasma membrane remodeling,
cytoskeletal rearrangements and changes
to cellular architecture. Autophagy is
implicated in the sequential removal of
erythroid organelles during erythropoiesis,
although how this is regulated during late
stages of erythroid differentiation, and the
potential contribution of autophagy during
reticulocyte maturation, remain unclear.
Using an optimized ex vivo differentiation
system for human erythropoiesis, we have
observed that maturing reticulocytes are
characterized by the presence of one or few
large vacuolar compartments. These label
strongly for glycophorin A (GYPA/GPA)
which is internalized from the plasma
membrane; however, they also contain
organellar remnants (ER, Golgi, mitochondria) and stain strongly for LC3, suggesting
that they are endocytic/autophagic hybrid
structures. Interestingly, we observed the
release of these vacuoles by exocytosis in
maturing reticulocytes, and speculate that
autophagy is needed to concentrate the
final remnants of the reticulocyte endomembrane system in autophagosome/
endosome hybrid compartments that are
primed to undergo exocytosis.
We are exploiting protocols for the in vitro
expansion and differentiation of circulating adult human CD34+ stem cells to
generate transfusable quantities of functional reticulocytes/erythrocytes. Using a
three-stage culture system, adapted to
include a leukofiltration step to eliminate
Autophagy
nucleated cells and cellular debris (Fig. 1),
we can achieve a 104-fold erythroid
precursor expansion, with an enucleation
rate of 55–95%. From a 24 L culture, we
can recover 3 ¾ 1010 cells after filtration,
equating to around 5 mLs of packed cells.
In comparison with the volume of packed
erythrocytes in a unit of blood (~220 mL),
this demonstrates that the objective to
generate transfusable blood products from
in vitro differentiation approaches is
achievable, but with the need for significant scaling up. As part of this program,
we have been testing the influence of
autophagy during human erythroid
differentiation, and will be publishing
our findings in the near future; however,
presently we have investigated the involvement of autophagy during the maturation
of reticulocytes into functional erythrocytes in vitro.
Reticulocytes are generated following
enucleation of orthochromatic erythroid
cells, but they themselves then undergo a
process of maturation. To begin with,
while in the bone marrow, so-called R1
reticulocytes are motile and multilobular,
and usually retain some membrane organelles. These are active in endocytosis
and lack the stability/deformability characteristics of erythrocytes. R1 reticulocytes
mature into R2 types, which are nonmotile and much more mechanically
stable, and these exit the bone marrow
and enter the peripheral circulation. The
reticulocytes obtained following leukofiltration of our cultures contain adult
hemoglobin and display oxygen binding/
release and membrane deformability/
stability parameters consistent with mature
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Autophagy and the endosome/exosome pathway
Figure 1. Schematic of the generation of R2 reticulocytes from human CD34+ precursors. Passage of enucleated cells through a leukofilter eliminates
immature forms, nuclei and other cell debris, enriching for mature R2 reticulocytes. These undergo continued remodeling involving the formation and
subsequent exocytosis of large autophagic structures (LC3 positive) that also label for plasma membrane-derived GYPA.
R2 reticulocytes. Importantly, recent data
demonstrate that cultured reticulocytes
can be transfused into adult human
recipients, and remain functional in vivo,
indicating that these cells are comparable
with their in vivo counterparts. The
continuing differentiation of the R1
reticulocyte to become a functional
erythrocyte involves a ~20% loss of plasma
membrane surface area, reduction in
volume and degradation/elimination of
residual cytoplasmic organelles. The endosome-exosome pathway for release of
multivesicular bodies is expected to contribute to plasma membrane shedding/
remodeling,
meanwhile
autophagy
remains as a candidate pathway for the
elimination of residual organelles. How
these pathways are coordinated and
whether they are coregulated had not been
previously examined.
On close inspection, in vitro differentiated R2 reticulocytes often contain large
vacuolar inclusions, typically one or two
per cell. Ultrastructural analysis suggests
that these vacuoles have a single limiting
membrane and usually contain undegraded cytoplasmic material, including
membranous structures. At the light
microscope level, these inclusions are
always positive for the surface marker
GYPA, and in live-cell antibody uptake
experiments we showed that the GYPA is
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internalized from the plasma membrane
and gradually accumulates at the surface of
the vacuolar compartment. Significantly,
these compartments also label for markers
of the ER, Golgi and mitochondria,
suggesting that they do indeed contain
cytoplasmic material. Consistent with this,
they also stain for the autophagosomal
marker LC3. In contrast to the staining for
GYPA, which is restricted to the limiting
membrane, the organelle markers and LC3
clearly decorate structures contained
within the vacuolar compartment.
An intriguing feature of organelle
clearance in the differentiating red blood
cell is that this must take place in the
background of a declining endomembrane
system. This suggests that the consumption of membranes must be tightly
coordinated with the biogenesis of the
final population of autophagosomes,
although how this is regulated remains a
mystery. A related puzzle concerns the fate
of these remaining autophagosomes, since
data from our lab and from several others
suggest that the lysosomal compartment is
largely absent in reticulocytes. Exocytosis
of the final bolus of cytoplasmic material
seems the most likely route for elimination, and the extrusion of large vacuoles
containing cytoplasmic material has previously been observed in reticulocytes. In
the in vitro cultured human reticulocyte
Autophagy
populations, we observed frequent exocytosis of these large vacuolar compartments.
Indeed, analysis of human R2 reticulocytes
cultured for 7 d post-filtration suggests
that cells continue to lose volume over this
time, and that while levels of endocytosis
decline, the incidence of exocytosis of large
vacuoles concomitantly increases. This
suggests that one important aspect of
ongoing reticulocyte maturation is the
release of this residual material via exocytosis. Interestingly, we also observed
plasma membrane blebbing and shedding
in these cell populations, which may also
contribute to the reduction in plasma
membrane area and cellular volume in
maturing reticuolcytes. It is formally
possible that exocytosis is functionally
coupled to the blebbing/shedding process
in vivo, such that immediately following
exocytosis a bleb is formed and subsequently shedded, further reducing cell size.
In summary, our study suggests that
during the final maturation of reticulocytes,
autophagosomes combine with endosomes
to form large, GYPA-positive autophagic
compartments that fuse with the plasma
membrane to release their contents by
exocytosis. This suggests an intriguing
cooperation between the endocytic-exocytic
and autophagic systems; however, the
molecular players required for this pathway
have not yet been described.
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