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RED CELLS
Mechanism of protein sorting during erythroblast enucleation: role
of cytoskeletal connectivity
James C.-M. Lee, J. Aura Gimm, Annie J. Lo, Mark J. Koury, Sharon W. Krauss, Narla Mohandas, and Joel A. Chasis
During erythroblast enucleation, nuclei
surrounded by plasma membrane separate from erythroblast cytoplasm. A key
aspect of this process is sorting of erythroblast plasma membrane components
to reticulocytes and expelled nuclei. Although it is known that cytoskeletal elements actin and spectrin partition to reticulocytes, little is understood about
molecular mechanisms governing plasma
membrane protein sorting. We chose glycophorin A (GPA) as a model integral
protein to begin investigating proteinsorting mechanisms. Using immuno-
fluorescence microscopy and Western
blotting we found that GPA sorted predominantly to reticulocytes. We hypothesized that the degree of skeletal linkage
might control the sorting pattern of transmembrane proteins. To explore this hypothesis, we quantified the extent of GPA
association to the cytoskeleton in erythroblasts, young reticulocytes, and mature
erythrocytes using fluorescence imaged
microdeformation (FIMD) and observed
that GPA underwent dramatic reorganization during terminal differentiation. We
discovered that GPA was more connected
to the membrane cytoskeleton, either directly or indirectly, in erythroblasts and
young reticulocytes than in mature cells.
We conclude that skeletal protein association can regulate protein sorting during
enucleation. Further, we suggest that the
enhanced rigidity of reticulocyte membranes observed in earlier investigations
results, at least in part, from increased
connectivity of GPA with the spectrinbased skeleton. (Blood. 2004;103:
1912-1919)
© 2004 by The American Society of Hematology
Introduction
During mammalian erythroid terminal differentiation, the plasma
membrane and cytoskeleton are in a state of dynamic reorganization. We and others have determined that changes in protein
expression and membrane protein assembly occur that involve both
integral and skeletal membrane components.1-7 At the conclusion of
terminal differentiation, erythroblasts expel their nuclei and become reticulocytes. During enucleation, nuclei surrounded by
plasma membrane separate from erythroblast cytoplasm. A key
aspect of this process is the sorting of erythroblast plasma
membrane components to the plasma membranes of the nascent
reticulocyte and the expelled nucleus. Although approximately 2
million reticulocytes are generated each second, amazingly little is
known about molecular mechanisms governing protein sorting
during enucleation. It is known that actin, spectrin, tubulin,
ankyrin, and protein 4.1 partition to young reticulocytes, leaving
extruded nuclei devoid of skeletal elements.3,8-10 Earlier studies
also report that nonsialated glycoproteins are enriched in membranes of extruded nuclei, while sialoglycoproteins are enriched in
membranes of young reticulocytes.11 However, the redistribution of
a large number of well-characterized integral membrane proteins
and the mechanism(s) underlying their redistribution are unexplored.
Since glycophorin A (GPA) is a well-defined, major sialoglycoprotein in the mature erythrocyte (as reviewed in Chasis and
Mohandas12), we chose it as a model integral membrane protein to
begin investigating protein-sorting mechanisms during erythroblast
enucleation. GPA reaches maximum expression at the early erythroblast stage and is maintained at a constant amount per cell
throughout further differentiation.13 However, during the later
stages of human erythroid differentiation14 the polypeptide undergoes structural changes characterized by increased O-glycosylation. In
mature erythrocytes, GPA is a class I transmembrane protein
(800 000 copies per cell) that forms homodimers by association
between hydrophobic membrane-spanning domains.12,15-19 Fluorescence recovery after photobleaching (FRAP) analyses have shown
that approximately 35% of GPA is linked to the spectrin-based
skeleton,20 whereas approximately 65% of GPA is mobile in the
lipid bilayer. Although the molecular linkage connecting GPA with
the skeleton has not been definitively characterized, data accumulated by us and others suggest that GPA does not link directly to the
skeleton, but rather associates with it via band 3 and ankyrin. With
its abundant sialic acid content, GPA is the predominant cell
surface carrier of negative charge and thus may play a pivotal role
in minimizing cell-cell interactions and preventing red cell
aggregation.
During reticulocyte maturation, dramatic remodeling of both
the plasma membrane and the spectrin-actin membrane skeleton
continues characterized, for example, by loss of transferrin
receptors21,22 and by synthesis of skeletal protein 4.1R capable
of forming ternary complexes with spectrin and actin.4 Although
changes in surface components and surface area are well
From the Department of Biological Engineering, University of MissouriColumbia, Columbia, MO; the Life Sciences Division, University of California
Lawrence Berkeley National Laboratory, Berkeley, CA; Department of
Medicine, Vanderbilt University, Nashville, TN; and the New York Blood Center,
New York, NY.
and DK26263, and by the Director, Office of Health and Environment Research
Division, US Department of Energy, under Contract DE-AC03-76SF00098.
Submitted March 26, 2003; accepted September 30, 2003. Prepublished
online as Blood First Edition Paper, October 16, 2003; DOI 10.1182/blood-200303-0928.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Supported in part by National Institutes of Health grants DK32094, DK56267,
© 2004 by The American Society of Hematology
1912
Reprints: Joel Anne Chasis, Lawrence Berkeley National Laboratory, Building
74, 1 Cyclotron Rd, Berkeley, CA 94720; e-mail: [email protected]
BLOOD, 1 MARCH 2004 䡠 VOLUME 103, NUMBER 5
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BLOOD, 1 MARCH 2004 䡠 VOLUME 103, NUMBER 5
documented, little information exists regarding reorganization
of membrane protein–skeletal protein interactions. However,
significant reorganization must occur since we have previously
shown that young reticulocyte membranes are rigid and mechanically unstable compared with highly deformable and mechanically durable mature red cell membranes.23 We speculate that
enhanced reticulocyte membrane rigidity could result from a
greater number of vertical interactions linking the lipid bilayer
to the underlying spectrin skeleton, thereby decreasing the
ability of the spectrin network to undergo necessary rearrangements for membrane deformation.24,25
In this report, we investigated the molecular reorganization of
GPA during terminal erythroid differentiation. Using immunofluorescence microscopy and Western blotting we first quantitated the
redistribution of GPA during erythroblast enucleation and found
that GPA sorted predominantly to plasma membranes of young
reticulocytes. In order to understand the molecular mechanism of
GPA sorting during erythroblast enucleation, we quantified the
extent of GPA association to the cytoskeleton in erythroblasts,
reticulocytes, and mature erythrocytes using fluorescence imaged
microdeformation (FIMD) and observed that GPA underwent
dramatic reorganization during terminal erythroid differentiation.
We discovered that GPA was more connected to the membrane
cytoskeleton in erythroblasts and young reticulocytes than in
mature cells. We conclude that skeletal protein association can
regulate protein sorting during enucleation. Further we suggest
that the enhanced rigidity of reticulocyte membranes results, at
least in part, from increased connectivity of GPA with the
spectrin-based skeleton.
PROTEIN SORTING DURING ERYTHROBLAST ENUCLEATION
1913
Pharmingen, San Diego, CA) diluted in the milk solution to 200 ng/mL,
anti–erythroid macrophage protein (EMP; a gift from Dr Manjit Hanspal, St
Elizabeth’s Medical Center, Boston, MA) diluted in the milk solution to 1
␮g/mL, or anti-CD29 (␤1 integrin; BD Pharmingen) diluted in the milk
solution to 0.5 ␮g/mL was added and incubated for one hour at room
temperature. Then the membrane was washed with 0.05% Tween20-PBS 4
times for 10 minutes and incubated for one hour with either horseradishconjugated donkey anti–rat immunoglobulin G (IgG) at 0.53 ␮g/mL
(Jackson Immuno Research, West Grove, PA) for Ter119 and anti-CD29 or
horseradish-conjugated donkey antirabbit IgG at 1 ␮g/mL (Jackson Immuno Research) for anti-EMP. The membrane was then washed as
previously and proteins were visualized on X-omat Blue xb-1 Kodak
imaging film (Perkin Elmer Life Sciences, Boston, MA) by chemiluminescence reagent (Western Lightning; Perkin Elmer Life Sciences).
Labeling of F-actin and lipid
To label the internal skeletal network, red cells were reversibly permeabilized by cold, hypotonic lysis allowing molecules in the lysis buffer to
diffuse into the permeabilized cell and bind.29-32 Labeling of skeletal actin
with rhodamine phalloidin (Molecular Probes, Eugene, OR) was accomplished by first air-drying 5 ␮L of 6.6-␮M rhodamine phalloidin in MeOH
and then redissolving the phalloidin in (10-15 ␮L) cold lysis buffer (10 mM
phosphate, pH 7.4). Cold, packed red cells (5 ␮L) were added and, after 5
minutes, the suspension was made 100 mM in KCl, 1 mM in MgCl2, and
then warmed at 37°C for 20 to 60 minutes. To label the lipid bilayer of cells,
15 ␮L of 6.6-␮M/mL Texas-red–1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (TR-DHPE; Molecular Probes) in MeOH was added to 1 mL
cell suspension in culture medium and incubated for 20 minutes at room
temperature. Excess TR-DHPE was removed by washing cells 3 times by
centrifugation with culture medium.
Labeling of GPA
Materials and methods
Bone marrow and FVA cells
Bone marrow cells were flushed with a 25-gauge needle from mouse femurs
and tibias into Iscove modified Dulbecco medium (GibcoBRL Products,
Rockville, MD) and 5% fetal calf serum (GibcoBRL Products) (hereafter
referred to as culture medium), and then washed by centrifugation. FVA
cells were isolated and cultured using methods established by Koury et
al.26,27 Briefly, CD2F1 or BALB/c mice were infected with 1 ⫻ 104 spleen
focus-forming units. Cells were harvested from spleens 2 weeks later and
separated by velocity sedimentation at unit gravity (pooled cells sedimenting at approximately 6 mm/h or greater). Cell separation yielded approximately 5 ⫻ 108 cells per spleen, more than 95% of which were proerythroblasts. When cultured with 1.0 U/mL recombinant erythropoietin over 48
hours, proerythroblasts proliferated and differentiated to yield approximately 2 ⫻ 109 late erythroblasts per spleen. Finally, a 1% to 2% albumin
gradient was used to purify the populations of young reticulocytes and
extruded nuclei by size and density fractionation.28 Approximately 3 ⫻ 108
young reticulocytes and 3.5 ⫻ 108 extruded nuclei were available from
each spleen. Total number of nucleated cells was determined using
methylene blue staining. Cell viability was quantified by trypan blue
exclusion.
GPA was labeled with either fluorescein-5-thiosemicarbazide (FTSC;
Molecular Probes) as previously described with minor modifications20,33 or
a GPA-specific antibody labeled with phycoerythrin (PE-Ter11934; BD
Pharmingen). To label GPA with FTSC, cells were suspended in 1 mL PBS.
A mild oxidation was produced by adding NaIO4 to a final concentration of
0.5 mM and the cell suspension was incubated at 0°C in the dark for 12
minutes. NaIO4 was removed by washing cells twice with culture medium,
followed by incubating cells for 30 minutes at room temperature in culture
medium containing 50 ␮g/mL FTSC. Excess FTSC was removed by
washing cells 3 times with culture medium. To label GPA with PE-Ter119,
cells were incubated in culture medium containing 1 ␮g/mL PE-Ter119.
Excess PE-Ter119 was removed by washing cells 3 times with culture medium.
Labeling of band 3
Band 3 was labeled in situ with eosin-5-maleimide (EMA; Molecular
Probes). To prevent echinocyte formation, 10 ␮L normal mouse red cells or
ankyrin-deficient mouse red cells from nb/nb mice (a gift from Dr Jane
Barker, The Jackson Laboratory, Bar Harbor, ME) were suspended and
washed 3 times at 4°C in 1 mL PBS with 0.05 g/percent bovine serum
albumin (BSA; Sigma, St Louis, MO). The cells were then incubated at
room temperature for 30 minutes in EMA (50 ␮g/mL) dissolved in
PBS/BSA and then washed 3 times in PBS/BSA buffer.35
Western blot analysis
Nuclei and reticulocytes were solubilized in sample buffer containing 3%
sodium dodecyl sulfate (SDS) and 10% dithiothreitol (Invitrogen, Carlsbad,
CA) and then boiled for 10 minutes. The fractions (5 ⫻ 105 nuclei or
reticulocytes per lane) were separated on a 10% to 15% SDS–
polyacrylamide gel (PAGE; BioRad, Hercules, CA) and blotted to either
nitrocellulose or polyvinylidene fluoride membrane. After blocking with
5% nonfat dry milk (Safeway, Pleasanton, CA), 0.05% Tween20–phosphatebuffered saline (PBS), pH 7.4, for one hour, primary antibody Ter119 (BD
Fluorescence imaged microdeformation
Fluorescence imaged microdeformation (FIMD), which couples fluorescence microscopy with micropipette aspiration of cells, was used to
examine the organization of membrane proteins. Detailed description of
this technique has been reported.32,36 In brief, micropipette cell deformation
was conducted in an open-sided chamber on a microscope stage. A
micropipette (diameter, approximately 1.5 ␮m) was inserted into the
chamber horizontally (ie, perpendicular to the optical axis) to aspirate a cell
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1914
LEE et al
BLOOD, 1 MARCH 2004 䡠 VOLUME 103, NUMBER 5
(diameter, approximately 5-8 ␮m). The micropipette was precisely controlled by a joystick manipulator. A manometer system equipped with a
sensitive pressure transducer was also built in the system to control and
monitor the pressure across the entrance of the micropipette.
The microscope system was based on an inverted Nikon TE300 (Nikon,
Tokyo, Japan), and the imaging device was a circulating-liquid–cooled,
charged-coupled device (CCD) camera controlled with a computer that ran
V⫹⫹ imaging software (Digital Optics, Auckland, New Zealand). Fluorescence and transmission illumination sources were 100 W mercury arc and
150 W metal halide lamps, respectively. The fluorescence source was
shuttered with a Uni-Blitz mechanical shutter, which was synchronous with
a second shutter exposing the CCD; the typical exposure time for
fluorescence microscopy was set between 200 and 300 milliseconds.
Background subtractions were done for all the images for quantitative analysis.
Results
Sorting pattern of GPA during enucleation
In order to determine the sorting pattern of GPA during erythroblast
enucleation, freshly obtained murine bone marrow cells were
fluorescently labeled with a GPA-specific antibody, PE-Ter119,34
and imaged by both bright field and fluorescence microscopy.
Under bright field illumination, the portion of the enucleating
erythroblast destined to become a reticulocyte, as well as the young
reticulocyte, appeared multilobular, while extruding and extruded
nuclei were spherical (Figure 1). Fluorescence microscopy of
PE-Ter119–labeled GPA in enucleating erythroblasts showed that
GPA localized to the portion of the erythroblast membrane destined
to become a reticulocyte (Figure 1). In the same samples multilobulated reticulocytes showed membrane-associated GPA (Figure 2A).
The observed clustering of fluorescence signal in the erythroblast
and reticulocyte membranes is most likely due to membrane
ruffling and folding since there were no patches of labeled GPA in
the reticulocyte (Figure 3) or erythroblast (data not shown) when
the membrane was smoothed out by micropipette aspiration.
Figure 1. Micrographs of PE-Ter119–labeled enucleating erythroblasts from
mouse bone marrow and FVA cell culture. (A) Bright field illumination of
enucleating erythroblasts, including nascent reticulocytes (black arrows) and extruding nuclei (white arrows). (B) Fluorescence microscopy of enucleating erythroblasts.
Fluorescent intensities in nascent reticulocytes were greater than those in extruding
nuclei, which indicated that GPA predominately partitions to reticulocytes during
erythroblast enucleation. For scale reference, the diameter of the nucleus is 4.4 ␮m.
Figure 2. Micrographs and Western blots of extruded nuclei and reticulocytes.
(A) Bright field illumination (top panels) and fluorescence microscopy (bottom panels)
of an extruded nucleus from FVA cell culture, a PE-Ter119–labeled reticulocyte from
FVA cell culture, and a PE-Ter119–labeled reticulocyte from bone marrow. Retic
indicates reticulocyte. For scale reference, the diameter of the nucleus is 4.4 ␮m.
(B) Nuclear and reticulocyte proteins from 5 ⫻ 105 nuclei and 5 ⫻ 105 reticulocytes
from FVA cell cultures, separated on a SDS-PAGE gel, were immunoblotted using
monoclonal antibody Ter119. Results from both fluorescence microscopy and
Western blot analysis showed that GPA sorted predominantly to reticulocytes.
(C) Nuclear and reticulocyte proteins from 5 ⫻ 105 nuclei and 5 ⫻ 105 reticulocytes
from FVA cell cultures, separated on a SDS-PAGE gel, were immunoblotted using
monoclonal antibodies specific for EMP or ␤1 integrin subunit. Both EMP and ␤1
partitioned predominantly to nuclei.
To confirm that GPA partitioned predominantly to reticulocytes,
it was critical to analyze extruded nuclei. Since expelled nuclei
rapidly undergo phagocytosis by marrow macrophages, they are
rarely observed in primary bone marrow cell suspensions. We,
therefore, analyzed extruded nuclei and young reticulocytes obtained from Friend virus–infected mouse proerythroblasts differentiated in culture (FVA cells). This carefully characterized model
system of terminal erythroid differentiation closely mimics in vivo
erythropoiesis.26,27 After 44 hours in culture, the cell population is
composed of well hemoglobinized and enucleating erythroblasts,
young reticulocytes, and expelled nuclei. GPA in FVA cells
cultured for 44 hours was fluorescently labeled with PE-Ter119,
Figure 3. Micrographs of a micropipette-aspirated, PE-Ter119–labeled reticulocyte. (A) Bright field illumination of a micropipette ready to aspirate a multilobular,
young reticulocyte. (B) Bright field illumination of aspirated reticulocyte showing that
the multilobular structure of the reticulocyte membrane could be smoothed out by
aspirating the cell into the micropipette. (C) Fluorescence microscopy of the aspirated
PE-Ter119–labeled reticulocyte showed a density gradient of GPA along the deformation projection. For scale reference, the diameter of the micropipette is 1.5 ␮m.
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BLOOD, 1 MARCH 2004 䡠 VOLUME 103, NUMBER 5
and expelled nuclei and young reticulocytes were analyzed. We
observed a marked difference in GPA staining with the sialoglycoprotein being present in young, multilobulated reticulocyte membranes but barely detected in extruded nuclei (Figure 2A).
To quantify the relative concentrations of GPA in plasma
membranes of reticulocytes and extruded nuclei, the fluorescence
intensity of PE-Ter119–labeled GPA in young reticulocytes and
extruding nuclei from primary mouse marrow was measured and
normalized per unit surface area. Micropipette aspiration was
applied to smooth out the multilobular reticulocyte membrane and
to estimate the average surface area37 (Figure 3). The area of the
aspirated reticulocyte was calculated using the following equation:
A ⫽ ␲[4Ro2 ⫺ RP2(1 ⫺ 2LP/RP)], where RP, Ro, and LP are the
inner radius of the micropipette, radius of the outer spherical part of
the cell, and the projection length of the aspiration, respectively.
Extruding nuclei were assumed to be spheres (ie, A ⫽ ␲D2, where
D is the diameter of the nucleus). The average surface area of
reticulocytes was calculated to be 83 ⫾ 3 ␮m2 (n ⫽ 10), whereas
the average surface area of nuclei was 60 ⫾ 1 ␮m2 (n ⫽ 10). After
normalization per unit surface area, the relative concentration of
GPA in plasma membranes of nascent reticulocytes was approximately 6-fold higher than in plasma membranes of extruding
nuclei. Of the total GPA, 85% distributed to reticulocytes. Results
from both freshly harvested bone marrow reticulocytes and reticulocytes from 44-hour FVA cell cultures were consistent.
In parallel experiments, we quantified relative amounts of GPA
in purified populations of extruded nuclei and immature reticulocytes by Western blotting. FVA cells cultured for 44 hours were
separated by size and density on an albumin gradient and then
proteins from an equivalent number (5 ⫻ 105) of nuclei and
immature reticulocytes were separated by SDS-PAGE and analyzed on Western blots probed with Ter119. When the densitometry
data were normalized with the surface area of nuclei and reticulocytes, we found that 75% of GPA partitioned to reticulocytes
(Figure 2B), consistent with the results from fluorescence microscopy. Together these data clearly show that GPA partitions predominantly to the reticulocyte during erythroblast enucleation. Its
sorting pattern is, therefore, similar to that of cytoskeletal
components.3,8
To determine whether there are significant variations in partitioning of different membrane proteins during enucleation, we quantified the relative amounts of several other erythroblast surface
components in Western blots of purified populations of extruded
nuclei and immature reticulocytes from cultured FVA cells. In
contrast to GPA, which partitioned predominantly to reticulocytes,
75% of EMP38 and 70% of ␤1 subunit of ␣4␤1 and ␣5␤1 integrins39
partitioned to nuclei (Figure 2C). These findings provide conclusive evidence that there is, indeed, differential sorting of membrane
proteins during enucleation.
PROTEIN SORTING DURING ERYTHROBLAST ENUCLEATION
1915
have different membrane properties, has not been systematically
studied. To validate the applicability of FIMD on mouse erythroid
cells, we analyzed membrane lipids and skeletal F-actin in mature
mouse red cells by FIMD and compared the results with those
previously obtained for human red cells.
The lipid bilayer component PE, labeled by incorporating a
Texas-red–labeled PE (TR-DHPE) in the mouse red cell membrane, exhibited a uniform and flat intensity profile along the
membrane projection (Figure 4A). The ratio of the fluorescence
intensity at the entrance to the fluorescence intensity at the cap
(␳e/␳c) was measured to be approximately 1.09 for different
projection lengths, indicating the fluidity of the lipid bilayer. In
contrast, the intensity profile of the solidlike cytoskeletal component, F-actin, exhibited a steep gradient along the projection (not
shown). When the cell is not deformed (ie, the normalized
projection length, L/Rp, is zero), ␳e/␳c is equal to one. With this
boundary condition, the empiric relationship between ␳e/␳c and
L/Rp can be obtained by fitting the data to the following equation:
␳e/␳c ⫽ 1 ⫹ ␥ (L/Rp) , where ␥ is the slope. ␥ of F-actin in mouse
red cells was measured to be ⫹ 0.47, approximately only 14%
different from that of human red cells (Figure 4B-C).36 In this
analysis the derived value for ␥ serves as an indicator of the degree
of connectivity between a membrane protein and the cytoskeleton;
as ␥ of a particular integral protein approaches ␥ of F-actin, a
higher connectivity of the protein to the cytoskeleton is indicated.
Since FIMD analyses showing the distribution of actin and lipid
following deformation of mouse cells were similar to that of human
cells, we applied this technique to investigate the connectivity of
GPA molecules in differentiating mouse erythroid cells.
Characterization of mouse red cell membranes by FIMD
Since we33 and others20 previously demonstrated that approximately only 35% of GPA is skeletal associated in the mature
erythrocyte, we reasoned that if skeletal association regulates
protein sorting during enucleation, then more GPA would be
skeletal associated in erythroblasts and young reticulocytes than in
mature erythrocytes. FIMD, a technique for quantitating connectivity of plasma membrane proteins to the cytoskeleton in situ, has
been applied to characterize the organization of human red cell
membranes.32,36 However, the application of this technique to
mouse cells, which are smaller than human erythrocytes and may
Figure 4. Application of fluorescence imaged microdeformation (FIMD) to
murine erythrocytes. (A) Erythrocyte with fluorescently labeled lipid bilayer is
aspirated by a micropipette (upper panel). For scale reference, the diameter of the
micropipette is 1.5 ␮m. The lipid bilayer exhibited a uniform density profile along the
deformation projection, indicating its fluidity (lower panel). ␳e and ␳c are defined as the
densities at the pipette entrance and at the cap of the projection, respectively.
(B) ␳e/␳c is plotted against the dimensionless projection length, L/Rp for the FIMD of
F-actin and the lipid bilayer of mouse red cells. L is the projection length and Rp is the
radius of the pipette. Data were fitted by straight lines with a y-intercept ⫽ 1. ␥ is
defined as the slope of the FIMD analysis and indicates the degree of cytoskeletal
association. As ␥ of a protein approaches ␥ of F-actin, a greater degree of
cytoskeletal association is indicated. (C) Histogram showing the ␥ of lipid bilayer and
cytoskeletal F-actin.
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1916
LEE et al
BLOOD, 1 MARCH 2004 䡠 VOLUME 103, NUMBER 5
Figure 5. Degree of cytoskeletal attachment of FTSC-labeled GPA. ␳e/␳c is plotted against the dimensionless projection length, L/RP for the FIMD of FTSC-labeled GPA in
erythroblasts (A), reticulocytes (B), and mature red cells (C). L is the projection length and Rp is the radius of the pipette. Data were fitted by straight lines to calculate ␥GPA of
FTSC-labeled GPA in erythroblasts (Eb), reticulocytes, and mature red cells. ␥GPA of FTSC-labeled GPA decreased progressively during red cell differentiation and reticulocyte
maturation (D).
Connectivity of GPA to the cytoskeleton during erythroid
differentiation
To compare the organization of GPA in the membrane in mouse
erythroblasts, young reticulocytes, and mature erythrocytes, we
determined the fluorescence intensity profiles of FTSC-labeled
GPA in these cell types in mouse bone marrow or FVA samples and
calculated the degrees of connectivity of GPA to the spectrin-based
skeleton. Although micropipette methods have been previously
used to study human reticulocytes,23 micropipette aspiration of
reticulocytes is technically challenging because of their rigidity,
multilobular form, and adhesiveness to glass surfaces. However
with carefully controlled aspiration pressures, the multilobular
structure of reticulocytes can be smoothed out (Figure 3), while
addition of approximately 20% BSA to the suspending medium
minimized adhesion. The fluorescence intensity profile of FTSClabeled GPA in erythroblasts (Figure 5A) exhibited a steeper
gradient than FTSC-labeled GPA in reticulocytes (Figure 5B) or
mature red cells (Figure 5C). We observed more heterogeneity in
the data set for erythroblasts than in the data sets for either
reticulocytes or mature cells, most likely reflecting differences in
erythroblast age. As illustrated in Figure 5D, FTSC-labeled GPA in
erythroblasts, reticulocytes, and mature red cells exhibited intensity
profiles with derived values of ␥GPA of 0.37, 0.25, and 0.05,
respectively. The ␥GPA of erythroblasts indicated a significant
connectivity between GPA and the cytoskeleton, and the value was
1.5-fold higher than that of reticulocytes from bone marrow and
FVA cells, which were identical. Moreover, more GPA molecules
were connected to the skeleton in young reticulocytes than in
mature cells (␥GPA ⫽ 0.25 and 0.05, respectively). These data
strongly suggest that GPA connectivity decreases during both
erythroblast differentiation and reticulocyte maturation.
To rule out the possibility that aggregates of GPA in erythroblasts might produce FIMD data interpreted as increased connectivity, we compared the intensity profiles of large molecular complexes with and without skeletal attachment. For these experiments
we analyzed band 3 as an example of a known component of a large
membrane protein complex, since recent data demonstrate the
presence of a band 3–based macrocomplex of integral and peripheral proteins linked to the skeleton via ankyrin.40 We, therefore,
determined the fluorescent intensity profile of EMA-labeled band 3
in normal mouse red cells and compared it with that in red cells
from nb/nb mice deficient in erythroid ankyrin.41 We observed that
in ankyrin-deficient mouse red cells EMA-labeled band 3 collected
at the cap of the membrane projection, as seen by the reverse
gradient and higher fluorescence intensity at the cap than at the
entrance of the pipette (Figure 6). These findings indicate a loss of
band 3 connectivity with the skeleton in ankyrin-deficient red cells.
The redistribution of band 3 is similar to that observed for
glycophorin C (GPC) on 4.1-deficient red cells.32 Thus, in the
absence of their skeletal binding partners, both band 3 and
glycophorin C accumulate at the cap of the membrane projection
upon micropipette aspiration. Our current data on band 3 illustrate
that large molecular complexes can decrease connectivity if
unlinked to the skeleton. These findings indicate that the observed
increased connectivity of GPA in erythroblasts does not result from
molecular aggregates containing GPA.
Effect of Ter119 binding on the connectivity of GPA to
the cytoskeleton
We have previously observed that antibody binding to GPA on
human red cells induces membrane rigidification and decreases the
mobile fraction of GPA.25,33,42 To explore whether antibody binding
Figure 6. Redistribution of band 3 on normal and ankyrin-deficient (nb/nb) red cells. (A) Fluorescence micrograph of normal erythrocyte with EMA-labeled band 3
aspirated by a micropipette. (B) Corresponding intensity profile of band 3 exhibited a steep gradient in concentration along the deformation projection, indicating that the density
of EMA-labeled band 3 increased markedly at the pipette entrance and subsequently decreased toward the aspiration cap. ␳e and ␳c are defined as the densities at the pipette
entrance and at the cap of the projection, respectively. (C) Fluorescence micrograph of ankyrin-deficient erythrocyte with EMA-labeled band 3 aspirated by a micropipette.
(D) Without ankyrin, band 3 collected at the cap as seen by the reverse gradient and higher fluorescence intensity at the cap. For scale reference for panels A and C, the
diameter of the micropipette is 0.8 ␮m.
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BLOOD, 1 MARCH 2004 䡠 VOLUME 103, NUMBER 5
Figure 7. Degree of cytoskeletal attachment of nonliganded and liganded GPA
in reticulocytes and mature red cells. (A) FTSC-labeled GPA in reticulocytes from
FVA cell culture; (B) FTSC-labeled, Ter119-liganded GPA in reticulocytes from FVA
cell culture; (C) PE-Ter119–liganded GPA in reticulocytes from FVA cell culture; (D)
FTSC-labeled GPA in mature red cells; and (E) PE-Ter119–liganded GPA in mature
red cells. Ligand binding increased ␥GPA in both reticulocytes and mature red cells.
to GPA in mouse red cells and reticulocytes has a similar effect, we
measured ␥GPA for Ter119-liganded mouse cells. ␥GPA of phycoerythrin-Ter119–liganded mature red cells was 8-fold greater than
that of nonliganded FTSC-labeled mature mouse red cells (Figure
7), indicating that the connectivity of GPA to the cytoskeleton in
mature mouse red cells can be enhanced by ligand binding of GPA
and that liganded GPA in mouse and human erythrocytes behaves
similarly. Interestingly, ␥GPA in FVA reticulocytes with either
PE-Ter119–liganded GPA or FTSC-labeled GPA liganded with
unlabeled Ter119 was approximately 1.6-fold greater than that of
nonliganded reticulocytes (Figure 7), indicating that antibody
ligand binding to GPA on murine reticulocytes increases the
association of GPA to the cytoskeleton. Of note, ␥GPA values of
liganded mature cells and liganded reticulocytes were similar even
though the preliganded connectivity of GPA was greater in
reticulocytes than in mature cells. The finding that ␥GPA values for
both liganded reticulocytes and mature red cells approach the value
for actin suggests that ligand binding induces the connectivity of
membrane GPA to the membrane skeleton.
Discussion
During the dramatic process of enucleation, erythroblasts’ integral
and GPI-linked proteins, as well as the spectrin-based skeleton, are
redistributed to the plasma membranes surrounding expelled nuclei
and young reticulocytes. A striking finding of the current report is a
quantitative documentation of the partitioning of the major integral
protein GPA to plasma membranes of young reticulocytes. Quantification of GPA in young reticulocytes and expelled nuclei by live
cell immunofluorescence microscopy of freshly harvested mouse
bone marrow or 44-hour FVA cell cultures and Western blot
analysis of purified populations of young reticulocytes and extruded nuclei gave consistent results—75% to 85% of GPA sorted
to reticulocytes. We are confident that Ter119 binding did not affect
GPA sorting in live cell studies of enucleating erythroblasts since
Western blot data from purified populations of nuclei and reticulocytes, which had enucleated in the absence of any Ter119 antibody,
were consistent with live cell data.
The molecular mechanisms of protein sorting that regulate
protein content of plasma membranes of reticulocytes and extruding nuclei have not been previously investigated. We hypothesized
that the presence or absence of skeletal linkage might control the
sorting pattern of transmembrane proteins. As earlier studies
document that components of the spectrin-based skeleton partition
PROTEIN SORTING DURING ERYTHROBLAST ENUCLEATION
1917
to reticulocytes,3,8 we predicted on the basis of our current data that
the majority of GPA molecules would be linked to the membrane
skeleton at the time of enucleation.
To test our hypothesis, FIMD was applied to monitor the
association between GPA and the skeleton at different stages of
differentiation. A major finding of our current study is that GPA
molecules undergo marked reorganization during terminal erythroid differentiation and reticulocyte maturation. Prior investigations documented the expression and membrane assembly of
integral and skeletal proteins during erythropoiesis, but did not
address issues of molecular organization and specific proteinprotein interactions in differentiating cells. The technique of FIMD
measures redistribution of integral and skeletal proteins relative to
each other in highly extended membranes, thereby providing
insight into in situ protein linkages. This method provides direct
evidence regarding the connectivity of membrane proteins with the
membrane skeleton.32,36 An important basis for this conclusion is
that we have studied the connectivity of glycophorin C (GPC),
which is normally skeletally associated via interaction with protein
4.1, and have been able to document complete loss of connectivity
of GPC in 4.1-deficient cells.32 Moreover, extracellular interconnections do not change connectivity as shown, for example, in studies
in which the connectivity of GPC was the same whether it was
bound with a monovalent or bivalent IgG.32,36 Although this
method has been elegantly applied to human erythrocytes, it has
not been used previously to study mouse cells. We, therefore,
validated the applicability of FIMD to mouse erythroid cells and
then used it to study the connectivity of GPA molecules to the
cytoskeleton in situ in mouse erythroblasts, young reticulocytes,
and mature red cells. Although FTSC labels all glycophorins in
proportion to their sialic acid content, mouse cells express only
homologs of human GPA and GPC. Based upon relative copy
number we calculate that more than 90% of FTSC labeling was
associated with GPA. Our findings that ␥GPA was highest in the
erythroblast, decreased 1.6-fold between the erythroblast and
young reticulocyte stages, and decreased a further 5-fold during
reticulocyte maturation clearly show that during terminal differentiation progressively fewer GPA molecules associate with the
spectrin-based skeleton. Importantly, the large proportion of GPA
molecules associating with the membrane skeleton in erythroblasts
and young reticulocytes strongly suggests that the GPA sorting
pattern during enucleation is regulated by GPA’s connectivity to the
skeleton. These data add credence to our hypothesis that skeletal
linkage controls, at least in part, the sorting pattern of transmembrane proteins.
An unresolved question is the identity of the protein linking
GPA to the membrane skeleton. Data collected by us and others
strongly suggest that GPA does not interact directly with the
skeleton but rather associates with band 3, which in turn binds to
the skeleton via ankyrin. Specifically, (1) during membrane biogenesis, membrane assembly of GPA and band 3 appear to be
interdependent since Xenopus oocytes43,44 and yeast45 cotransfected with band 3 and GPA cDNAs assemble more band 3 on their
surface than those transfected with band 3 alone; (2) Wrb antigen
expression requires the presence of both GPA and band 3 on the
erythrocyte surface46,47 and appears to involve an interaction
between E658 of band 3 and amino acid residues within the
extracellular ␣-helical domain and transmembrane junction of
GPA48,49; (3) band 3 null mice fail to assemble GPA on their
membranes,50 and surface expression levels of GPA and band 3 are
tightly coupled in human GPA transgenic mice34; and (4) antibody
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1918
BLOOD, 1 MARCH 2004 䡠 VOLUME 103, NUMBER 5
LEE et al
binding to the extracellular domain of human GPA decreases the
lateral mobility of both GPA and band 3.33 Our current data show
that antibody binding to the extracellular domain of mouse GPA
increases GPA connectivity to the skeleton, indicating that liganding GPA has a similar effect in mouse and human cells. Remarkably, the ␥GPA of liganded mature mouse cells and liganded mouse
reticulocytes was identical despite the fact that in the absence of
antibody binding, connectivity of GPA was 5-fold greater in
reticulocytes than in mature cells.
It is well documented that levels of expression of integral
proteins change late in erythropoiesis with, for example, beginning
expression of the laminin receptor, Lutheran glycoprotein,51 and
decreased expression of ␣4␤1, ␣5␤1 integrins.52 Our GPA findings
emphasize that the membrane remodeling occurring during terminal differentiation involves not only gain and/or loss of integral
proteins but also modifications in linkages of continuously expressed proteins. Moreover, these data illustrate the dynamic state
of vertical interactions between the lipid bilayer and membrane
skeleton during differentiation.
Vertical interactions between the lipid bilayer and membrane
skeleton regulate membrane deformability by affecting the ability
of the spectrin network to undergo necessary rearrangements for
membrane deformation.24 Our current data on GPA connectivity in
young reticulocytes elucidate a reasonable molecular mechanism
for decreased reticulocyte membrane deformability observed in
earlier investigations, although other mechanisms may be operative. We have previously studied membrane material properties of
reticulocytes during their maturation and observed dramatically
different membrane deformability in reticulocytes of varying age.23
Young reticulocytes, containing large amounts of RNA, had
marked membrane rigidity. With increasing maturation, we found a
progressive increase in membrane deformability that in older
reticulocytes, containing little RNA, approached that of mature red
cells. Our current data showing a greater degree of connectivity of
GPA with the spectrin-based skeleton in reticulocytes compared
with mature cells support a molecular mechanism whereby enhanced rigidity of reticulocyte membranes results, at least in part,
from increased vertical interactions between the reticulocyte lipid
bilayer and membrane skeleton.
In sum, we have shown that GPA molecules are predominantly
connected to the membrane skeleton late in terminal differentiation
and partition almost exclusively to the reticulocyte during enucleation. We speculate that the presence of negatively charged GPA on
the surface of young reticulocytes may, along with CD47,53 protect
reticulocytes from phagocytosis, while extruded nuclei, lacking the
negative charge imparted by GPA, are readily engulfed by macrophages following enucleation.
Acknowledgment
We are very grateful to Dr Jane Barker (The Jackson Laboratory,
Bar Harbor, ME) for generously providing the nb/nb mouse
red cells.
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2004 103: 1912-1919
doi:10.1182/blood-2003-03-0928 originally published online
October 16, 2003
Mechanism of protein sorting during erythroblast enucleation: role of
cytoskeletal connectivity
James C.-M. Lee, J. Aura Gimm, Annie J. Lo, Mark J. Koury, Sharon W. Krauss, Narla Mohandas
and Joel A. Chasis
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