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RED CELLS, IRON, AND ERYTHROPOIESIS
Imaging of the diffusion of single band 3 molecules on normal and mutant
erythrocytes
Gayani C. Kodippili,1 Jeff Spector,2 Caitlin Sullivan,2,3 Frans A. Kuypers,4 Richard Labotka,5 Patrick G. Gallagher,6
Ken Ritchie,2 and Philip S. Low1
Departments of 1Chemistry and 2Physics, Purdue University, West Lafayette, IN; 3Department of Physics, University of Illinois at Urbana-Champaign;
4Children’s Hospital Oakland, Research Institute, CA; 5Division of Pediatric Hematology/Oncology, University of Illinois, Chicago; and 6Department of Pediatrics,
Yale University School of Medicine, New Haven, CT
Membrane-spanning proteins may interact with a variety of other integral and
peripheral membrane proteins via a diversity of protein-protein interactions. Not
surprisingly, defects or mutations in any
one of these interacting components can
impact the physical and biological properties on the entire complex. Here we use
quantum dots to image the diffusion of
individual band 3 molecules in the plasma
membranes of intact human erythrocytes
from healthy volunteers and patients with
defects in one of their membrane compo-
nents, leading to well-known red cell
pathologies (hereditary spherocytosis, hereditary elliptocytosis, hereditary hydrocytosis, Southeast Asian ovalocytosis,
and hereditary pyropoikilocytosis). After
characterizing the motile properties of
the major subpopulations of band 3 in
intact normal erythrocytes, we demonstrate that the properties of these subpopulations of band 3 change significantly in diseased cells, as evidenced by
changes in the microscopic and macroscopic diffusion coefficients of band 3
and in the compartment sizes in which
the different band 3 populations can diffuse. Because the above membrane abnormalities largely arise from defects in
other membrane components (eg, spectrin, ankyrin), these data suggest that
single particle tracking of band 3 might
constitute a useful tool for characterizing
the general structural integrity of the human erythrocyte membrane. (Blood. 2009;
113:6237-6245)
Introduction
In the well-studied erythrocyte membrane, a complex of membranespanning proteins comprised of band 3, Rh proteins, CD47,
glycophorin A, and several other polypeptides is linked to a
spectrin/actin cortical cytoskeleton via the bridging protein, ankyrin
(labeled “ankyrin complex” in Figure 1A).1,2 In the same membrane, a second major complex comprised of band 3, glycophorin
C, the glucose transporter, and perhaps Rh proteins is linked to a
complex of spectrin, actin, and protein 4.1 via bridging proteins,
p55, adducin, and dematin (labeled “junctional complex” in Figure
1A).2-5 A third population of band 3 is hypothesized to be
unencumbered by attachment to a cytoskeletal protein (labeled free
band 3)6 and might therefore be expected to display increased
lateral diffusion, at least over long time periods.7
Despite the network of interactions among erythrocyte membrane proteins, even polypeptides attached to the hexagonally
tethered junctional complex would be predicted to display some
degree of lateral mobility (Figure 1B). Discher and colleagues8
determined that thermal fluctuations in the actin hub can lead to
movements of up to 40 nm in the anchored actin filaments without
release of the actin from the membrane. Furthermore, since the
distance between junctional complexes (⬃ 70 nm) is much smaller
than the contour length of spectrin tetramers (⬃ 200 nm),8,9 it
might be expected that the band 3 molecules attached to ankyrin
near the center of spectrin tetramers should also be capable of
considerable movement without dissociating from the spectrin
skeleton (Figure 1B). Finally, the spectrin tetramers may form
fences that define triangular compartments within the hexagonal
spectrin lattice, and within these compartments, unattached proteins (eg, free band 3) might be anticipated to diffuse up to
approximately 70 nm without encountering a spectrin barrier. Long
range diffusion might then proceed via slow intercompartment
movement, perhaps when the spectrin tetramers transiently dissociate, thereby opening a gap in the putative “fence” that would
otherwise prevent diffusion to neighboring compartments.
Because of extensive interconnections among membrane proteins, defects in one protein can often destabilize multiprotein
complexes and thereby affect global membrane properties such as
cell morphology, mechanical stability, and function. Thus, deficiencies or defects in ankyrin,10,11 certain domains of spectrin, band
3,12-14 or protein 4.215 can lead to a collection of pathologies,
termed hereditary spherocytosis (HS),16-18 where membrane shedding, reduced cell deformability, and loss of biconcave disc
morphology lead to early removal of the cell from circulation.11,19-21 Similarly, defects or deficiencies in proteins that stabilize the 2-dimensional spectrin/actin lattice can result in hereditary
elliptocytosis (HE)21-23 or in more severe cases to hereditary
pyropoikilocytosis (HPP)21,24 Other morphologic abnormalities can
derive from other molecular lesions, including Southeast Asian
ovalocytosis (SAO) from deletion of residues 400-408 in band
325-27 and hereditary stomatocytosis/hydrocytosis (HHy) from
mutations within the membrane-spanning domain of band 328 or
RhAG.29,30 Because of this strong interdependence of membrane
Submitted February 12, 2009; accepted April 9, 2009. Prepublished online as
Blood First Edition paper, April 15, 2009; DOI 10.1182/blood-2009-02-205450.
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 USC section 1734.
The online version of this article contains a data supplement.
© 2009 by The American Society of Hematology
BLOOD, 11 JUNE 2009 䡠 VOLUME 113, NUMBER 24
6237
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KODIPPILI et al
BLOOD, 11 JUNE 2009 䡠 VOLUME 113, NUMBER 24
Figure 1. Models of human erythrocyte membrane showing
major populations of band 3 and their likely models of lateral
diffusion. (A) Lateral view of erythrocyte membrane showing
major membrane-spanning proteins including: glycophorin C
(GPC), Rh protein (Rh), band 3 (3), the glucose transporter (GLUT
1), Rh-associated glycoprotein (RhAG), CD47, and glycophorin A
(GPA). Labeled peripheral membrane proteins include: spectrin,
ankyrin, protein 4.1 (4.1) and protein 4.2 (4.2), adducin, P55, and
dematin. Note that the 3 hypothesized populations of band 3
include freely diffusing band 3, band 3 in an ankyrin complex, and
band 3 in the junctional complex. (B) Top view of hexagonal
spectrin network showing expected mobilities of band 3. Three
types of band 3 interactions are hypothesized, each with its
predicted mobility: (1) free band 3 should exhibit high mobility over
short time scales, but lower mobility with periodically confined
diffusion (hop diffusion) over longer time scales, (2) band 3 bound
to the spectrin-actin junctional complex through adducin should
also show a limited (possibly even more limited than case 3)
diffusion over short time periods and similarly restricted long time
diffusion and (3) band 3 linked to spectrin tetramers through
ankyrin should display limited mobility over short time periods and
significantly restricted motion over long time periods.
proteins, we hypothesized that a protein such as band 3, that
participates in both major membrane complexes and also exists in
free form, might serve as a useful reporter of the structural integrity
of the membrane. To test this hypothesis, we labeled band 3
covalently with a quantum dot (q-dot) and characterized its
mobility in both intact normal erythrocytes and erythrocytes with a
variety of structural defects (HS, HE, SAO, HHy, and HPP) using
single particle imaging methodology. We report here that major
differences exist among the above pathologic red cells in both the
rate and extent that band 3 diffuses. These diffusion and compartment size data suggest that characterization of the motile properties
of a prominent membrane protein such as band 3 can serve as a tool
for interrogating the structural properties of multiple domains of a
complex biological membrane.
Methods
Labeling of band 3 in intact erythrocytes with DIDS-biotin
Materials used for the synthesis of DIDS-biotin are listed in supplemental
materials (available on the Blood website; see the Supplemental Materials
link at the top of the online article). Details regarding the synthesis (Figure
S1) and characterization (Figure S2) are also provided in supplemental
materials. Blood samples from patients with hereditary hemolytic anemias
(see Figure S3) were drawn following informed consent in accordance with
the Declaration of Helsinki and institutional review board (IRB) approval
obtained from Purdue University and centrifuged at 1000g to pellet the
erythrocytes. The plasma and white cell fraction (buffy coat) were
discarded. Erythrocytes were washed 3 times with phosphate-buffered
saline (PBS), pH 7.4, containing 5 mM glucose, and again the white cell
fraction was removed. Red blood cells (RBCs) diluted to 5% hematocrit in
the same buffer were incubated with 10⫺11 M to 10⫺13 DIDS-biotin at 37°C
for 1.5 hours to allow covalent reaction of the compound with band 3. This
low concentration was determined from binding studies, where approximately 50% binding of DIDS-biotin to RBCs was found to occur at 30 ␮m.
We used such low concentrations to react approximately with 1 biotin linker
per cell. Under these experimental conditions, we observed that approximately 70% of the cells were labeled with a single q-dot when incubated
with excess streptavidin q-dots. Cells with 2 or 3 q-dots attached were
rarely seen. DIDS-biotin–labeled cells were then washed 3 times with PBS
containing 0.1% bovine serum albumin (BSA) to remove unbound reagent,
and the cells were incubated at room temperature with streptavidin Q-dot
525 or streptavidin Alexa Fluor 488 for 30 minutes. Unbound streptavidin
conjugates were removed by washing twice with 0.1% BSA in PBS, and
cells were allowed to settle onto a precleaned, polylysine-coated coverslip
located within a custom built chamber ready for imaging. Unattached cells
were washed off of the coverslip with PBS containing 0.1% BSA. Finally,
500 ␮L PBS were added to the chamber, and the cells were imaged as described below.
Single q-dot fluorescence video microscopy
Observation was performed on an Olympus IX-71 inverted microscope
(Olympus, Center Valley, PA). Oblique angle fluorescence imaging was
used to excite single q-dots on the apical surfaces of immobilized
erythrocytes. The entire microscope was maintained at 37°C by enclosure
in a temperature-controlled environment. The excitation laser (argon ion,
488 nm emission; Newport, Irvine, CA) was expanded, filtered (488/10-nm
line width bandpass filter; Chroma Technology, Rockingham, VT) and
directed toward the microscope objective (100⫻ PlanApo, 1.45 NA TIRFM
oil immersion; Olympus) parallel but off the optical axis through a dichroic
mirror (500 nm cutoff; Chroma Technology). The resultant fluorescence
image was projected through the dichroic mirror and an emission filter
(525/50-nm bandpass; Chroma Technology), and the image was collected
with a dual microchannel plate (MCP)–intensified, cooled charge-coupled
device (CCD) camera (XR/Turbo-120z; Stanford Photonics, Palo Alto,
CA). The excitation beam was set such that it was just outside of the
condition for total internal reflection, thus allowing for a deeper excitation
while still reducing background due to fluorescent matter in solution. Data
were collected from the dots attached to the top of the cell at 120 frames/s.
Each movie was recorded for 1000 frames. Only q-dots whose trajectories
were at least 40 frames long were chosen for analysis, however, because
even trajectories recorded for 1000 frames revealed band 3 diffusion for
maximally 8 seconds, all data report only a brief snapshot of band
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BLOOD, 11 JUNE 2009 䡠 VOLUME 113, NUMBER 24
3 behavior in the membrane. The trajectories were collected on a random
selection of erythrocytes in each sample. In general, 100 to 500 RBCs were
analyzed in each sample.
Analysis of mobility
The apparent position of the q-dot in the video image was determined as
described by Gelles et al.31 Briefly, a kernel was developed from a
Gaussian distribution which was then cross-correlated with each video
frame in the neighborhood of the label of interest. For each frame, the
resulting cross-correlation function was thresholded, and the particle
position was found as the center-of-mass of the thresholded correlation
intensity.
Analysis of the mobility was carried out based on the measured
mean-squared displacement (MSD) of the observed label as described
previously.7,32-34 For each trajectory, the MSD for every time interval was
calculated according to the formula34,35:
N⫺1⫺n
MSD(n␦t) ⫽ (N ⫺ 1 ⫺ n) ⫺ 1
兺
{[ x( j␦t ⫹ n␦t) ⫺ x( j␦t)] 2
j⫽1
⫹ [ y( j␦t ⫹ n␦t) ⫺ y( j␦t)] 2 }
where ␦t is the time resolution and (x(j␦t ⫹ n␦t), y(j␦t ⫹ n␦t)) describes the
particle position following a time interval n␦t after starting at position
(x(j␦t), y(j␦t)), N is the total number of frames in the sequence, n is a
positive integer.
The MSD versus time plots (MSD-t plots) were analyzed and classified
as describing simple Brownian, confined, or hop diffusion, as described in
Fujiwara et al.36 and Umemura et al.37 All plots were fit with an
approximation to the exact result for diffusion through an infinite array of
partially permeable barriers.33 From this analysis, estimates of the shorttime diffusion coefficient, D␮, the asymptotic long time diffusion coefficient, DM, and, if applicable, the average spacing between barrier (referred
to as the compartment size) L were obtained. Gaussian fits were used on the
histograms of the distributions of diffusion coefficients/compartment sizes
to determine the mean values of the distributions. The number of Gaussian
distributions required to fit the histograms was chosen using the F test with
a 95% confidence level.
Results
Labeling of band 3
Characterization of the movement of single band 3 molecules in
whole erythrocytes required the ability to specifically label band 3,
the erythrocyte anion transporter (AE1), in a nonperturbing manner. Anti-band 3 antibodies were not selected for this purpose
because of their potential to cross-link band 3 into unnatural
aggregates or displace band 3 from its natural protein partners.
Therefore, DIDS, an inhibitor of band 3-mediated anion transport,
which reacts covalently with Lys539 of band 3,38 was selected for
the design of a band 3–specific labeling reagent. Importantly, DIDS
binding to intact erythrocytes follows a noncooperative, single
site-binding isotherm, suggesting that it does not preferentially
label 1 band 3 population over another.39
To convert DIDS from a simple anion transport inhibitor to a
selective anchor for q-dots on band 3, DIDS was linked via a
peptide spacer to biotin, a high-affinity ligand of streptavidin, as
described in “Methods” in the supplementary material. The peptide
spacer was constructed to contain 4 anionic amino acids (to
facilitate binding to the anion transporter) and a lysine residue (for
attachment of biotin). The resulting construct was found to react
covalently with whole erythrocytes and mediate binding of fluorescent streptavidin, as demonstrated in Figure 2A.
LATERAL DIFFUSION OF BAND 3
6239
The specificity of the DIDS-biotin conjugate was established by
reacting the conjugate with intact erythrocytes, isolating their
membranes, separating component proteins by sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE), blotting
the proteins onto nitrocellulose paper, and visualizing the location
of biotinylated polypeptides by staining with streptavidin–
horseradish peroxidase. As seen in Figure 2C, only 1 polypeptide
with apparent Mr approximately 100 kDa was labeled. Because
band 3 is the only known receptor for DIDS on human erythrocytes, and because band 3 has the molecular weight of the protein
stained in Figure 2C (ie, ⬃ 100 kDa), we conclude that band 3 is
selectively labeled by the DIDS-biotin conjugate in intact erythrocytes. Further, because streptavidin-linked q-dots do not bind
erythrocytes that have not been reacted with DIDS-biotin (Figure
2B), we suggest that DIDS-biotin labeling enables selective
imaging of band 3 diffusion in intact erythrocytes.
Single-particle tracking experiments were invariably performed
on whole cells reacted with very few q-dots to avoid confusion due
to crossing trajectories. To derivatize a single or at most 2 or 3 band
3 molecules per intact erythrocyte, a standard DIDS-biotin binding
curve was obtained by evaluating the fluorescence of bound
streptavidin-Alexa Fluor 488–labeled erythrocytes as a function of
the concentration of DIDS-biotin added. Half maximal binding was
observed at 30 ␮M DIDS-biotin (Figure 3), and derivatization of
only 1 or 2 molecules/cell was achieved at 10⫺11 M DIDS-biotin.
Therefore, all subsequent erythrocyte labeling was performed
using 10⫺11 M DIDS-biotin. We do not believe that this concentration of DIDS-biotin significantly affects the diffusion of band
3 in the red cell.
Analysis of band 3 mobility on normal and fixed erythrocytes
Due to the noise inherent in the measurement of the position of a
single q-dot during single particle tracking, even stationary labels
were found to report a nonzero diffusion coefficient. This nonzero
diffusion coefficient for immobilized labels sets the level of
accuracy in determining meaningful mobilities. To determine this
level in the current study, the mobility of band 3 on acrolein-fixed
RBCs was monitored at 37°C using the usual methodology. Figure
4 shows a typical trajectory of the labeled band 3 on these fixed
intact RBCs. From analysis of individual band 3 trajectories on
greater than 130 of these fixed cells, 2 diffusion coefficients were
measured corresponding to a diffusion coefficient at short times
(termed microscopic diffusion coefficient, D␮) and the asymptotic
diffusion coefficient at long times (termed macroscopic diffusion
coefficient, DM). The top row of Figure 5 displays the distribution
of D␮ and DM values measured for labeled band 3 molecules on
fixed human RBCs. Fitting a single Gaussian distribution to each of
these sets of measurements yields a mean D␮ of 6.8 ⫻ 10⫺14 cm2/s
and a mean DM of 5.5 ⫻ 10⫺14 cm2/s. These values establish the
smallest meaningful D␮ and DM diffusion coefficients that can
be measured with our imaging system. Importantly, D␮ and DM
values for q-dots attached directly to polylysine-coated glass slides
were not significantly different (D␮ ⫽ 6.2 ⫻ 10⫺14 and DM ⫽
3.2 ⫻ 10⫺14 cm2/s).
In comparison to fixed cells, labeled band 3 displays a higher
mobility on intact, unfixed normal human RBCs at 37°C. A typical
trajectory is shown in Figure 4, while the distributions of measured
D␮ and DM are shown in row 2 of Figure 5 (see individual data in
Figure S4). Note that the observed diffusion coefficients are
significantly larger than those measured on fixed cells. The
distribution of microscopic diffusion coefficients fits well with
2 Gaussian curves centered at 1.4 ⫻ 10⫺11 and 2.2 ⫻ 10⫺10 cm2/s
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BLOOD, 11 JUNE 2009 䡠 VOLUME 113, NUMBER 24
KODIPPILI et al
B
A
C
1 2
3 4
250
125
98
64
50
O
D
Biotin
HN
NH
H
O
O
NH
O
NH3+
O
O
HN
O
NCS
O
-
HN
S
O
S
O
O
-
H
H
N
N
H
O
O
O
N
H
O
O
S
O
O-
DIDS
Anionic spacer
O-
Figure 2. Structure and specificity of DIDS-biotin reagent for band 3. (A) Fluorescent image of whole erythrocytes reacted with a subsaturating concentration of
DIDS-biotin reagent (10 ␮M) and then labeled with Alexa Flour 598–derivatized streptavidin. (B) Image of unlabeled (control) whole erythrocytes that were similarly incubated
with Alexa Fluor 598–derivatized streptavidin. (C) SDS-PAGE analysis of the specificity of DIDS-biotin reagent for band 3. Erythrocytes were either not reacted with DIDS-biotin
reagent (lane 1) or reacted with 1 ␮M DIDS-biotin reagent (lane 2) before separation by SDS-PAGE. After electrophoretic transfer onto nitrocellulose, the molecular weights of
DIDS-biotin–labeled polypeptides were determined by staining with streptavidin–horseradish peroxidase (1:1500). Lane 3 contains Coomassie blue–stained erythrocyte
membranes, and lane 4 contains molecular weight markers. Vertical line(s) have been inserted to indicate a repositioned gel lane. (D) Structure of the DIDS-biotin reagent.
(Table 1), which represent of 77% and 23% of the labeled band
3 population, respectively. In contrast, the macroscopic diffusion coefficient displays a single broad peak at 1.7 ⫻
10⫺12 cm2/s (Table 1).
Band 3 mobility in erythrocytes with HE and HPP
A typical trajectory of band 3 on intact erythrocytes with HE
(commonly thought to derive from a defect in one of the horizontal
interactions that stabilizes the membrane skeleton) is shown in
Figure 4. Although the specific mutations responsible for this and
the other altered RBCs evaluated in this publication were not
determined, it is conceivable that at least 2 of the 3 HE samples
examined (Figures 5 and S5) represent different molecular defects,
because their osmotic deformability scans were significantly different
Normal (77%)
HS-5 (58- 80%)
HPP (100%)
Fixed cell (100%)
HE (60-80%)
HHy (60%)
58
SAO( 84%)
56
278
280
100nm
Figure 3. Analysis of DIDS-biotin binding to intact human erythrocytes. Washed
normal human erythrocytes were reacted with DIDS-biotin reagent as described in
Methods, labeled with streptavidin–Alexa Fluor 488, washed 3 times in buffered
saline, and analyzed for bound Alexa Fluor 488 by flow cytometry. The intensity of the
bound Alexa Fluor 488 fluorescence is plotted as a function of the concentration of
DIDS-biotin reagent added. Fifty percent saturation was observed at 30 ␮M
DIDS-biotin reagent.
Figure 4. Representative trajectories of DIDS-biotin–labeled band 3 on normal
and mutant intact human erythrocytes. After labeling with DIDS-biotin conjugate,
the diffusion of the labeled band 3 was imaged for 100 consecutive frames at
120 frames/s on fixed normal, unfixed normal, HS, HPP, HE, HHy, and SAO
erythrocytes. Because most of these cell types have multiple populations of band 3,
the trajectory of only the most abundant population is displayed (percent of total band
3 is indicated in parentheses).
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BLOOD, 11 JUNE 2009 䡠 VOLUME 113, NUMBER 24
LATERAL DIFFUSION OF BAND 3
Dmicro
Compartment size
Dmacro
30
Fixed cells
30
20
20
10
10
0
0
60
35nm
71nm
40
40
40
Normal RBC
20
20
20
0
0
0
60
40
12
20
20
0
0
6
0
18
12
12
8
12
HS-2
HE-1
Number of cells
8
HS-1
58 nm
93 nm
122 nm
18
40
HPP
4
6
0
0
20
20
10
10
0
0
36nm
4
0
12
40nm
6
0
8
10
12
8
36nm
4
5
4
0
0
0
40
16
20
8
0
0
20
20
10
10
0
0
20
12
20
HE-2
6241
35 nm
10
0
18
42 nm
12
HHy
6
0
20
SAO
42 nm
8
10
10
4
0
-16
0
0
-12
-8
-16
-12
-8
Dµ
DM
Log Dµ or DM (cm2/s)
0 100
200 300
nm
Figure 5. Distributions of the logarithms of the microscopic (D␮) and macroscopic (DM) diffusion coefficients, and the compartment sizes determined by analysis of
individual trajectories of labeled band 3 molecules on intact fixed normal, unfixed normal, HE, HPP, HHy, SAO, and HS erythrocytes.
(see Figure S6). Consistent with this interpretation, analysis of the
distribution of diffusion coefficients of band 3 at 37°C in the first HE
sample (HE-1) revealed a single Gaussian distribution for D␮ values, but
2 Gaussian distributions for DM values; however, the converse was
true for sample HE-2 (Figure 5 and Table 1). Thus, although the
major population of band 3 in both patient’s samples exhibited
similar values of D␮ and DM of approximately 4 ⫻ 10⫺11 and
approximately 9 ⫻ 10⫺13 cm2/s, respectively, the “extra” band 3
population in the HE-1 sample displayed a faster DM of 2.0 ⫻ 10⫺10
cm2/s, whereas the additional population of band 3 in the HE-2
sample had a slower D␮ of 1.2 ⫻ 10⫺12cm2/s. Because approximately 150 cells were examined from each patient, we interpret
these behaviors to indicate that the 2 HE pathologies are molecularly different.
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KODIPPILI et al
Table 1. Microscopic and macroscopic diffusion data of healthy and disease RBCs
RBC type
D␮, cm2/s
10⫺14§
Fixed (n ⫽ 134)‡
(6.8 ⫾ 0.1) ⫻
Normal (n ⫽ 626)
(1.4 ⫾ 0.4) ⫻ 10⫺11 (77%)¶
HPP (n ⫽ 349)
HS-1 (n ⫽ 107)
DM, cm2/s
(5.5 ⫾ 0.2) ⫻
Percent immobile*
10⫺14
Percent constrained†
Percent free
95储
0
5
(1.7 ⫾ 0.1) ⫻ 10⫺12
24
30
46
(7.9 ⫾ 0.5) ⫻ 10⫺11
(1.5 ⫾ 0.1) ⫻ 10⫺11
7
37
56
(2.7 ⫾ 1.3) ⫻ 10⫺12 (27%)
(1.4 ⫾ 0.1) ⫻ 10⫺12
34
32
34
(2.2 ⫾ 0.1) ⫻ 10⫺12
20
37
43
(9.8 ⫾ 1.7) ⫻ 10⫺13 (69%)
23
31
46
(8.8 ⫾ 0.5) ⫻ 10⫺13
19
25
56
(8.2 ⫾ 0.4) ⫻ 10⫺13 (20%)
(8.3 ⫾ 0.3) ⫻ 10⫺13 (78%)
26
22
52
(2.6 ⫾ 0.4) ⫻ 10⫺11 (80%)
(8.1 ⫾ 1.1) ⫻ 10⫺11 (22%)
(1.5 ⫾ 1.2) ⫻ 10⫺12 (40%)
(8.1 ⫾ 0.4) ⫻ 10⫺13
31
30
39
(1.8 ⫾ 0.1) ⫻ 10⫺12
21
31
48
(6.8 ⫾ 0.3) ⫻ 10⫺12
34
28
38
(2.2 ⫾ 0.1) ⫻ 10⫺10 (23%)
(5.5 ⫾ 0.7) ⫻ 10⫺11 (73%)
HS-2 (n ⫽ 124)
(3.8 ⫾ 1.1) ⫻ 10⫺12 (42%)
(9.6 ⫾ 1.2) ⫻ 10⫺11 (58%)
HE-1 (n ⫽ 133)
(5.4 ⫾ 0.8) ⫻ 10⫺11
(2.0 ⫾ 0.4) ⫻ 10⫺10 (31%)
HE-2 (n ⫽ 212)
(1.2 ⫾ 0.1) ⫻ 10⫺12 (37%)
(3.0 ⫾ 0.3) ⫻ 10⫺11 (63%)
HE-3 (n ⫽ 168)
HHy (n ⫽ 171)
(4.4 ⫾ 1.4) ⫻ 10⫺11 (60%)
SAO (n ⫽ 173)
(2.1 ⫾ 0.1) ⫻ 10⫺12 (16%)
(4.5 ⫾ 0.3) ⫻ 10⫺11 (84%)
HS-unknown defects
HS-3 (n ⫽ 231)
(1.2 ⫾ 1.9) ⫻ 10⫺12 (25%)
(2.9 ⫾ 0.3) ⫻ 10⫺11 (75%)
HS-4 (n ⫽ 173)
(4.9 ⫾ 0.3) ⫻ 10⫺11
(4.0 ⫾ 0.2) ⫻ 10⫺13
21
33
46
HS-5 (n ⫽ 206)
(1.8 ⫾ 0.3) ⫻ 10⫺12 (17%)
(1.8 ⫾ 0.1) ⫻ 10⫺12
25
35
40
(3.2 ⫾ 0.1) ⫻ 10⫺12
20
30
50
(6.7 ⫾ 0.4) ⫻ 10⫺11 (82%)
HS-6 (n ⫽ 136)
(2.7 ⫾ 0.5) ⫻ 10⫺12 (11%)
(1.0 ⫾ 0.2) ⫻ 10⫺10 (89%)
*The totally immobile fraction is defined as the fraction of band 3 molecules that was found to have a DM value less than or equal to the slowest 95% of DM values measured
for band 3 on fixed erythrocytes (DMImm); that is, the fraction of band 3 that diffuses similar to band 3 on fixed cells.
†Percent confined includes all trajectories that were classified as undergoing confined or hop diffusion and for which DM ⬎ DMImm.
‡Total number of trajectories analyzed.
§Errors are given as the SEM.
储Defined as such.
¶Reflects the fraction of total area contained in each peak of the Gaussian fit to the distribution. For those distributions that fit best with a single peak it is understood that
100% of the area is contained within that peak
The other major morphologic disorder ascribed to defects in
horizontal interactions within the membrane skeletal network is
termed HPP. Although analysis of band 3 diffusion in these cells at
37°C revealed a relatively normal D␮ of 7.9 ⫻ 10⫺11 cm2/s
(compared with 1.4 ⫻ 10⫺11 cm2/s [77%] and 2.2 ⫻ 10⫺10 cm2/s
[23%] in normal RBCs), the value for DM was the fastest yet
measured (Figures 4 and 5 and Table 1). Thus, DM in HPP cells was
determined to be 1.5 ⫻ 10⫺11 cm2/s (ie, 10 times faster than
normal), suggesting that long-range band 3 diffusion in HPP cells is
relatively unimpeded by physical barriers. Moreover, all band 3
molecules in these cells appeared to diffuse homogeneously (ie,
only a single Gaussian distribution was obtained for both D␮ and
DM), suggesting that the 2 distinct band 3 populations seen in
healthy cells (Figure 5) are either indistinguishable or absent in
HPP cells.
Band 3 mobility in erythrocytes with HS
HS cells are collectively characterized by a defect in one of several
interactions connecting the phospholipid bilayer to the spectrin
skeleton. Six HS samples were analyzed in this study. SDS-PAGE
and Western blot analysis revealed that HS sample 1 may have an
abnormal band 3 characterized by a slightly lower molecular
weight, whereas HS sample 2 may suffer from a slight deficiency in
spectrin (see Figure S7). However, no molecular defect could be
identified in any of the other 4 HS samples other than their
spherocytic morphologies, lower mean cell volumes (MCV), and
altered osmotic scans on an ektacytometer. In general, the diffusion
behavior of band 3 at 37°C in each of the 6 HS samples was similar,
with each sample yielding 2 distinct Gaussian distributions of D␮
and one distribution of DM (Figure 5, rows 4 and 5, and Figure S8
and Table 1). Although similar to normal cells in this gross
description, the behavior of band 3 in the HS patients was
otherwise very different. Thus, the distribution of band 3 molecules
between faster and slower diffusing populations of D␮ in intact HS
erythrocytes was significantly shifted toward the faster diffusing
population (ie, 80% of band 3 in HS cells was in the faster diffusing
population vs 23% of band 3 in normal cells; Figures 5 and S8),
suggesting a smaller fraction of band 3 molecules in HS erythrocytes is restricted by attachment to the membrane skeleton. Further,
the slower diffusing population in HS cells was seen to move
considerably slower than the corresponding slow diffusing population in normal RBCs (average of 2.4 ⫻ 10⫺12 in HS vs 1.4 ⫻ 10⫺11
cm2/s in normal RBCs). Taken together, these data suggest that
short term constraints on band 3 diffusion in HS cells are
significantly different from those in normal cells.
Because average values of DM did not significantly differ
between HS and normal RBCs (2.6 ⫻ 10⫺12 vs 1.7 ⫻ 10⫺12 cm2/s,
respectively; Table 1), we would assume that the rate of diffusion
from one spectrin compartment to another is not significantly
different between normal and HS samples.
Band 3 mobility in erythrocytes with HHy
Analysis of the trajectories of band 3 in intact HHy cells (Figure 4)
revealed a microscopic diffusion coefficient (D␮) characterized by
2 Gaussian distributions with mean values of 1.5 ⫻ 10⫺12 (ie,
10 times slower than the slower population in normal RBCs) and
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BLOOD, 11 JUNE 2009 䡠 VOLUME 113, NUMBER 24
LATERAL DIFFUSION OF BAND 3
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4.4 ⫻ 10⫺11 cm2/s (ie, 5 times slower than the faster population
in normal RBCs; Table 1). The distribution of macroscopic
diffusion coefficients showed only a single peak with a mean of
8.1 ⫻ 10⫺13 cm2/s (Figure 5 and Table 1). This macroscopic diffusion
coefficient was 2 times slower than that found in normal RBCs.
exhibiting “confined” diffusion (Table 1). Except for band 3 in HPP
cells, the fraction of this relatively unrestricted band 3 appears to be
moderately constant from 1 pathologic cell to the next.
Band 3 mobility in SAO erythrocytes
Discussion
Approximately half of the band 3 molecules in SAO erythrocytes
contain a deletion of residues 400-408, the remaining copies of
band 3 being normal.26,27,40 Because only the “normal” population
of band 3 can be labeled with DIDS,26 analysis of diffusion data
from SAO cells must be interpreted with caution, because the
mobility of the mutated band 3 is never measured. With this caveat
in mind, 2 Gaussian distributions of D␮ with mean values of
2.1 ⫻ 10⫺12 and 4.5 ⫻ 10⫺11 cm2/s were fitted, both 5 times slower
than the corresponding populations on normal RBC (Table 1).
These data would suggest that diffusion of even the normal
population of band 3 in SAO membranes is significantly impacted
by the presence of the mutant isoform. In contrast, only a single
population of band 3 molecules was detected by analysis of DM,
and the measured value (1.8 ⫻ 10⫺12 cm2/s) was similar to that
found on normal RBCs.
To study the mobility of band 3 in essentially unperturbed intact
erythrocytes, we required a probe that would: (1) react covalently
with band 3 (to prevent probe hopping among band 3 molecules
during the measurement), (2) derivatize no other membrane
protein, (3) avoid promoting aggregation or clustering of band 3,
and (4) enable imaging of band 3 with highly fluorescent particles.
For this purpose, we designed and synthesized a DIDS-biotin
conjugate that not only met all of the above criteria, but also
permitted the reproducible derivatization of only one or few band 3
molecules/cell, thereby avoiding confusion over which particle/
band 3 was being monitored. Because single fluorophores (eg,
fluorescein) were strongly quenched by the high concentration of
proximal hemoglobin, the DIDS-biotin probe proved to be especially valuable because it allowed labeling of band 3 with q-dots
that could be easily tracked using a high speed CCD camera.
As noted in the introduction, we set out to determine whether
the mobility of a prominent membrane protein such as band 3
might be exploited to detect defects in other major membrane
components. Although the molecular mutations in the 12 different
hemolytic anemia samples examined were not determined, we
suggest that the answer to this question remains in the affirmative,
because band 3 diffusion in all of the pathologic samples displayed
unequivocally abnormal properties. Thus, unlike normal erythrocytes, where 77% of the band 3 diffused with a slow D␮ of
1.4 ⫻ 10⫺11 cm2/s, HPP cells contained no band 3 that diffused
with such slow kinetics (Table 1 and Figure 5). Although HS cells,
like normal erythrocytes, exhibited a bimodal distribution of D␮
values, the relative abundances of the slow and fast populations
differed markedly from normal, with the slow population dominating the behavior in healthy cells (73% of the total band 3), but the
fast population assuming prominence in HS samples (80% of total
band 3). Moreover, a substantial fraction of band 3 in all HE
samples was found to redistribute from the slower to faster
diffusing population, and DM values for 2 of the 3 HE samples were
approximately 10 times faster than in normal cells. Perhaps equally
interesting was the observation that even normal copies of band 3
in cells containing mutated band 3 (ie, SAO cells) were able to
sense the impact of their mutated counterparts (ie, the slow and fast
D␮ values in SAO cells were both 10 times slower than normal).
Because most of the above pathologies do not involve a defect in
band 3, we suggest that a prominent membrane component such
as band 3 can be used as a sentinel to determine whether mutations in other cellular components compromise the integrity of
the membrane.
It was interesting that many (but not all) of the changes in band
3 properties in the hemolytic anemias were consistent with current
understandings of the underlying defects in these membrane
abnormalities. Thus, as noted above, a substantial fraction of band
3 in all HS cells shifted toward a more rapidly diffusing population.
If one or both of the D␮ populations were to correspond to ankyrinand/or adducin-attached band 3, then this increase in band 3
mobility would be consistent with observations that linkages
connecting the bilayer (primarily via band 3) to the spectrin
skeleton are compromised in HS.11-14 Similarly, the complete loss
of the slow diffusing population of band 3 in HPP and the
Analysis of compartment size distributions
Roughly half of the band 3 population in each cell type was
determined by our analysis algorithm to diffuse within a confined
area during the observation period of the imaging experiment (see
“Analysis of mobility”). The final column of Figure 5 shows the
measured distribution of calculated compartment sizes for the
fraction of band 3 that exhibited this temporarily confined diffusion
in the various cell types (see also Figure S9). In normal cells,
roughly half of this confined population diffused within an apparent
compartment of 35 nm diameter, while the other half diffused
within a compartment of approximately 70 nm. Except for HPP
erythrocytes (and 3 of 6 HS samples shown in Figure S8), the best
fits of the data on all of the pathologic cells yielded only a single
compartment size of 35-42 nm. However, diffusion of band 3 on
HPP erythrocytes was characterized by compartment sizes of 58,
93, and 122 nm, and diffusion on HS samples was highly variable,
with 2 of the 6 showing multiple compartment sizes similar to HPP
cells. Based on these limited data, it would appear that the anion
transporter experiences significantly fewer barriers while diffusing
on HPP and some HS cells than on normal erythrocytes.
While it is tempting to begin to draw conclusions on the
dimensions of the spectrin network from the above data, because
only half of the band 3 population exhibits this temporarily
confined diffusion, any complete interpretation of compartment
size data must also consider those band 3 molecules that do not
experience “confined diffusion.” Included in this category are band
3 polypeptides that display any of the following 3 modes of
diffusion: (1) molecules that see no barriers within the time frame
of 1 analysis (usually 8.0 s), (2) molecules whose “hopping”
movement between compartments is fast relative to the frequency
of image acquisition, and (3) molecules that are confined to
compartments so small that their compartments/diffusion coefficients cannot be accurately determined.41 Because those band 3
molecules that exhibit unrestrained diffusion might also provide
information on the integrity of the spectrin network, we have
calculated the percent of total band 3 that diffuses in a relatively
unrestricted manner by subtracting the fraction of band 3 diffusing
according to mode 3 above from the total fraction of band 3 not
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BLOOD, 11 JUNE 2009 䡠 VOLUME 113, NUMBER 24
KODIPPILI et al
concomitant nearly complete loss of totally immobilized band 3 in
the same cells would be consistent with published data demonstrating that the spectrin skeleton is extensively fragmented in HPP and
that band 3-ankyrin affinity is influenced by spectrin tetramer
formation42; ie, in agreement with previous fluorescence recovery
after photobleaching (FRAP) data from Golan and coworkers43
showing that band 3 diffuses 6 times faster and has a smaller
immobilized fraction in HPP cells than in any HS or normal sample
examined (see Table 1). Moreover, the slower D␮ values of band 3
in the SAO cells is consistent with the fact that band 3 forms linear
aggregates in these cell types,26,44 and the faster D␮ in HE cells is
not incompatible with the view that the spectrin network is
weakened in this pathology.22,23
In contrast, except for results on normal erythrocytes, the
compartment size data were generally not anticipated. Rationalization of compartment sizes measured in normal cells might be
possible if the larger compartments (ie, approximately 70 nm) were
to correspond to open areas between putative “spectrin fences,” and
the smaller compartments were to derive from cytoskeletally
attached band 3. Thus, because the length of an extended spectrin
tetramer is reported at approximately 200 nm,8,9 and because
spectrin tetramers are thought to be compacted to approximately
one-third of this length in situ,9 the size of a compartment in which
free band 3 can diffuse might be approximately 70 nm. The smaller
compartments of 35 nm might then correspond to spectrinanchored band 3, because as Discher and coworkers8 note, even
spectrin-attached actin hubs move/oscillate within a compartment
of approximately 40 nm. How then might the common compartment size of 35-42 nm seen in most of the remaining hemolytic
anemias be explained? While no definitive interpretation can be
offered, we speculate that this population of band 3 might also
correspond to those polypeptides that are anchored to spectrin via
the junctional complex or ankyrin. Thus, our data analysis algorithm calculates a compartment size for only those band 3
molecules that exhibit confined diffusion over the time period of a
measurement (⬃ 8 s), and thus those band 3 molecules that
encounter no barriers within this time frame will not contribute to
the compartment size measurement. In this regard, it is interesting
that HPP cells and 2 of the HS samples display several distinct
compartment sizes of significantly larger dimensions, suggesting
that the defects in these membranes somehow enable band 3 to
diffuse within larger putative “corrals.”
Previous studies of the rotational and lateral diffusion of band 3
in erythrocyte membranes have consistently identified 2 populations of the polypeptide, one attributed to a cytoskeletally anchored
fraction and the other to a freely diffusing population. Rotational
relaxation studies of Cherry and colleagues45 demonstrated that
both populations were mobile, but that one exhibited relatively
unrestricted rotation and the other displayed restrained, yet measurable rotation. Subsequent studies by Beth and coworkers46 showed
that the skeletally attached band 3 can rotate through an angle of
73°, probably because of the flexibility of the junction between the
cytoplasmic and membrane-spanning domains of band 3. Data of
Golan et al47 have further demonstrated that depletion of ankyrin
releases the less mobile population from its rotational restriction
and that the rotational and lateral diffusion of band 3 are
independently regulated, the former by protein interactions at the
cytoplasmic surface of the membrane, and the latter by the spectrin
content of the cell.43,48 Additional studies from the Golan laboratory49 have also identified 2 populations of band 3 based on lateral
diffusion measurements using FRAP. Finally, Kusumi and colleagues7,50 introduced single-particle imaging methodologies to
explore band 3 diffusion in erythrocyte ghosts, and they also
identified 2 diffusing populations, albeit the diffusion coefficient of
only the more mobile fraction was reported.
We have also found evidence for 2 major populations of band 3
in normal human erythrocytes. Thus, analysis of over 600 cells
from 8 different donors yields 2 D␮ values and 2 compartment sizes
for band 3. The conundrum, however, lies in the fact that recent
membrane models (Figure 1A) display 3 populations of band 3 (ie,
an ankyrin bound, an adducin bound, and a free population of band
3). Whether band 3 in both the ankyrin and junctional complex
diffuse with the slower diffusion coefficients and within the smaller
compartments will have to await further scrutiny, but it is tempting
to speculate that the faster diffusing band 3 that moves within the
larger compartment might correspond to freely diffusing band 3.
Ongoing studies on mice deficient in major proteins of both the
ankyrin complex and junctional complex will help to resolve this
uncertainty.
Acknowledgments
The authors thank Steven C. Habicht, Derek Doorneweerd, Sumith
Kalaratne, Prasad Rajendra Bandari, and Estela Campanella for
helpful discussions.
P.S.L. acknowledges funding from the National Institutes of
Health (Grant GM24417-29). K.R. acknowledges funding from the
National Science Foundation (Grant 0646633).
Authorship
Contribution: G.C.K. and P.S.L. designed research; G.C.K. performed research; G.C.K., synthesized and characterized the DIDSbiotin; J.S., C.S., and G.C.K. contributed microscopic analysis;
G.C.K., J.S., and K.R. analyzed data; F.A.K., R.L., and P.G.G.
supplied blood specimens; and P.S.L. and K.R. wrote the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Philip S. Low, Department of Chemistry,
Purdue University, 560 Oval Dr, West Lafayette, IN 47907; e-mail:
[email protected] or Ken Ritchie, Department of Physics, Purdue
University, 525 Northwestern Ave, West Lafayette, IN 47907;
e-mail: [email protected].
References
1. Bruce LJ, Ghosh S, King MJ, et al. Absence of
CD47 in protein 4.2-deficient hereditary spherocytosis in man: an interaction between the Rh
complex and the band 3 complex. Blood. 2002;
100:1878-1885.
3. Khan AA, Hanada T, Mohseni M, et al. Dematin and
adducin provide a novel link between the spectrin
cytoskeleton and human erythrocyte membrane by
directly interacting with glucose transporter-1. J Biol
Chem. 2008;283:14600-14609.
5. Mohandas N, Chasis JA. Red-blood-cell deformability, membrane material properties and
shape—regulation by transmembrane, skeletal
and cytosolic proteins and lipids. Semin Hematol.
1993;30:171-192.
2. Bruce LJ, Beckmann R, Ribeiro ML, et al. A band
3-based macrocomplex of integral and peripheral
proteins in the RBC membrane. Blood. 2003;101:
4180-4188.
4. Low PS. Structure and function of the cytoplasmic domain of band-3: center of erythrocyte
membrane-peripheral protein interactions. Biochim Biophys Acta. 1986;864:145-167.
6. Che A, Morrison IEG, Pan RJ, Cherry RJ. Restriction by ankyrin of band 3 rotational mobility in human erythrocyte membranes and reconstituted
lipid vesicles. Biochemistry. 1997;36:9588-9595.
From www.bloodjournal.org at UT SOUTHWESTERN MED CTR on November 9, 2009. For personal use only.
BLOOD, 11 JUNE 2009 䡠 VOLUME 113, NUMBER 24
LATERAL DIFFUSION OF BAND 3
7. Tomishige M, Sako Y, Kusumi A. Regulation
mechanism of the lateral diffusion of band 3 in
erythrocyte membranes by the membrane skeleton. J Cell Biol. 1998;142:989-1000.
lar basis and haplotyping of the alpha II domain
polymorphisms of spectrin: application to the
study of hereditary elliptocytosis and pyropoikilocytosis. Am J Hum Genet. 1996;59:351-359.
8. Lee JCM, Discher DE. Deformation-enhanced
fluctuations in the red cell skeleton with theoretical relations to elasticity, connectivity, and spectrin unfolding. Biophys J. 2001;81:3178-3192.
24. Tolpinrud W, Maksimova YD, Forget BG,
Gallagher PG. Nonsense mutations of the alphaspectrin gene in hereditary pyropoikilocytosis.
Haematologica. 2008;93:1752-1754.
9. Paramore S, Ayton GS, Mirijanian DT, Voth GA.
Extending a spectrin repeat unit. I: linear forceextension response. Biophys J. 2006;90:92-100.
25. Sarabia VE, Casey JR, Reithmeier RAF. Molecular characterization of the band-3 protein from
Southeast Asian ovalocytes. J Biol Chem. 1993;
268:10676-10680.
39.
26. Moriyama R, Ideguchi H, Lombardo CR, Vandort
HM, Low PS. Structural and functional characterization of band 3 from Southeast Asian ovalocytes. J Biol Chem. 1992;267:25792-25797.
40.
27. Liu SC, Palek J, Yi SJ, et al. Molecular basis of
altered red blood cell membrane properties in
Southeast Asian ovalocytosis: role of the mutant
band 3 protein in band 3 oligomerization and retention by the membrane skeleton. Blood. 1995;
86:349-358.
41.
10. Bennett V, Healy J. Organizing the diseases fluid
membrane bilayer: diseases linked to spectrin
and ankyrin. Trends Mol Med. 2008;14:28-36.
11. Lux SE, Tse WT, Menninger JC, et al. Hereditary
spherocytosis associated with deletion of human
erythrocyte ankyrin gene on chromosome-8.
Nature. 1990;345:736-739.
12. Agre P, Casella JF, Zinkham WH, McMillan C,
Bennett V. Partial deficiency of erythrocyte spectrin in hereditary spherocytosis. Nature. 1985;
314:380-383.
13. Bruce LJ. Red cell membrane transport abnormalities. Curr Opin Hematol. 2008;15:184-190.
14. Butler J, Mohandas N, Waugh RE. Integral protein linkage and the bilayer-skeletal separation
energy in red blood cells. Biophys J. 2008;95:
1826-1836.
15. Bustos SP, Reithmeier RAF. Structure and stability of hereditary spherocytosis mutants of the cytosolic domain of the erythrocyte anion exchanger 1 protein. Biochemistry. 2006;45:10261034.
38.
42.
28. Bruce L. Mutations in band 3 and cation leaky red
cells. Blood Cells Mol Dis. 2006;36:331-336.
29. Gallagher PG, Chang SH, Rettig MP, et al. Altered erythrocyte endothelial adherence and
membrane phospholipid asymmetry in hereditary
hydrocytosis. Blood. 2003;101:4625-4627.
30. Bruce L, Guizouarn H, Burton N, et al. The monovalent cation leak in overhydrated stomatocytic
red blood cells results from amino acid substitutions in the Rh-associated glycoprotein. Blood.
2009;113:1350-1357.
43.
44.
45.
16. An XU, Mohandas N. Disorders of red cell membrane. Br J Haematol. 2008;141:367-375.
31. Gelles J, Schnapp BJ, Sheetz MP. Tracking
kinesin-driven movements with nanometre-scale
precision. Nature. 1988;331:450-453.
17. Goodman SR, Shiffer KA, Casoria LA, Eyster ME.
Identification of the molecular defect in the erythrocyte-membrane skeleton of some kindreds with
hereditary spherocytosis. Blood. 1982;60:772784.
32. Sako Y, Kusumi A. Compartmentalized structure
of the plasma membrane for receptor movements
as revealed by a nanometer-level motion analysis. J Cell Biol. 1994;125:1251-1264.
46.
33. Powles JG, Mallett MJD, Rickayzen G, Evans
WAB. Exact analytic solution for diffusion impeded by an infinite array of partially permeable
barriers. Proc R Soc London. 1992;A:391-403.
47.
18. Perrotta S, Gallagher PG, Mohandas N. Hereditary spherocytosis. Lancet. 2008;372:1411-1426.
19. Hassoun H, Vassiliadis JN, Murray J, et al. Characterization of the underlying molecular defect in
hereditary spherocytosis associated with spectrin
deficiency. Blood. 1997;90:398-406.
20. Costa FF, Agre P, Watkins PC, et al. Linkage of
dominant hereditary spherocytosis to the gene for
the erythrocyte membrane-skeleton protein
ankyrin. N Engl J Med. 1990;323:1046-1050.
21. Yawata Y. Cell membrane: The red blood cell as a
model. Weinheim: WILEY-VCH Verlag GmbH &
Co. KGaA; 2003.
34. Kusumi A, Sako Y, Yamamoto M. Confined lateral
diffusion of membrane receptors as studied by
single particle tracking (nanovid microscopy). Effects of calcium-induced differentiation in cultured
epithelial cells. Biophys J. 1993;65:2021-2040.
35. Sheetz MP, Turney S, Qian H, Elson EL.
Nanometer-level analysis demonstrates that lipid
flow does not drive membrane glycoprotein
movements. Nature. 1989;340:284-288.
22. Gallagher PG. Hereditary elliptocytosis: spectrin
and protein 4.1R. Semin Hematol. 2004;41:142164.
36. Fujiwara T, Ritchie K, Murakoshi H, Jacobson K,
Kusumi A. Phospholipids undergo hop diffusion in
compartmentalized cell membrane. J Cell Biol.
2002;157:1071-1081.
23. Gallagher PG, Kotula L, Wang YP, et al. Molecu-
37. Murase K, Fujiwara T, Umemura Y, et al. Ultrafine
48.
49.
50.
6245
membrane compartments for molecular diffusion
as revealed by single molecule techniques. Biophys J. 2004;86:4075-4093.
Salhany JM. Mechanistic basis for site-site interactions in inhibitor and substrate binding to band
3 (AE1): evidence distinguishing allosteric from
electrostatic effects. Blood Cells Mol Dis. 2001;
27:901-912.
Janas T, Bjerrum PJ, Brahm J, Wieth JO. Kinetics
of reversible DIDS inhibition of chloride self exchange in human erythrocytes. Am J Physiol.
1989;257:C601-C606.
Schofield AE, Reardon DM, Tanner MJA. Defective anion transport activity of the abnormal
band-3 in hereditary ovalocytic red blood cells.
Nature. 1992;355:836-838.
Ritchie K, Shan XY, Kondo J, Iwasawa K,
Fujiwara T, Kusumi A. Detection of non-Brownian
diffusion in the cell membrane in single molecule
tracking. Biophys J. 2005;88:2266-2277.
Giorgi M, Cianci CD, Gallagher PG, Morrow JS.
Spectrin oligomerization is cooperatively coupled
to membrane assembly: a linkage targeted by
many hereditary hemolytic anemias? Exp Mol
Pathol. 2001;70:215-230.
Corbett JD, Agre P, Palek J, Golan DE. Differential control of band-3 lateral and rotational mobility
in intact red cells. J Clin Invest. 1994;94:683-688.
Mirchev R, Karnchanaphanurach P, Lam A,
Golan DE. Single particle tracking of membrane
proteins on intact Southeast Asian ovalocytosis
red blood cells (SAO RBCs). Biophys J. 2003;84:
526A.
Nigg EA, Cherry RJ. Anchorage of a band-3
population at the erythrocyte cytoplasmic membrane surface: protein rotational diffusion measurements. Proc Natl Acad Sci U S A. 1980;77:
4702-4706.
Blackman SM, Hustedt EJ, Cobb CE, Beth AH.
Flexibility of the cytoplasmic domain of the anion
exchange protein, band 3, in human erythrocytes.
Biophys J. 2001;81:3363-3376.
Yi SJ, Liu SC, Derick LH, et al. Red cell membranes of ankyrin-deficient nb/nb mice lack band
3 tetramers but contain normal membrane skeletons. Biochemistry. 1997;36:9596-9604.
Cho MR, Eber SW, Liu SC, Lux SE, Golan DE.
Regulation of band 3 rotational mobility by
ankyrin in intact human red cells. Biochemistry.
1998;37:17828-17835.
Golan DE, Veatch W. Lateral mobility of band-3 in
the human erythrocyte membrane studied by fluorescence photobleaching recovery: evidence for
control by cytoskeletal interactions. Proc Natl
Acad Sci U S A. 1980;77:2537-2541.
Tomishige M, Kusumi A. Compartmentalization of
the erythrocyte membrane by the membrane
skeleton: intercompartmental hop diffusion of
band 3. Mol Biol Cell. 1999;10:2475-2479.