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Blood First Edition Paper, prepublished online April 15, 2009; DOI 10.1182/blood-2009-02-205450
Imaging of the diffusion of single band 3
molecules on normal and mutant erythrocytes
Gayani C. Kodippili1, Jeff Spector2, Caitlin Sullivan2,3, Frans A. Kuypers4, Richard Labotka5,
Patrick G. Gallagher6, Ken Ritchie2, Philip S. Low1
1
Department of Chemistry, Purdue University, 560 Oval Dr., West Lafayette, IN 47907;
2
Department of Physics, Purdue University, 525 Northwestern Ave, West Lafayette, IN, 47907;
3
Department of Physics, University of Illinois at Urbana-Champaign, 1110 West Green, Urbana,
IL, 61801; 4Children's Hospital Oakland, Research Institute, 5700 Martin Luther King Jr. Way,
Oakland, CA, 94609; 5Univ. Illinois, Chicago, Division of Pediatric Hematology/Oncology, 840
S. Wood St. (M/C 856), Chicago, IL 60612 ; 6Department of Pediatrics, Yale Univ. School of
Medicine, 333 Cedar St., P.O. Box 208064, New Haven, CT 06520-8064
Address correspondence to Philip S. Low ([email protected]) or Ken Ritchie
([email protected])
1
Copyright © 2009 American Society of Hematology
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Abstract
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 components, 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 sub-populations of band 3 in intact normal erythrocytes, we demonstrate
that the properties of these sub-populations 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 (e.g. spectrin,
ankyrin, etc.), 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.
2
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Introduction
In the well-studied erythrocyte membrane, a complex of membrane-spanning 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 Fig. 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
Fig. 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 (Fig. 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. Further, 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 (Fig. 1B). Finally, the spectrin tetramers may form fences that define
triangular compartments within the hexagonal spectrin lattice, and within these compartments,
unattached proteins (e.g. free band 3) might be anticipated to diffuse up to ~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
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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 multi-protein 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 two dimensional
spectrin/actin lattice can result in hereditary elliptocytosis (HE)21-23 or in more severe cases to
hereditary pyropoikilocytosis (HPP)21,24 Other morphological abnormalities can derive from
other molecular lesions, including southeast asian ovalocytosis (SAO) from deletion of residues
400-408 in band 3 25-27 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 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 and characterized its mobility in both intact normal
erythrocytes and erythrocytes with a variety of structural defects (hereditary spherocytosis,
hereditary elliptocytosis, southeast asian ovalocytosis, hereditary hydrocytosis and hereditary
pyropoikilocytosis) 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
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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.
Materials and Methods
Labeling of band 3 in intact erythrocytes with DIDS-biotin
Materials used for the synthesis of DIDS-biotin are listed in the supplemental materials. Details
regarding the synthesis (Supplementary Fig.1) and characterization (Supplementary Fig. 2) are
also provided in supplemental materials. Blood samples from patients with hereditary hemolytic
anemias (see Supplementary Fig. 3) were drawn following informed consent in accordance with
the Declaration of Helsinki and 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 3x with phosphate buffered saline, pH 7.4 containing 5mM
glucose and again the white cell fraction was removed. RBCs diluted to 5% hematocrit in the
same buffer were incubated with 10-11M to 10-13 DIDS-biotin at 37oC for 1.5h to allow covalent
reaction of the compound with band 3. This low concentration was determined from binding
studies, where ~50% binding of DIDS-biotin to RBCs was found to occur at 30µm. We used
such low concentrations in order to react approximately with one biotin linker per cell. Under
these experimental conditions, we observed that ~70% of the cells were labeled with a single
quantum dot when incubated with excess streptavidin-quantum dots. Cells with two or three qdots attached were rarely seen. DIDS-biotin labeled cells were then washed 3x with PBS
containing 0.1% BSA to remove unbound reagent and the cells were incubated at room
temperature with streptavidin Q-dot 525 or streptavidin AlexaFluor 488 for 30min. Unbound
streptavidin conjugates were removed by washing twice with 0.1% BSA in PBS, and cells were
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allowed to settle onto a pre-cleaned, poly-lysine-coated cover slip located within a custom built
chamber ready for imaging. Unattached cells were washed off of the cover slip with PBS
containing 0.1% BSA. Finally, 500µl of PBS were added to the chamber and the cells were
imaged as described below.
Single Quantum dot fluorescence video microscopy
Observation was performed on an Olympus IX-71 inverted microscope. Oblique angle
fluorescence imaging was used to excite single quantum 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, 488nm emission,
Newport) was expanded, filtered (488/10 nm line width bandpass filter, Chroma) and directed
towards the microscope objective (100x PlanApo, NA 1.45 TIRFM oil immersion, Olympus)
parallel but off the optical axis through a dichroic mirror (500 nm Cutoff, Chroma). The
resultant fluorescence image was projected through the dichroic mirror and an emission filter
(525/50 nm bandpass, Chroma), and the image was collected with a dual MCP intensified,
cooled CCD camera (XR/Turbo-120z, Stanford Photonics, Inc., 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 per
second. Each movie was recorded for 1000 frames. Only quantum dots whose trajectories were
at least 40 frames long were chosen for analysis, however, since even trajectories recorded for
1000 frames revealed band 3 diffusion for maximally 8 seconds, all data report only a brief
snapshot of band 3 behavior in the membrane. The trajectories were collected on a random
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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 quantum 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 found as the centerof-mass of the thresholded correlation intensity.
Analysis of the mobility was carried out based on the measured mean-squared displacement of
the observed label as described previously.7,32-34 For each trajectory, the MSD for every time
interval was calculated according to the formula:34,35
MSD(nδt) = (N-1-n)−1
N-1-n
∑ {[x(jδt + nδt)-x(jδt)]
2
+ [y(jδt + nδt)-y(jδt)]2 }
j=1
where δt is the time resolution and (x(jδt + nδt), y(jδt + nδt)) describes the particle position
following a time interval nδt after starting at position (x(jδt), y(jδt)), N is the total number of
frames in the sequence, n is a positive integer.
The MSD vs. 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 short-time diffusion
coefficient, Dµ , the asymptotic long time diffusion coefficient, DM, and, if applicable, the average
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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, that reacts covalently with
Lys539 of band 338, 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 one band 3 population over another.39
To convert DIDS from a simple anion transport inhibitor to a selective anchor for quantum dots
on band 3, DIDS was linked via a peptide spacer to biotin, a high affinity ligand of streptavidin,
as described in Methods. 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 Fig. 2A.
The specificity of the DIDS-biotin conjugate was established by reacting the conjugate with
intact erythrocytes, isolating their membranes, separating component proteins by SDS-PAGE,
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blotting the proteins onto nitrocellulose paper, and visualizing the location of biotinylated
polypeptides by staining with streptavidin-horseradish peroxidase. As seen in Fig. 2C, only one
polypeptide with apparent Mr ~100 kDa was labeled. Because band 3 is the only known receptor
for DIDS on human erythrocytes, and since band 3 has the molecular weight of the protein
stained in Fig. 2C (i.e. ~100 kDa), we conclude that band 3 is selectively labeled by the DIDSbiotin conjugate in intact erythrocytes. Further, since streptavidin-linked quantum dots do not
bind erythrocytes that have not been reacted with DIDS-biotin (Fig. 2B), we suggest that DIDSbiotin 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 quantum dots in order to avoid confusion due to crossing trajectories. To derivatize a single
or at most two or three 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 µM DIDS-biotin (Figure 3) and derivatization of only one or two 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 quantum dot during
single particle tracking, even stationary labels were found to report a non-zero diffusion
coefficient. This non-zero diffusion coefficient for immobilized labels sets the level of accuracy
in determining meaningful mobilities. To determine this level in the current study, the mobility
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of band 3 on acrolein-fixed RBC’s was monitored at 37◦C using the usual methodology. Figure
4 shows a typical trajectory of the labeled band 3 on these fixed intact RBC’s. From analysis of
individual band 3 trajectories on >130 of these fixed cells, two diffusion coefficients were
measured corresponding to a diffusion coefficient at short times (termed microscopic diffusion
coefficient, Dμ) and the asymptotic diffusion coefficient at long times (termed macroscopic
diffusion coefficient, DM). The top row of Figure 5 displays the distribution of Dμ and DM values
measured for labeled band 3 molecules on fixed human RBC’s. Fitting a single Gaussian
distribution to each of these sets of measurements yields a mean Dμ of 6.8×10-14 cm2/s and a
mean DM of 5.5×10-14 cm2/s.
These values establish the smallest meaningful Dμ and DM
diffusion coefficient that can be measured with our imaging system. Importantly, Dμ and DM
values for quantum dots attached directly to polylysine-coated glass slides were not significantly
different (Dμ = 6.2 x 10-14 and DM =3.2 x 10-14 cm2/s).
In comparison to fixed cells, labeled band 3 displays a higher mobility at on intact, unfixed
normal human RBCs at 37◦C. A typical trajectory is shown in Figure 4, while the distributions
of measured Dμ and DM are shown in row 2 of Figure 5 (see individual data in Supplementary
Fig. 4). Note that the observed diffusion coefficients are significantly larger than those measured
on fixed cells. The distribution of microscopic diffusion coefficients fits well with two Gaussian
curves centered at 1.4×10-11 and 2.2×10-10 cm2/s (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).
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Band 3 mobility in erythrocytes with hereditary elliptocytosis and hereditary pyropoikilocytosis
A typical trajectory of band 3 on intact erythrocytes with hereditary elliptocytosis (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 two of the three HE samples examined (Fig. 5 and Supplementary Fig. 5) represent
different molecular defects, since their osmotic deformability scans were significantly different
(see Supplementary Fig. 6). 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 Dμ values, but two Gaussian distributions for DM values; however, the converse
was true for sample HE-2 (Fig. 5 and Table 1). Thus, although the major population of band 3 in
both patient’s samples exhibited similar values of Dμ and DM of ~4×10-11 and ~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 Dμ of
1.2×10-12cm2/s.
Because ~150 cells were examined from each patient, we interpret these
behaviors to indicate that the two HE pathologies are molecularly different.
The other major morphological disorder ascribed to defects in horizontal interactions within the
membrane skeletal network is termed hereditary pyropoikilocytosis (HPP). Although analysis of
band 3 diffusion in these cells at 37◦C revealed a relatively normal Dμ of 7.9×10-11 cm2/s
(compared to 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 (Figs. 4 and 5, and Table 1). Thus, DM in HPP cells was
determined to be 1.5×10-11 cm2/s (i.e. 10x faster than normal), suggesting that long range band 3
diffusion in HPP cells is relatively unimpeded by physical barriers. Moreover, all band 3
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molecules in these cells appeared to diffuse homogeneously (i.e. only a single Gaussian
distribution was obtained for both Dμ and DM), suggesting that the two distinct band 3
populations seen in healthy cells (Fig. 5) are either indistinguishable or absent in HPP cells.
Band 3 mobility in erythrocytes with hereditary spherocytosis
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. SDSPAGE and Western blotting 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 Supplementary Fig. 7). However, no molecular defect could be
identified in any of the other four 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 six HS samples was similar, with each sample
yielding two distinct Gaussian distributions of Dμ and one distribution of DM (Rows 4 and 5 of
Fig. 5, Supplementary Fig. 8, 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 Dμ in intact
HS erythrocytes was significantly shifted towards the faster diffusing population (i.e. 80% of
band 3 in HS cells was in the faster diffusing population vs. 23% of band 3 in normal cells; Fig.
5 and Supplementary Fig. 8), 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
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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 versus 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 hereditary hydrocytosis (HHy)
Analysis of the trajectories of band 3 in intact HHy cells (Fig. 4) revealed a microscopic
diffusion coefficient (Dμ) characterized by two Gaussian distributions with mean values of
1.5x10-12 (i.e. 10x slower than the slower population in normal RBCs) and 4.4x10-11 cm2/s (i.e.
5x 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 (Fig. 5, Table 1).
This macroscopic diffusion coefficient was 2x slower than that found in normal red blood cells.
Band 3 mobility in South East Asian Ovalocytosis erythrocytes
Approximately half of the band 3 molecules in SAO erythrocytes contain a deletion of residues
400-408, the remaining copies of band 3 being normal26,27,40.
Because only the “normal”
population of band 3 can be labeled with DIDS26, analysis of diffusion data from SAO cells must
be interpreted with caution, since the mobility of the mutated band 3 is never measured. With
this caveat in mind, two Gaussian distributions of Dμ with mean values of 2.1x10-12 and 4.5x1011
cm2/s were fitted, both 5x 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
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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 red blood cells.
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 Methods). 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 Supplemental Fig. 9). 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 ~70 nm. Except for HPP erythrocytes (and three of
six HS samples shown in Supplementary Fig. 8), 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 two of the six 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 three modes of diffusion: 1) molecules that see no
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barriers within the time frame of one analysis (usually 8.0 seconds), 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 determined41. 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 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 one pathologic cell to the next.
Discussion
In order 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 (e.g. fluorescein) were strongly quenched by
the high concentration of proximal hemoglobin, the DIDS-biotin probe proved to be especially
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valuable because it allowed labeling of band 3 with quantum 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, since 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 Dμ of 1.4x10-11 cm2/s, HPP cells contained no band 3 that diffused with such slow kinetics
(Table 1, Fig. 5). Although HS cells, like normal erythrocytes, exhibited a bimodal distribution
of Dμ 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 two of the three HE samples were ~10x
faster than in normal cells. Perhaps equally interesting was the observation that even normal
copies of band 3 in cells containing mutated band 3 (i.e. SAO cells) were able to sense the
impact of their mutated counterparts (i.e. the slow and fast Dμ values in SAO cells were both
10X 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.
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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 towards a more rapidly diffusing population. If one or both of the Dμ populations were
to correspond to ankyrin- and/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 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; i.e. in agreement with previous FRAP data from
Golan and coworkers43 showing that band 3 diffuses 6x faster and has a smaller immobilized
fraction in HPP cells than in any HS or normal sample examined (see Table 1). Moreover, the
slower Dμ 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 Dμ 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 (i.e. ~70nm) 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 ~200
nm,8,9 and since spectrin tetramers are thought to be compacted to ~1/3 of this length in situ,9 the
size of a compartment in which free band 3 can diffuse might be ~70 nm.
The smaller
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compartments of 35 nm might then correspond to spectrin-anchored band 3, since as Discher and
coworkers8 note, even spectrin-attached actin hubs move/oscillate within a compartment of ~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 seconds), 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 two 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 two populations of the polypeptide, one attributed to a cytoskeletallyanchored 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
18
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spectrin content of the cell.43,48 Additional studies from the Golan lab49 have also identified two
populations of band 3 based on lateral diffusion measurements using fluorescence recovery after
photobleaching. Finally, Kusumi and colleagues7,50 introduced single particle imaging
methodologies to explore band 3 diffusion in erythrocyte ghosts, and they also identified two
diffusing populations, albeit the diffusion coefficient of only the more mobile fraction was
reported.
We have also found evidence for two major populations of band 3 in normal human erythrocytes.
Thus, analysis of over 600 cells from eight different donors yields two Dμ values and two
compartment sizes for band 3. The conundrum, however, lies in the fact that recent membrane
models (Fig. 1A) display three populations of band 3, i.e. 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.
Acknowledgements
The authors would like to thank Steven C Habicht, Derek Doorneweerd, Sumith Kalaratne,
Prasad Rajendra Bandari, Estela Campanella for helpful discussions. P.S.L. would like to
acknowledge funding from the National Institute of Health (GM24417-29). K.R would like to
acknowledge funding from the National Science Foundation (Grant #0646633).
19
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Author contributions: G.C.K., and P.S.L., design research; G.C.K performed research, G.C.K.,
synthesized and characterized the DIDS-biotin, J.S., C.S., G.C.K contribute microscopic
analysis., G.C.K., J.S., K.R., analyzed data., F.A.K., R.L and P.G.G., supply blood specimens.,
P.S.L., and K.R., wrote the paper.
The authors declare no conflict of interest
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Table 1: Microscopic and macroscopic diffusion data of healthy and disease RBCs
RBC
type
Dμ (cm2/s)
Fixed (6.8 ± 0.1) ×10-14 ††
DM (cm2/s)
-14
Percent
immobile
Percent
constrained
†
‡
Percent
free
95 ‡‡
0
5
-12
24
30
46
-11
7
37
56
-12
34
32
34
(2.2 ± 0.1) ×10-12
20
37
43
(5.5 ± 0.2) ×10
(n= 134)¶
Normal
(n= 626)
(1.4 ± 0.4) ×10
(2.2 ± 0.1) ×10
-11
-10
(77%) ¶¶
(1.7 ± 0.1) ×10
(23%)
HPP (7.9 ± 0.5) ×10-11
(1.5 ± 0.1) ×10
(n=349)
HS-1
(n=107)
HS-2
(2.7 ± 1.3) ×10
(5.5 ± 0.7) ×10
-12
-11
(27%)
(1.4 ± 0.1) ×10
(73%)
(3.8 ± 1.1) ×10-12 (42%)
(n=124) (9.6 ± 1.2) ×10-11 (58%)
24
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HE-1
11
13
(5.4 ± 0.8) ×10-
(9.8 ± 1.7) ×10- (69%)
(n=133)
HE-2
(n=212)
23
31
46
19
25
56
26
22
52
-13
31
30
39
-12
21
31
48
-12
34
28
38
-13
21
33
46
-12
25
35
40
-12
20
30
50
10
(2.0 ± 0.4) ×10- (31%)
(1.2 ± 0.1) ×10
(3.0 ± 0.3) ×10
-12
-11
(37%)
(8.8 ± 0.5) ×10
-13
(63%)
(8.2 ± 0.4) ×10-13 (20%)
(8.3 ± 0.3) ×10-13 (78%)
(n=168) (2.6 ± 0.4) ×10-11 (80%)
(8.1 ± 1.1) ×10-11 (22%)
HE-3
HHy (1.5 ± 1.2) ×10-12 (40%)
(n=171)
(4.4 ± 1.4) ×10
-11
(60%)
SAO (2.1 ± 0.1) ×10-12 (16%)
(n=173)
(4.5 ± 0.3) ×10
-11
(8.1 ± 0.4) ×10
(1.8 ± 0.1) ×10
(84%)
HS- unknown defects
HS-3
(n=231)
HS-4
(1.2 ± 1.9) ×10
(2.9 ± 0.3) ×10
(4.9 ± 0.3) ×10
-12
-11
(25%)
(6.8 ± 0.3) ×10
(75%)
-11
(4.0 ± 0.2) ×10
(n=173)
HS-5
(n=206)
HS-6
(n=136)
(1.8 ± 0.3) ×10
(6.7 ± 0.4) ×10
(2.7 ± 0.5) ×10
-12
-11
-12
(1.0 ± 0.2) ×10
(17%)
(1.8 ± 0.1) ×10
(82%)
(11%)
-10
(3.2 ± 0.1) ×10
(89%)
†
The totally immobile fraction is defined as the fraction of band 3 molecules that were 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); i.e. the fraction of band 3 that diffuses similar to band 3 on fixed cells.
25
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‡
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 standard error of the mean.
‡‡
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
Figure Legends
Figure 1. a) Model of human erythrocyte membrane showing major populations of band 3 and
associated proteins. Labeled membrane-spanning proteins include: 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) 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 linked to spectrin tetramers through
ankyrin should display limited mobility over short time periods and restricted significantly
restricted motion over long time periods, and (3) band 3 bound to the spectrin-actin junctional
complex through adducin should also show a limited (possibly even more limited than case (2))
diffusion over short time periods and similarly restricted long time diffusion.
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 (10µM)
26
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and then labeled with AlexaFlour 598-derivatized streptavidin. b) Image of unlabeled (control)
whole erythrocytes that were similarly incubated with AlexaFlour 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 1µM DIDS-biotin reagent
(lane 2) prior to 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.
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-AlexaFluor 488, washed 3x in buffered saline, and analyzed for bound AlexaFluor
488 by flow cytometry. The intensity of the bound AlexaFluor 488 fluorescence is plotted as a
function of the concentration of DIDS-biotin reagent added. 50% saturation was observed at
30µM 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/sec 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
(% of total band 3 is indicated in parenthesis).
27
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Figure 5. Distributions of the logarithms of the microscopic (Dμ) 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.
28
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Figure 2 – Kodippili et al.
a)
c)
b)
3 4
250
125
98
64
50
O
Biotin
d)
HN
NH
O
-
O
S
H
H
O
S
O
-
O
HN
NH3+
O
O
O
H
N
O
O
NCS
NH
N
H
O
S
N
H
O
O
O-
DIDS
O
HN
O
O-
O
Anionic spacer
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1 2
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Figure 4 – Kodippili et al.
HS-5 (58- 80%)
HPP (100%)
Fixed cell (100%)
HE (60-80%)
HHy (60%)
58
56
278
280
100nm
SAO( 84%)
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Normal (77%)
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Figure 5 – Kodippili et al.
Dmicro
30
Fixed cells
Compartment size
Dmacro
30
20
20
10
10
0
0
60
35nm
71nm
40
40
40
Normal RBC
20
20
20
0
0
0
60
40
40
HPP
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
4
6
0
0
20
20
36nm
4
0
12
10
10
0
0
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
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
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Prepublished online April 15, 2009;
doi:10.1182/blood-2009-02-205450
Imaging of the diffusion of single band 3 molecules on normal and mutant
erythrocytes
Gayani C Kodippili, Jeff Spector, Caitlin Sullivan, Frans A. Kuypers, Richard Labotka, Patrick G.
Gallagher, Ken Ritchie and Philip S. Low
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