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2006-11-30
The Influence of Membrane Lipid Order on Cell
Shape and Microvesiculation in Human
Erythrocytes
Laurie Jackson Gonzalez
Brigham Young University - Provo
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(2006). All Theses and Dissertations. Paper 1058.
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THE INFLUENCE OF MEMBRANE LIPID ORDER ON CELL SHAPE AND
MICROVESICULATION IN HUMAN ERYTHROCYTES
by
Laurie J. Gonzalez
A thesis submitted to the faculty of
Brigham Young University
in partial fulfillment of the requirements for the degree of
Master of Science
Department of Physiology and Developmental Biology
Brigham Young University
December 2006
BRIGHAM YOUNG UNIVERSITY
GRADUATE COMMITTEE APPROVAL
of a thesis submitted by
Laurie J. Gonzalez
This thesis has been read by each member of the following graduate committee and by
majority vote has been found to be satisfactory.
______________________________
Date
____________________________________
John D. Bell, Chair
______________________________
Date
____________________________________
William S. Bradshaw
______________________________
Date
____________________________________
Allan M. Judd
BRIGHAM YOUNG UNIVERSITY
As chair of the candidate’s graduate committee, I have read the thesis of Laurie J.
Gonzalez in its final form and have found that (1) its format, citations, and
bibliographical style are consistent and acceptable and fulfill university and department
style requirements; (2) its illustrative materials including figures, tables, and charts are in
place; and (3) the final manuscript is satisfactory to the graduate committee and is ready
for submission to the university library.
__________________________
Date
__________________________________________
John D. Bell
Chair, Graduate Committee
Accepted for the Department
__________________________________________
James P. Porter
Chair. Physiology and Developmental Biology
Accepted for the College
__________________________________________
Alan R. Harker
Associate Dean, College of Biology and Agriculture
ABSTRACT
THE INFLUENCE OF MEMBRANE LIPID ORDER ON CELL SHAPE AND
MICROVESICULATION IN HUMAN ERYTHROCYTES
Laurie J. Gonzalez
Department of Physiology and Developmental Biology
Master of Science
Exposure of human erythrocytes to elevated intracellular calcium causes
alterations in cell shape and stimulates shedding of the cell membrane in the form of
microvesicles. We hypothesized that both the shape transition and microvesiculation are
influenced by microscopic membrane physical properties such as lipid order. To test this
hypothesis, membrane properties were manipulated by varying the experimental
temperature, membrane cholesterol content, and the internal ionic environment. Changes
in membrane order were assessed using steady-state fluorescence spectroscopy with an
environment-sensitive probe, laurdan. Our observations led us to the following
conclusions: 1) the modest temperature dependence of membrane structure observed with
laurdan is shifted to lower temperatures and becomes more cooperative upon removal of
membrane cholesterol, 2) the calcium-induced shape change observed in erythrocytes
requires a decrease in membrane order, 3) the influence of membrane order is not limited
to shape transitions induced only by calcium, and 4) decreased order is also a permissive
factor for microvesicle shedding. Our data suggest that while the mechansims that
regulate the shape transition and the release of microvesicles are different, they both
require a state of membrane disorder.
ACKNOWLEDGMENTS
Gratitude is expressed to Dr. John Bell for his role as an excellent mentor and
advisor. Not only did he provide me with the opportunity to have a fine graduate
experience, but through his leadership this project was also the means of training many
undergraduate scientists, including: Jeremy Fairbourn, Taylor Brueseke, Mai Vu, Quinn
Bahm, Erin Olson, Rachel Bailey, Chisako McLemore, and Brian Thurber. I appreciate
their assistance in completing the experiments described in this paper. Thanks to
Samantha Smith for her input on the Laurdan experiments. I am deeply grateful to my
family for their encouragment and hospitality, and above all to my husband, Casey, for
his support and patience while I completed this work.
TABLE OF CONTENTS
1. INTRODUCTION…………………………………………………………..1
2. MATERIALS AND METHODS…………………………………………...3
3. RESULTS…………………………………………………………………...8
4. FIGURES AND FIGURE LEGENDS……………………………………..14
5. DISCUSSION……………………………………………………………....27
6. LITERATURE REVIEW…………………………………………………..35
7. REFERENCES…..………………………………………………………….50
vii
INTRODUCTION
Human erythrocytes are characterized by their resting discoid shape and
deformability, which allows them to pass through tiny capillaries to facilitate gas
exchange. Physical and chemical properties of the plasma membrane contribute to these
characteristics. The membrane consists of 52% protein, 40% lipid, and 8% carbohydrate
(Nakao, 2002). Intrinsic proteins of the membrane serve as a connection to an underlying
cytoskeleton and include band 3 (AE1), glycophorin C and transport adenosine
triphosphatases. The cytoskeleton is located just below the membrane and is composed
of spectrin fibers arranged in a hexagonal array, along with actin, protein 4.1, adducin,
ankyrin, p55, tropomyosin, tropomodulin, calmodulin and various others.
The primary lipid components of the membrane are phospholipids and
approximately equimolar cholesterol arranged in a bilayer (Lange et al., 1980). In
erythrocytes, the understanding of an asymmetrical arrangement of phospholipids
between the two leaflets of the bilayer has been well established. Most of the cholinecontaining lipids are found in the outer leaflet, while the majority of phosphatidylserine
(PS), phosphatidylethanolamine (PE), and phosphatidylinositol (PI) are in the inner
leaflet (Zachowski, 1993). Carbohydrates are localized on the external face of the
membrane covalently bound to lipids and proteins designated as glycolipids and
glycoproteins.
The individual behavior and interactions of the lipids and proteins in the
membrane contribute to its ability to deform. In addition to typical circulation stresses,
the cell shape is sensitive to the well-being of the cell and as erythrocytes become aged or
damaged, the shape changes. Furthermore, under these conditions the cells also exhibit
1
microvesicle release, or “blebbing” of the plasma membrane. These changes are similar
to what happens during platelet activation, and in other cell types during apoptosis and
necrosis (Chukhlovin, 1996).
Experimentally, the shape change and concomittant microvesicle release can be
induced by lowering intracellular ATP or elevating intracellular calcium. Moreover,
increased cytoplasmic calcium causes other physical changes to the membrane including
increased exposure of PS in the outer leaflet (flip-flop), susceptibility of the membrane to
hydrolysis via secretory phospholipase A2, and increased ordering of membrane lipids
(Henseleit et al., 1990; Smith et al., 2001; Vest et al., 2004).
Efforts have been made to understand more specifically the effects of calcium on
erythrocyte shape change and microvesicle release (Allan and Thomas, 1981; Bucki et
al., 1998; Comfurious et al., 1990; Gedde et al., 1997; Greenwalt, 2006; Greco et al.,
2006; Liu et al., 2005; Muller et al., 1981; Pasquet et al., 1998; Wolfs et al., 2003). What
remains to be elucidated, however, is an explanation of the potential relationship between
these phenomena and membrane fluidity. In order to accomplish this purpose, we have
manipulated membrane properties by varying the experimental temperature, membrane
cholesterol content, and the internal ionic environment of erythrocytes. We then assessed
the effects of these manipulations on the release of microvesicles and an erythrocyte
shape transition initiated by treatment with the ionophore, ionomycin.
Membrane cholesterol was removed from the erythrocytes with the cholesterolbinding agent methyl-ß-cyclodextrin (MBCD). Cyclodextrins have been used in
pharmacology research for years in delivery of lipophilic drugs and have recently become
important in membrane studies mainly because they have been shown to selectively
2
extract membrane cholesterol in multiple cell types, including erythrocytes (Klein et al.,
1995; Lange et al., 2002; Ohtani et al., 1989; Wolkers et al., 2002; Yancey et al., 1996).
Ohtani et al. showed that MBCD neither bonds to nor incorporates into the membrane
(1989). MBCD acts at the membrane surface extracting cholesterol into a central, nonpolar cavity of cyclic oligomers of glucopyranoside (Pitha et al., 1988).
Membrane order was assessed by steady-state fluorescence spectroscopy with an
environment-sensitive probe, laurdan. Laurdan is sensitive to the presence and mobility
of water molecules within the bilayer, yielding information on membrane order and
fluidity (Parasassi et al., 1991, 1993, 1997). It has been used previously to study effects
of temperature and divalent cations on membrane lipid order in erythrocytes (Best et al.,
2002; Smith et al., 2001; Vest et al., 2004). Harris et al. used laurdan fluorescence to
study the effects of membrane cholesterol concentration on fluidity in artificial
membranes (2002). Subsequently, we used this data to aid in our interpretation of the
measurements of microvesicle shedding and shape change with cholesterol-depleted
cells.
MATERIALS AND METHODS
Materials - Erythrocytes were obtained from healthy individuals undergoing
routine physicals at Brigham Young University McDonald Health Center. The samples
were fresh or stored up to 2 days at 4 °C in EDTA vacutainers from which patient
identification was removed. Control experiments comparing fresh blood with stored
samples demonstrated that the storage conditions did not influence the results (Smith et
al., 2001). Erythrocytes were isolated by centrifugation, plasma and buffy coat removed,
3
washed, centrifuged a second time, and resuspended to the original hematocrit in a lowcalcium, low-magnesium cell medium (LCCM) (NaCl = 134 mM, KCl = 6.2 mM, Hepes
= 18.0 mM, glucose = 13.6 mM, pH 7.4, 37 °C). Cell preparations for experiments using
indo-1 used MBSS as a cell medium (NaCl = 134 mM, KCl = 6.2 mM, CaCl2 = 1.6 mM,
MgCl2 = 1.2 mM, Hepes = 18.0 mM, and glucose = 13.6 mM, pH 7.4, 37 °C).
Laurdan (6-dodecanoyl-2-dimethylaminonapthalene), indo-1 (acetoxymethyl ester form),
and the Amplex Red Cholesterol Assay Kit were purchased from Invitrogen (Carlsbad,
CA). Ionomycin was purchased from Calbiochem (La Jolla, CA) and MBCD from
Sigma-Aldrich (St. Louis, MO). All other reagents are from standard sources. Laurdan
(250 µM), ionomycin (120 µM) and indo-1 (2.5 µM) were dissolved in
dimethylsulfoxide (DMSO). MBCD (0.1 mM, 0.3 mM, 1.0 mM) was dissolved in
LCCM. BaCl2, CaCl2, CoCl2, MgCl2, MnCl2, NiSO4, and Sr(NO3)2 were dissolved at
1.0 M stock concentrations in deionized water and then used at appropriate
concentrations. Control experiments indicate that the addition of these ions to LCCM at
the concentration used in this study (0.6 mM) does not significantly alter pH (Vest et al.,
2004).
Fluorescence spectroscopy - Steady-state fluorescence was monitored for
fluorescent probes laurdan and indo-1 using photon-counting spectrofluorometers
(Fluoromax and Fluoromax-3 from Jobin Yvon, Edison, NJ, and PC1 from ISS,
Champaign, IL). All three instruments maintain sample homogeneity by continuous
gentle stirring with a magnetic stir bar. Control experiments (not shown) show that stir
bar speed had no effect on light scatter noise. Sample temperature was controlled and
maintained using circulating water baths and equilibrated for at least 5 minutes to achieve
4
temperature stability. In both Fluoromax instruments, simultaneous assessment of
fluorescence intensity at multiple excitation and emission wavelengths was obtained by
rapid sluing of monochromator mirrors using control software provided with the
instrument. Band pass was set at 4 nm for both monochromators. In all instruments,
erythrocyte preparations were suspended in 2 ml of MBSS or LCCM in a quartz
fluorometer sample cell to a final density of about 3 - 4 x 106 cells/ ml.
Rayleigh light scattering - Light scattering was assessed in samples of
erythrocytes prepared as described for fluorescence spectroscopy. No fluorescent probes
were included in the experiments and monochromator settings were set to 500 nm
excitation and 510 nm emission.
Intracellular calcium influx - Intracellular calcium concentrations at 25 °C, 37 °C
and 45 °C were assessed using indo-1 as described previously (Wilson et al., 2004).
Erythrocyte samples were prepared as described above using MBSS and further diluted to
a density of 1.5 - 2 x 106 cells/ ml. Indo-1 (final concentration = 2.5 µM) was added and
incubated for 60 minutes at 37 °C in a shaking water bath. Cells were washed,
resuspended in MBSS, and placed in cuvette (final volume = 2 ml). Fluorescence was
continually monitored with excitation set at 350 nm and emission at 405 to 480 nm. A
baseline was established for 3 minutes, followed by the addition of ionomycin (final
concentration = 0.3 µM) and then a continuation of the time course for 13 additional
minutes.
Laurdan generalized polarization (GP) - Erythrocytes were prepared with LCCM
as described above for fluorescence experiments. Samples were incubated with varying
concentrations of MBCD (0.1 mM, 0.3 mM, or 1.0 mM) or a deionized water control for
5
30 minutes. After washing and resuspending cells to original concentrations, CaCl2 (final
concentration = 0.6 mM) and laurdan (final concentration = 2.5 µM) were added to bring
the total volume to 2 ml and placed in a quartz cuvette. In order to allow the laurdan to
fully equilibrate into the membrane, samples were processed through a successive
temperature series (25 °C, 30 °C, 35 °C, 40 °C, and 45 °C) recording the emission
spectrum (excitation at 350 nm and emission from 360 to 600 nm) for each temperature
to monitor incorporation. Incorporation did not change during the third series, so in the
fourth series data were collected for the desired temperatures. Results were quantified by
calculating the generalized polarization (GP) of laurdan at 435 nm and 500 nm as
described by Parasassi et al. (1991).
Quantification of cholesterol extraction - Washed erythrocytes were suspended in
MBSS to a final volume of 1 ml and incubated in the presence or absence of varying
concentrations (0.1 mM, 0.3 mM or 1.0 mM) of MBCD for 30 minutes at 37 °C. Cells
were washed and separated by centrifugation (13,500 rpm for 120 seconds) in a
microcentrifuge. The supernatant was removed and cholesterol was extracted from it by
adding 900 ml chloroform, vortexing, removing organic layer and drying under a
nitrogen stream.
Cell pellets were frozen in liquid nitrogen to lyse the cells. Samples were quickly
thawed, and lipids were extracted with chloroform and methanol using the method of
Bligh and Dyer (1959). In brief, 150 µl of chilled MBSS was added to suspend the pellet
followed by 125 µl chloroform and 250 µl methanol. After vortexing the tubes for 10
seconds, 125 µl of deionized water was added. The samples were vortexed and
centrifuged for 30 seconds at 13,500 rpm. The residual organic layer was separated and
6
dried under a nitrogen stream. Samples were stored at –20 °C until utilized for the
cholesterol assay.
Reagents and basic protocol for quantification of cell and supernatant cholesterol
were from the Amplex Red Cholesterol Assay Kit (Invitrogen). Stock solutions of
reagents were prepared and stored at -20 °C. Briefly, 50 µl of distilled water and 50 µl of
amplex red working solution was added to the dried supernatant and cell samples.
Samples were incubated for 30 minutes at 37 °C in a shaking water bath, after which 50
µl was removed and added to deionized water in a quartz cuvette (final volume = 2 ml).
Fluorescence emission spectrum were collected using excitation at 560 nm and emission
from 570 to 700 nm and values for experimental samples were compared to a standard
curve to determine concentrations.
Microscopy - Erythrocytes were suspended in 2 ml LCCM in a 24-well plate dish
at a final density of 3 - 4 x106 cells/ ml and placed in a shaking water bath and warmed to
the determined temperature. After temperature equilibration, divalent ions (BaCl2, CaCl2,
CoCl2, MgCl2, MnCl2, NiSO4) were added to different wells at a final concentration of 0.6
mM and allowed to mix for 5 minutes. Ionomycin (final concentration = 0.3 µM) was
added and after 10 minutes, the plate was removed from the bath and photomicrographs
collected on a Canon IX70 inverted light microscope. Data were collected for individual
well plates at 25 °C, 30 °C, 33 °C, 37 °C, 40 °C and 45 °C. Photomicrographs of
cholesterol-depleted cells were also collected at the same temperatures. LCCM-prepared
erythrocytes were incubated with varying concentrations of MBCD (0.1 mM, 0.3 mM, or
1.0 mM) at 37 °C for 30 minutes. After incubation, the temperature of bath was changed
to the desired experimental temperature. After reaching the set point, CaCl2 was added
7
(final concentration = 0.6 mM), followed 5 minutes later by ionomycin (final
concentration = 0.3 µM). After a 10 minute incubation, the plate was removed for image
collection.
RESULTS
At 45 °C, the addition of ionomycin to human erythrocytes at in the presence of
calcium caused an increase in the intensity of light scattered by the sample concurrent
with a decrease in the signal noise (Figure 1). Previous studies have demonstrated that the
elevated scattering intensity results from an increase in the number of particles available
to reflect light due to microvesicle shedding (Smith et al., 2001). Furthermore, changes in
light-scatter noise apparently report the transition in cell shape from discocyte to a
smaller rounded shape called spherocyte (Hoffman, 1987; Smith et al., 2001). As
discocytes, erythrocytes aggregate as rouleaux in circulation (Derganc et al., 2003).
These large structures reflect light irregularly as they move in a stirred suspension. As the
shape changes, the rouleaux break apart, and the noise decreases as the suspension
becomes more homogenous in terms of particle size. Figure 2 displays visual
confirmation of the shape changes associated with the light scattering data.
Figure 1 also shows that these changes in light scattering depended on the
experimental temperature. They were clearly evident at 45 °C, reminiscent of similar
effects observed previously at 37 °C (Vest et al., 2004). Conversely, neither the increase
in intensity nor the reduction in signal noise was observed at 25 °C. Photomicrographs of
treated cells at 25 °C do, however, show some changes. However, these alterations in
shape represent an early transition to irregular discocytes and early echinocytes compared
8
to the advanced spherocytes and spheroechinocytes seen at 45 °C. While not
quantitative, the images provide a more complete understanding of what is happening to
the cell during these changes.
In order to establish that the observed temperature dependence was not due to the
ability of ionomycin to transport ions from the extracellular environment, we used the
fluorescent probe indo-1 to report relative internal calcium ion concentration at 25 °C,
37 °C, and 45 °C. As illustrated in Figure 3, there was no statistical difference among the
treatments. Although calcium flux was slower at 25 °C than at higher temperatures, there
was no statistical difference in the end point level of calcium achieved after 600 seconds.
This result was further validated by examining longer time courses (3000 seconds, data
not shown).
The temperature effect suggests the possibility that membrane physical properties
are relevant to the processes involved in microvesicle release and the transition in cell
shape. Although microvesicle shedding stimulated by calcium influx requires specific
proteins such as calcium-dependent enzymes and ion channels (Allan and Thomas, 1981;
Smith et al., 2001), recent evidence suggests that the effect of calcium on cell shape may
involve direct interactions of the ion with the inner face of the cell membrane (Vest et al.,
2004). Specifically, it was found that while calcium uptake causes shedding at 37 °C, a
broader range of ions are capable of initiating the shape transition. Introduction of cobalt,
nickel or manganese ions (but not strontium, magnesium, or barium) into erythocytes
produces effects on cell shape similar to those caused by calcium (Vest et al., 2004). If
the hypothetical link between membrane structure and the shape transition suggested by
9
Figure 1 is real, it should be manifest for each of the ions capable of causing that
transition.
Accordingly, the experiments in Figure 1 were repeated using erythrocytes treated
with other divalent cations: manganese, cobalt, nickel, magnesium and barium (Figure 4).
As expected, the level of signal noise diminished at 45 °C upon addition of ionomycin in
each case except for magnesium and barium (data for barium not shown). Only
manganese showed any evidence for microvesicle release at 45 °C. As observed with
calcium, no changes were apparent with any ion for either parameter at 25 °C. Once
again, the photomicrographs offer additional insight. Manganese, cobalt and even
magnesium show early changes in the cell shape at 25 °C (Figure 5). As indicated in the
figure, cells treated with magnesium at 25 °C are irregular and may be due to a
nonspecific effect; repetition would be required to confirm this. At 45 °C, magnesium
had no effect, but the effect of other ions on cell shape is advanced compared to the
changes seen at 25 °C in agreement with the light scattering data.
Figures 6 & 7 show summaries of light scattering data obtained from multiple
samples like those shown in Figure 4. Figure 6 shows the effect of both low and high
temperature on light scatter intensity (MV release) in the presence of calcium,
manganese, cobalt, or nickel. At low temperatures, no ion caused a significant change.
On the contrary, at 45 °C calcium treatment produced a large increase in intensity while
cobalt resulted in a minor decrease. Magnesium and barium were also assayed and had no
effect at either temperature, consistent with recent studies (Vest et al., 2004). Figure 7
illustrates the temperature effect on signal noise. In general, the average trend at 25 °C
was toward a decrease in noise. However, this trend was sufficiently consistent to be
10
statistically significant only in the case of manganese. None of the groups were
distinguishable from each other by analysis of variance. The trend towards a decrease in
noise supports the early changes seen in the photomicrographs (Figures 2 & 5). In
contrast, at 45 °C all ions caused a reproducible diminution in noise, manganese being
the most obvious.
To further explore possible relationships between membrane physical properties
and calcium stimulation of the shape transition and microvesicle release, we repeated the
experiments of Figure 1 at many temperatures and compared the results to measurements
of laurdan fluorescence (Figure 9). Figure 8A represents the temperature dependence of
the change in average signal noise following 600 seconds of incubation with ionomycin.
In the range of 20 to 27 °C the change in noise increased steadily with temperature.
Between 27 and 43 °C, the effect of temperature was more modest. The slope became
more steep again from 43 to 48 °C. The change in light scatter intensity (Figure 8B)
followed a similar trend, although the details were somewhat different. The intensity
change rose very little from 20 to 30 °C, followed by a steep increase to 33 °C, a shallow
rise until 37 °C, and finally a sharp increase to 48 °C.
Figure 8C illustrates the effect of temperature on membrane order detected by the
fluorescent probe laurdan. Laurdan is a fluorescent probe sensitive to the presence and
mobility of water molecules trapped within the lipid bilayer (Parasassi et al., 1991, 1993,
1997) and has been shown to detect lipid order in the membrane (Harris et al., 2002).
These data were analyzed by calculating GP, a quantitative method of evaluating shifts in
laurdan spectra due to the solvent relaxation effect (Parnassi et al., 1991). Decreased GP
values indicate increased solvent relaxation due to an increase in membrane water
11
content. As the temperature increased, the GP value decreased, indicating decreased
membrane order. These results were consistent with previous observations made in our
laboratory (Best et al., 2002).
If the changes observed in shape transition and microvesicle shedding are
dependent on membrane physical properties, then altering membrane cholesterol content
should affect the results in a predictable manner. Figure 9 shows 5 panels, the upper 4
each representing samples treated with different concentrations of MBCD, followed by a
summary of superimposed data (for clarity of presentation, only means are shown). Panel
A is the same data illustrated in Figure 8C. As the concentration of MBCD increased, the
GP value decreased concurrent with a shift in the curve toward lower temperatures
suggesting a reduction in membrane lipid order. In cells with no MBCD, a mild
transition towards decreased order occurred between 35 to 40 °C. By comparison, the
cells treated with 1.0 mM MBCD saw a more dramatic transition toward decreased order
between 25 to 30 °C, showing reduced cholesterol allowed the transition to occur at a
lower temperature than in the control cells.
Data in Figure 10 justify normalizing the GP values for the initial value at 25 °C,
showing that the GP at the starting temperature of 25 °C is not significantly different for
any given concentration of MBCD. While the absolute value of GP is not distinguishable
from sample to sample because of donor effect, we wanted to know if the shape of the
temperature curve was altered by MBCD concentration. A two-way analysis of variance
was performed for the normalized data to see whether the temperature or the MBCD
concentration had a significant effect on the results (p < 0.0001 for both). The interaction
between the two variables was not significant (p = .99).
12
Visual inspection of the data in Figure 11 shows that removal of membrane
cholesterol resulted in greater differences in light scatter intensity before and after the
addition of ionomycin for both 0.3 mM and 1.0 mM MBCD. The data at 0.1 mM MBCD
do not appear very different from those obtained for the control condition. A two-way
analysis of variance showed that both MBCD and temperature had a significant effect on
the results (p < 0.001 for both), and that the interaction between the two was also
significant (p = 0.012), n = 3-4. The interaction between temperature and MBCD
concentration is not unexpected, given that the data for both variables follow the same
trend towards an increase of light scatter.
Figure 12 shows the effect of cholesterol depletion on the shape transition. Photos
and analysis of the light scattering of cells treated with 1.0 mM MBCD illustrate that the
shape change occurred prior to ionomycin treatment; cells were almost all spherocytes
regardless of whether they were treated without calcium, calcium only, calcium +
ionomycin, or ionomycin only. This effect was similar for all temperatures. The results
at 0.1 mM and 0.3 mM MBCD (not shown) also show changes in cell shape prior to
ionomycin treatment, though not as dramatic as the 1.0 mM treatment. This further
suggests that the shape transition was already occurring prior to the addition of
ionomycin.
Figure 13 illustrates the effectiveness of MBCD in removing cellular cholesterol
by comparing the amount of cholesterol in the supernatant and the cell pellet after the 30
minute incubation. Treatment with 1.0 mM MBCD removed 69% ± 9% of cellular
cholesterol compared to 6% ± 5% for cells treated without MBCD.
13
FIGURES AND FIGURE LEGENDS
600000
45C
500000
400000
300000
25C
200000
100000
0
600
800
1000
1200
Time (s)
FIGURE 1. Light scattering by erythrocytes treated with calcium and ionomycin at
25 °C and 45 °C. Samples were treated with calcium as described in Materials and
Methods. Ionomycin was added at the arrow. Fluorescence intensity is meaured as
counts per second (“cps”).
14
calcium only
calcium + ionomycin
25 °C
~100% discocytes
~50% discocytes, 50%
early echinocytes
earlyspherocytes
45 °C
~100% discocytes
~25% echinocytes,
75% spherocytes
FIGURE 2. Photomicrographs of erythrocytes treated with calcium and ionomycin
at 25 °C and 45 °C. Images are portions of field, while notes are approximations
determined by visual inspection of entire field.
15
0.3
0.2
0.1
0.0
25C
37C
45C
Temperature (°C)
FIGURE 3. Effects of temperature on transport of internal erythrocyte calcium
concentration by ionophore. Data were obtained using indo-1 to indicate change in
internal calcium concentration (see Materials and Methods). Raw intensities were
normalized using the indo-1 excitation/emission ratio to show net relative changes in
calcium concentration. The treatment groups are not significantly different by analysis
of variance (p = 0.71, n = 2-3). Data are expressed as mean ± SEM.
16
60000
60000
Manganese
Cobalt
50000
50000
45C
40000
45C
40000
30000
20000
200
60000
30000
25C
20000
400
600
800
300000
Nickel
250000
50000
400
600
800
1000
Magnesium
200000
45C
40000
150000
100000
30000
20000
200
25C
10000
200
1000
600
800
25C
50000
25C
400
45C
different
samples
25C
0
1000
600
800
1000
1200
time (s)
FIGURE 4. Light scattering by erythrocytes treated with divalent cations and
ionomycin at 25 °C and 45 °C. Samples were treated with divalent cations as described
in Materials and Methods. Ionomycin was added at the arrow.
17
45 °C
~20% discocyte, 80% early
echinocyte or beyond
Mixed group,
abundant spherocytes
~100% discocytes
~50% discocyte,
50% echinocyte
~50% discocyte, 50%
early echinocyte
~50% discocyte,
50% echinocyte
Magnesium
Cobalt
Nickel
Manganese
25 °C
~50% discocyte, 50%
echinocyte or beyond
(strange shape, may be artifact)
~100% discocytes
FIGURE 5. Photomicrographs of erythrocytes treated with various divalent cations
and ionomycin at 25 °C and 45 °C. Images are portions of field, while notes are
approximations determined by visual inspection of entire field.
18
1.5
A
1.0
0.5
0.0
-0.5
Ca
Mn
Co
Ni
1.5
B
1.0
0.5
0.0
-0.5
Ca
Mn
Co
Ni
FIGURE 6. Change in intensity of scattered light after the addition of ionomycin at
25 ºC and 45 ºC for selected cations. Data (mean ± SEM) express the difference in the
average intensity before the addition of ionomycin compared to that measured 600 s later
(normalized to intitial average intensity). Panel A, microvesicle release at 25 ºC. Means
are significantly different from each other using ANOVA (p = 0.015). The results for
each group (cation) were indistinguishable from zero based upon one sample t-tests (Ca2+,
p = 0.20, n = 4; Mn2+, p = 0.08, n = 5; Co2+, p = 0.07, n = 5; Ni2+, p = 0.26, n = 5). Panel
B, microvesicle release at 45 ºC. Means are significantly different from each other using
ANOVA (p = 0.0001). Individual differences measured with the one sample t-test show
that Ca2+and Co2+ are different from 0 (p = 0.0001, n = 6; p = 0.0011, n = 5 respectively)
while Mn2+ and Ni2+ are not (p = 0.35, n = 5; p = 0.10, n = 4, respectively).
19
0.00
-0.25
-0.50
-0.75
A
Ca
Mn
Co
Ni
0.00
-0.25
-0.50
-0.75
B
Ca
Mn
Co
Ni
FIGURE 7. Change in noise of light scattering after the addition of ionomycin at 25
ºC and 45 ºC for selected cations. Data (mean ± SEM) express the difference in the
average standard deviation (SD) before the additon of ionomycin compared to that
measured 600 s later (normalized to intital average SD). Panel A, erythrocyte shape
change (Ca2+, n = 4; Mn2+, n = 5; Co2+, n = 5; Ni2+, n = 5) at 25 ºC. Means are not
significantly different from each other using ANOVA (p = 0.87). Only Mn2+ was
significantly different from 0 based upon a one sample t-test (p = 0.02), (Ca2+, p = 0.67;
Co2+, p = 0.16; Ni2+, p = 0.16). Panel B, erythrocyte shape change at 45 ºC. Means are
significantly different from each other using ANOVA (p = 0.04). Individual differences
measured with the one sample t-test show that all ions are different from 0 (Ca2+, p =
0.0003, n = 6; Mn2+, p = 0.0003, n = 5; Co2+, p = 0.03, n = 4; Ni2+, p = 0.01, n = 4).
20
14000
11000
A
8000
5000
2000
-1000
20
30
40
50
Temperature (°C)
1.00
0.75
B
0.50
0.25
0.00
20
30
40
50
Temperature (°C)
1.1
1.0
0.9
0.8
0.7
20
C
30
40
50
Temperature (°C)
FIGURE 8. The effect of temperature on fluorescence intensity, noise and
membrane order. Panel A, raw light scattering data (like those seen in Fig. 1) were
analyzed using nonlinear regression. Pre-ionomycin and pre-ionomycin + 600 s standard
deviations were calcuated from the residuals and averaged. The data show the difference
between the two and are expressed as mean ± SEM (n = 3-15). Panel B, the same data as
Panel A were analyzed in a similar method, only here showing the difference between the
minimum post-ionomycin intensity and minimum + 240 s. Panel C, GP was calculated
as described in Materials and Methods and normalized for the value at 25 °C. Error bars
represent mean ± SEM (n = 9 for each temperature). Lines drawn in all panels have no
theoretical significance.
21
1.1
1.1
A
1.0
0.9
0.9
0.8
0.8
0.7
B
1.0
0.7
0 mM MBCD
0.6
0.6
1.1
1.1
C
1.0
0.9
0.1 mM MBCD
D
1.0
0.9
0.8
0.7
0.8
0.6
0.5
0.4
0.7
0.3mM MBCD
20
25
30
35
40
45
0.6
50
1.0 mM MBCD
20
25
30
35
40
45
50
Temperature (°C)
1.1
E
1.0
0.9
O MBCD
0.1 MBCD
0.3 MBCD
1.0 MBCD
0.8
0.7
0.6
0.5
0.4
20
Summary
25
30
35
40
45
50
Temperature (°C)
FIGURE 9. Effect of cholesterol depletion on laurdan GP. Erythrocyte samples were
prepared with MBCD to remove cholesterol as described in Materials and Methods. GP
was calculated as described in Materials and Methods and normalized for the value at 25
°C. Panel A, n = 9; Panel B, n = 10; Panel C, n = 10; Panel D, n = 9 for each
temperature. Data are expressed as mean ± SEM. Panel E illustrates the same data
expressed in the individual panels but without error bars and curves were generated by
nonlinear regression using an arbitrary function.
22
0.6
0.4
0.2
0.0
FIGURE 10. Relationship between laurdan GP at initial temperature of 25 °C and
MBCD concentration. GP values shown are the original un-normalized data from the
25 °C data point for each group in Fig. 9. Data were compared using ANOVA. The initial
GP values for the treated cells are not significantly different from each other (p = .36, n =
5-8). Data are expressed as mean ± SEM.
23
1.25
0.1 mM MBCD
1.00
0.75
0.50
0.25
0.00
-0.25
-0.50
-0.75
20
25
30
35
40
45
50
Temperature (°C)
1.25
0.3 mM MBCD
1.00
0.75
0.50
0.25
0.00
-0.25
-0.50
-0.75
20
30
40
50
Temperature (°C)
1.25
1.0 mM MBCD
1.00
0.75
0.50
0.25
0.00
-0.25
-0.50
-0.75
20
30
40
50
Temperature (°C)
FIGURE 11. Relationship between temperature and light scattering displacement
using cholesterol-depleted erythrocytes. Raw light scattering data (like those seen in
Fig. 1) were analyzed using nonlinear regression and the average intensity pre-ionomycin
and pre-ionomycin + 600 s were calculated. These data show the difference between the
two and are expressed as mean ± SEM (n = 3-15). Data are normalized for the yintercept. Control data (no MBCD) are shown as black boxes in each panel.
24
calcium only
calcium + ionomycin
no drug
~100% discocytes
~5% discocytes, 95%
echinocytes or beyond
~5% echinocytes,
95% spherocytes
~100% spherocytes
1.0 mM
MBCD
FIGURE 12. Photomicrographs of erythrocytes treated with and without MBCD in
the presence of calcium ± ionomycin at 37 °C.
25
A
100
B
75
50
25
0
0.00
0.25
0.50
0.75
1.00
[MBCD] (mM)
FIGURE 13. Cholesterol content of MBCD treated erythrocytes. Cells and
supernatant were assayed to measure cholesterol content using cholesterol
oxidase/Amplex Red assay as described in methods. Panel A shows a theoretical graph
of relative MBCD and cholesterol concentrations assuming the MBCD was able to
remove all the cellular cholesterol. Panel B shows cholesterol concentrations in cell
pellet and supernatant treated with various concentrations of MBCD (no MBCD, 0.1
mM, 0.3 mM and 1.0 mM), n = 4 for all conditions. Data are expressed as mean ± SEM.
26
DISCUSSION
Our purpose in this work was to characterize the shape transition and
microvesiculation of ionophore-treated erythrocytes in terms of microscopic membrane
physical properties such as lipid order. We reasoned that manipulation of these physical
properties by altering cholesterol content and the experimental temperature would affect
these processes in ways that can be interpreted using known lipid phase behavior
obtained from artificial bilayers (Harris et al., 2002). Our observations led us to the
following conclusions: 1) the modest temperature dependence of membrane structure
observed with laurdan is shifted to lower temperatures and becomes more cooperative
upon removal of membrane cholesterol, 2) the calcium-induced shape change observed
in erythrocytes requires a decrease in membrane order, 3) the influence of membrane
order is not limited to shape transitions induced only by calcium, and 4) decreased order
is also a permissive factor for microvesicle shedding.
1) Erythrocyte membranes display significant temperature dependence of several
membrane properties including membrane capacitance, K+-permeability inducement via
amphotericin-B derivatives, deformability, transmembrane potential, membrane order
and susceptibility to PLA2 (Bao, 1992; Best et al., 2002; Galla, 1980; Glaser, 1992;
Wietserbin, 1990; Williamson et al., 1975). Figure 9 shows a clear temperature
dependence of laurdan GP in human erythrocytes. As the temperature increases, the GP
decreases reflecting a more disordered membrane. This data are consistent with reports
from artificial membranes (Sheffield et al., 1995) as well as erythrocytes (Best et al.,
2002; Moore et al., 1999; Wolkers et al., 2002). Furthermore, the temperature
dependence of control cells (no MBCD) demonstrates a mildly cooperative phase
27
transition between 35 to 40 °C. Using FTIR to measure fluidity, Wolkers et al. also noted
low-cooperativity phase transitions at 14 °C and 34 °C in non-depleted erythrocytes
(2002), while Moore et al. saw only a monotonic increase in temperature-dependent
fluidity (1999).
The cells treated with 1.0 mM MBCD (Figure 9) saw a more drastic transition
toward decreased order between 25 to 30 °C. That the removal of cholesterol increases
the cooperativity of the transition is not surprising. One of the roles of cholesterol in the
membrane is to function as a fluidity buffer, fluidizing the bilayer in the gel state and
adding stability in the fluid phase by reducing lateral mobility of phospholipids. These
data agree with studies performed in artificial membranes (Harris et al., 2002; Silvius,
1992; 1996). Harris et al. demonstrate how incorporation of exogenous cholesterol into
artificial membranes attenuates the temperature dependent decrease in membrane order
above the the phase transition of approximately 40 °C (2002).
The fluidity buffer concept has been further corroborated in biological systems.
FTIR studies in cholesterol-depleted membranes show evidence that cholesterol
modulates membrane fluidity by decreasing the lipid order at low temperatures and
increasing the lipid order at high temperatures (Moore et al., 1999; Wolkers et al., 2002).
Our data support the concept that at higher temperatures cholesterol rigidifies the
membrane. However, at lower temperatures our temperature range does not discriminate;
or in other words, the variation in laurdan GP between donors is as great or greater than
the variation between MBCD treatments (Figure 10).
2) While it is well known that increased intracellular calcium causes a reversible
shape transition in erythocytes from discocyte to echinocyte or crenated sphere (Smith et
28
al., 2002; Smith et al., 1981; Vest et al., 2004), the mechanisms responsible for the
change are not well understood. We conclude that one of the requirements for the
calcium-induced shape transition is a decrease in membrane order. The shape transition
is clearly temperature dependent in both light scattering data and photomicrographs
(Figures 1, 2, 7, & 8) . The results at higher temperatures are reminiscent of similar
effects observed previously at 37 °C (Vest et al., 2004).
An alternative explanation for the data in the figures cited above, that the
temperature dependence of the shape transition is due to the ability of ionomycin to
transport calcium, is refuted in Figure 3. While the kinetics of the reaction may lag
somewhat at lower temperatures, when allowed to equilibrate as described in Materials
and Methods, the overall calcium influx is the same. Therefore, any explanation of
observed changes due to efficacy of ionomycin transport may be abandoned.
We reasoned that if membrane order was necessary for the shape transition to
occur, removing cholesterol from the erythrocytes would affect the shape transition in a
manner similar to temperature. However, considering the amount of cholesterol we
removed in treatment with MBCD we cannot rule out the possibility that the effects of
cholesterol depletion may be due partly or completely to changes in membrane surface
area (Shattil, 1975).
3) By utilizing other divalent cations in our experiments, we were able to
distinguish between calcium-specific effects and general effects of cations on the shape
transition. While not all cations caused a change in shape, manganese, cobalt, and nickel
were able to cause a shape change that exhibited a similar temperature dependence to that
induced by calcium (Figures 4 & 5). Therefore, we build on the observations that Vest et
29
al. (2004) made at 37 ºC by concluding that the temperature dependence that permits the
shape change is not calcium-specific. Interestingly, Vest et al. (2004) also reported that
the presence of cobalt, nickel or manganese elicited the highest responses to ionomycininduced membrane ordering when compared to the alkaline earth metals. There was a
high correlation seen between maximal response and ionic radius or electronegativity.
Thus, the unique characteristics of each ion could contribute to their ability to bind to the
membrane and cause the variations in effect that we observe with the selected ions.
The effect of various ions on the shape transition raises interesting questions. On
the one hand, we show that a shape transition is most likely to occur when the membrane
order is decreased. On the other hand, all of the ions that stimulate the transition in shape
also cause an increase in membrane order (Vest et al., 2004). So how does a decrease in
membrane order permit the shape transition? There are three possible explanations. 1)
The shape transition may require the actual conversion of a disordered to an ordered
membrane in order to change. 2) We may assume that the transition itself causes major
disruptions in the protein scaffolding network and the membrane enough to cause
ordering of the membrane through altered lipid-protein interactions. 3) The ordering of
the membrane may be a parallel event that is unrelated to the shape transition.
While our data can not distinguish among these three possibilities, limited
evidence suggests that that the first explanation is preferred. An interesting concept
introduced by Glaser and Leitmannova in 1975 called the “rigidity-pattern hypothesis”
proposes a model of shape transformation based upon lateral inhomogeneities in the
erythrocyte membrane. They suggest that shape dynamics of erythrocytes must be
considered in the context of occurrence of microphases and local lipid domains (see
30
Literature Review for more detail). Furthermore, studies with two-photon microscopy
that looked at the spatial distribution of order across the membrane demonstrate that the
effect of calcium on membrane order is markedly different from ordering caused by
decreasing temperature. Lowering the temperature increases order in a general,
homogeneous fashion while increased internal calcium (or other ions) expanded regions
or more ordered domains. This suggests the possiblity, since ionomycin causes ordering
heterogenously on top of a fluid background, that the ordering caused by the introduction
of the cations may directly influence the shape transition.
4) The connection of membrane physical properties with microvesicle release is
not well-established. Our data illustrate that calcium-induced microvesicle release shows
a clear temperature dependence (Figures 1, 6, & 8B). In addition, removal of membrane
cholesterol created an environment more willing to release microvesicles (Figure 11).
These observations strongly suggest that decreased membrane order is an important
component of the mechanism that regulates microvesicle shedding.
To address the possibility of a non-specific artifact of MBCD, we included a
control experiment in which we pre-loaded the MBCD molecules with erythrocyte
cholesterol, and then assessed its effect on calcium-induced microvesicle release (data not
shown). The pre-loaded MBCD treatment showed an significant attenuation of
microvesicle shedding when compared to 1.0 mM MBCD treated cells, illustrating that
MBCD alone is not responsible for the shedding. Care should be noted that cells treated
with 1.0 mM MBCD are considered fragile and have, at time, undergone hemolysis at
high temperatures.
31
An alternate interpretation of the data that reflect temperature dependence of
microvesicle release and shape change is that changes in activation energy of
enzymatically driven processes or other protein lipid interactions may play a role.
However, the fact that MBCD treatment yielded similar results as temperature
manipulation in the case of microvesicle release suggests that changes in membrane
properties are a more likely explanation than activation energies, although we cannot rule
out that some of the temperature dependence may be due to enzymatic function.
Clearly, proteins have been implicated in the mechanisms that regulate
microvesicle release. Interactions between calcium and specific proteins such as calpain,
the Gardos channel and scramblase are well documented as being linked to the
phenomenon (Allan and Thomas, 1981; Smith et al., 2001). However, the data presented
herein show that the physical properties of membrane lipids are also involved. For
example, Best et al. (2002) notes that the “flip-flop” which causes the asymmetrical
distribution of lipids (involving scramblase) is not temperature sensitive. This reinforces
the idea that the effects we are observing are also membrane-dependent, rather than
exclusively protein-mediated.
In contrast to the results regarding the shape transition, we show in Figure 6 that
only calcium was able to produce a robust microvesicle response at 45 ºC (manganese did
cause an effect, however the reaction was greatly reduced compared to calcium). This is
in agreement with the observations made by Vest et al., 2004. While the shape transition
from discocyte to echinocyte is often accompanied by the release of microvesicles, the
ion specificity of the response suggests that the mechanisms of the two events are
different.
32
How would the order of the lipid bilayer affect the ability of the membrane to
produce vesicles? Membrane domains of varying fluidity, i.e. lipid rafts, may provide
insight to the relationship. A study by Salzer et al. concludes that lipid rafts are involved
in the vesiculation process because the calcium-induced microvesicles contain high
amounts of lipids and proteins from rafts, specifically cholesterol, gangliosides, somatin,
flotillin, and AchE. They suggest that elevated calcium induces segregation of different
rafts selectively and that membrane fusion proteins such as synexin may also play a role
(2003). This idea is further supported by two-photon data that suggest that the plasma
membranes of ionophore-treated erythrocytes also exhibit an increase in liquid-ordered
domains (Vest et al., 2006). Whether MBCD removes cholesterol selectively within
domains or generally throughout the cell is not well established (Hoessli et al., 1998;
Steck et al., 2002; Rothblat et al., 1999).
The data presented in this paper offer new insight as to the reason that the
cholesterol:phospholipid ratio (normally approximately 0.8 mol/mol of phospholipid,
(Lange et al., 1980)) in erythrocyte membranes is tightly regulated. When the membrane
cholesterol pool is depleted, cells undergo a change in the average order of the membrane
that makes them susceptible to vesiculation and possibly a shape change. At
physiological temperature, the cholesterol:phospholipid ratio provides just enough
rigidity to give the cell strength and stability in its discoid shape, yet allows the cell to be
fluid enough to be flexible while moving through the microcirculation. Moreover, by
illustrating the requirement of a disordered membrane for the cell to shed microvesicles,
and changes in shape, we provide compelling evidence that membrane order is a critical
33
component of the mechanisms that regulate both of these processes in human
erythrocytes.
34
LITERATURE REVIEW
Regulation of Erythrocyte Shape
“…a classic problem in cell biology”
The normal resting shape of the erythocyte is a flattened, biconcave disk called a
discocyte (or normocyte) 8 µm in diameter and possesses a unique ability to deform
enough to pass through capillaries as small as 2 µm wide. In vitro, various treatments are
known to change the normal resting shape reversibly into other shapes such as
echinocytes (spiculated cells), stomatocytes (cup-shaped cells) and spherocytes (small,
round cells). Standardized classification of the morphological details of these shapes and
their various stages was established in work by M. Bessis after the introduction of the
scanning electron microscope (1972). For example, a type II echinocyte maintains a
disk-like appearance with cellular projections that vary in length while the type III looks
more like a spherical erythrocyte with numerous spicules.
While the morphological details have been well characterized, to this day the
mechanisms responsible for the transformation of discocyte to echinocyte, or discocyte to
stomatocyte are less understood. This remains a “classic problem in cell biology”
(Mukhopadhyay et al., 2002) and is the first of eight questions posed by Joseph Hoffman
for red blood cell physiologists to ponder in this millenium (2001).
Implications from clinical studies
Hereditary disorders of the erythrocyte membrane can offer important clues to
the mechanisms of shape regulation. Hereditary spherocytosis, hereditary elliptocytosis,
hereditary pyropoikilocytosis, and hereditary stomatocytosis comprise an important
35
group of hemolytic anemias that can be diagnosed by looking at the various shapes
present in blood smears. Some disorders are linked to deficiencies in or inactive
cytoskeletal proteins such as hereditary spherocytosis and elliptocytosis (Liu et al., 1990;
Mohandas et al., 1992). Sickle-cell anemia (HbS) is caused by the auto-oxidation of
membrane proteins such as band 3, which disrupts the assembly of the cytoskeleton
leading to sickling (Platt and Falcone, 1995). Other types of anemias may have a variety
of causes that are less understood or unknown, such as the molecular basis for hereditary
stomatocytosis. For more information regarding these hemolytic anemias, refer to a
recent review on the subject (Gallagher, 2005).
More generally, pathologies such as vascular disease, diabetes (type II), childhood
leukemia, and liver disease have been shown to alter the morphology of red blood cells
(Perotta et al., 2001; Turchetti et al., 1997). Administration of certain drugs, such as
cyclosporine or lidocaine, can cause morphological changes (Nishiguchi et al., 1989;
Ohsako, et al. 1998). Erythrocytes from patients with chronic obstructive pulmonary
disease, Down syndrome, and those who have suffered an acute myocardial infarction all
show evidence of decreased fluidity of red blood cell membranes (Przybylska et al.,
2000; Saldhana et al., 1999; Santini et al., 1997). While review of these pathologies is
helpful in determining various protein and membrane components that may be
responsible for altering cell shape and membrane properties, it is important to note that
the mechanisms may be different than the experimentally-induced, reversible changes in
shape on which this report focuses.
Perturbations that can cause shape changes
Normal, healthy erythrocytes can undergo shape transformations in vitro to
36
echinocytes or stomatocytes (or the final stage for both, spherocyte) upon addition or
variation in: electrolyte concentrations (Deutike, 1968; Herrman et al., 1985; Rasia,
1998) including calcium (which will be addressed in more detail at the end of this
section), pH (Gedde et al.,1997; Libera et al., 1997; Rasia, 1998), ATP concentration
(Gov and Safran, 2005; Nakao et al., 1960, Weed et al.,1969), amphiphilic compounds
such as lipids, fatty acids, various drugs, etc. (Chen et al., 2003; Deutike, 1968; Iglic et
al., 1998; Isomaa et al., 1997; Sheetz and Singer 1974,) and the rearrangement of
asymmetrically organized membrane lipids (reviewed by Zachowski, 1993). A
relationship exists between erythrocyte shape and the transmembrane electric potential;
consequently, the effect of pH and and other compounds can also be attributed to the
transmembrane potential (Bifano et al., 1984; Glaser, 1992, 1998). See citations for an
interesting debate on this topic.
The effect of elevated intracellular calcium on the shape of erythrocytes
It is well known that increased intracellular calcium causes a reversible shape
transition in erythrocytes from discocyte to echinocyte or crenated sphere (Smith et al.,
2001; Smith et al., 1981;Vest et al., 2004). In normal erythrocytes, the concentration of
calcium is kept at levels below 1.0 mM (via a calcium-ATPase pump and calmodulin),
however these levels can be increased due to ATP depletion (inhibition of outward
calcium pumping) or introduction of calcium via ionophore (Wiley and McCullough,
1982). Elevated intracellular calcium with ionophore causes rapid echinocytosis,
decreased potassium levels, dehydration and volume decrease (Dreher et al., 1980;
Gedde, 1997; Kirkpatrick et al., 1975). This is commonly known as the Gardos effect.
37
Chukhlovin carefully reviewed the connection between echinocytosis and
apoptosis, saying “the transition of the normal discocyte to echinocyte is often induced by
the same factors that cause apoptosis of blood cells (i.e. ionizing radiation and other ROIinducing agents, exogenous oxidants, in vitro aging conditions, cytosolic calcium
increase, etc. (1996).” He also cited three mechanisms that may explain the calciuminduced changes in shape. Briefly, 1) altered transverse asymmetry of membrane
phospholipids caused by the inhibition of aminophospholipid translocase, 2) activation
of PKC, disruption of the bonds between proteins 4.1 and band 3 and 3) decrease in
phosphotidylinositol bi and monophosphates which also cause alterations in membrane
cytoskeleton contacts (1996). These proposals all implicate protein-dependent regulation
of cell shape.
Moreover, it is known that the membrane structure and stability of the cell is
especially sensitive to the interaction of calcium and proteins. In the presence of calcium
levels higher than 0.1 mM, the calcium-calmodulin complex reduces the affinity of
protein 4.1 to the spectrin-actin region of the cytoskeleton, weakening the system
(Takakuwa and Mohandas, 1998). However, similar concentrations of calcium also
reduce the affinity of protein 4.1 to the cytoplasmic domain of band 3, and increases the
binding of band 3 to ankyrin (Takakuwa, 2000). Liu et al. suggest the opposing effects of
these events can regulate membrane structure and stability through an unknown
mechanism (2003). Furthermore, using atomic force microscopy, Liu et al. showed that
extra calcium binding on the cytoplasmic surface of the membrane increases cytoskeleton
rigidity and prevented spectrin aggregation (2005).
38
Alternatively, Sikorski et al. emphasizes that protein 4.1 and spectrin are capable
of binding to the lipid portion of the membranes, not only to other proteins (2000). The
interaction of protein 4.1 with PS-containing membranes was inhibited by calcium (Sato
and Ohnishi, 1983). Sikorski et al. postulate whether lipid binding can substitute for the
regulatory processes involved with protein 4.1, i.e. phosphorylation and calciumcalmodulin binding (2000). Other studies with artificial membranes (Binder et al., 2001;
Binder and Zschornig, 2002; Hauser, 1991) and erythrocytes (Vest et al., 2004) claim that
divalent cations can alter cell membrane physical properties by interacting directly with
the bilayer, instead of through protein-mediated regulation.
Bilayer Couple Theory
In 1974, Sheetz and Singer proposed the bilayer couple theory that explains shape
change based upon the relative surface area of the outer membrane leaflet compared to
the inner leaflet, and that the bending elasticity of the membrane is caused by the area
elasticity of the two lipid layers and their fixed distance. For example, a positivelycharged amphiphile added to the membrane will penetrate and remain in the relatively
negative inner leaflet. Consequently, the surface area of the inner layer expands slightly
while the outer layer remains the same, causing the membrane to form cup-shaped
cavities (stomatocytosis) to accommodate the extra area. Alternatively, a factor which
leads to the expansion of the outer leaflet forms convex structures like the spicules of the
echinocyte (also called crenate cells). Although many studies support the bilayer couple
theory (Chen et al., 2003; Farge and Devaux, 1992; Lange and Steck, 1982; Sheetz and
Singer, 1974; Tachev et al., 2004), many phenomena argue against the bilayer theory
being the only valid hypothesis explaining the change in shape.
39
Protein Scaffolding
The protein scaffolding theory maintains that is it the organization of the intrinsic
and cytoskeletal proteins that largely determine cell shape, and the asymmetrical
distribution of the bilayer may be a result, but not a cause, of a shape transition (see
review by Nakao, 2002). For example, the amphipathic molecule 2,4-dinotrophenol
(DNP); a cup-former, and chlorpromazine; a crenator, are still able to induce shape
changes in ghosts that demonstrate a large hole (hence, a discontinuous membrane) in the
lipid layer at 4 °C (Lieber, 1982). Furthermore, after the cytoskeleton was destroyed
using proteinases, the amphipaths were unable to cause morphological changes (Jinbu,
1982). Anderson and Lovrien fixed the cytoskeletal proteins of intact cells with wheat
germ agglutination antibodies, and found that the cell shape remained unchanged upon
addition of ionophore and calcium (1981). The bilayer hypothesis and studies that
support protein regulation of shape are not mutually exclusive, and a complete
description of the mechanism most likely requires elements of both ideas. As mentioned
previously, Sikorski et al. reviewed the interaction of membrane skeletal proteins with
membrane lipid domain and pointed out a variety of cases where structural proteins such
as spectrin and protein 4.1 can bind directly to the lipid layer instead of other membrane
proteins to provide a “scaffolding” (2000). These lipid-protein interactions may prove to
be a connection between the two theories.
Band 3 conformation hypothesis
Band 3 (Anion exchanger 1, AE1) was one of the first erythrocyte membrane
proteins to be characterized, mainly because of its abundance (1.2 x 106 copies per cell,
representing 10% of the surface area and 25% of total membrane protein) (Fairbanks et
40
al., 1971). It has been the subject of many reviews in its role in maintaining cell
homeostasis and regulating cell shape (Inaba et al., 1996; Jay and Cantley, 1986; Tanner
1993). However, it was Wong who proposed a specific mechanism of eythrocyte shape
control in which Band 3 plays a central role and claims that it explains several
observations related to human erythrocyte shape (Wong, 2006 – see article for history of
other Wong papers).
Briefly, transport of an anion changes the conformation of the transmembrane
domain of Band 3, which in turn changes the conformation of the cytoplasmic domain, as
both are flexibly linked. Decrease and increase of the outward/inward band 3 ratio folds
and unfolds spectrin. The conformational change in spectrin contracts and relaxes the
cytoskeleton to promote echinocytosis and stomatocytosis, respectively. Alternatively,
Gimsa and Reid (1995) proposed the Band 3 conformation-controlled bilayer couple
hypothesis, which maintains that the change of the ratio of inward and outward binding
sites in turn significantly alters the membrane monolayer area ratio, the external
orientation increasing the area of the outer leaflet.
Rigidity-pattern hypothesis
Glaser and Leitmannova (1975) proposed a model of shape transformation based
upon lateral inhomogeneities in the erythrocyte membrane called the rigidity-pattern
hypothesis. It considers a changing pattern of membrane areas with different bending
properties. The size of the areas may be governed by the metabolic state of the cell, and
the location by the bending of the membrane. For example, rigid areas may be extruded
from strongly bent regions into flat areas of the membrane. Glaser further postulates that
it is likely that membrane composition in strongly bent regions differs from that of other
41
areas (1992). For support, Glaser cites Bessis and Prenant (1972) for establishing that a
membrane “memory” exists: spicules from echinocytes occur in the same places after
reversing and restarting the process. Thus, the shape dynamics of erythrocytes must be
considered in the context of the occurrence of microphases and local lipid domains.
Cell volume can change shape
Hereditary stomatocytosis is a group of disorders associated with abnormalities in
cation permeability that lead to changes in cell volume, whether increased (hydrocytosis)
or decreased (xerocytosis). Hydrocytosis is characterized by significant stomatocytosis,
severe hemolysis, and macrocytosis, along with elevated cytosolic sodium concentration,
reduced potassium, and increased total cation content…predictably elevating cell water
content. Interestingly, xerocytosis is characterized by echinocytes and variable numbers
of stomatocytes and is linked to hyperkalemia (see review by Gallagher, 2005).
Relationship between domains, order and shape
A few papers have given some insight to the general principles associated with
how physical properties of the bilayer may be involved in the erythrocyte shape
transition. Typical stomatocyte ⇔ discocyte ⇔ echinocyte shape changes based on the
bilayer couple hypothesis have been used to study the transmembrane motion of lipids, as
pointed out by Zachowski in a comprehensive review of the subject (1993). He also
notes that the two leaflets of the bilayer have different physical properties due to the
degree of saturation in the fatty acid chains of resident phospholipids, i.e. lower local
microviscosity and faster lateral diffusion in the inner leaflet. Fisher (1993) claims that
monolayer bending must be taken into consideration to accurately describe shape
changes.
42
A possible connection between order, shape, and membrane proteins exists due to
the fact that spectrin can partially penetrate the lipid bilayer. Data show that the removal
of spectrin induced an increase in the spin-labeled order parameter and a decrease when it
was reassociated (Sikorski et al., 1987). Similar results were achieved when the
fluorescent probe, diphenylhexatriene, was used to measure fluidity (Michalak et al.,
1993).
In an effort to understand the existence of lipid domains in the erythrocyte
membrane, Moore et al. published a series of papers based upon a novel FTIR method to
study the conformational order and likelihood of membrane domain formation of specific
phospholipids (1996, 1997, 1999). They used acyl chain perdeuterated lipid species that
acted as “reporter molecules,” providing structural, phase transition, and miscibility info
similar to their proteated counterparts based upon a well-established correlation between
the methylene stretching frequencies and acyl chain conformational order. Briefly, they
concluded that 1) echinocytic erythrocytes have domains of PC and PE in the outer leaflet
of the bilayer. The phase transitions were broadened in erythrocytes compared to pure
lipids, indicating a relatively small domain size or mixing of other components into the
domain. 2) The order of lipids that commonly reside in the inner leaflet (PE and PS) is
altered when they are forced to reside in the outer layer. PE appears to be a mostly pure
lipid domain (cooperative transition) and PS shows no cooperative change but has higher
conformational order than when it is located in the inner leaflet. 3) The observed phase
behavior of incorporated species appears to be a strong function of cell morphology.
When erythrocytes were incubated with 1:1 mixtures of PS-d54/PC or PS/PC-d54
(d54 indicating perdeuteration, or the reporter lipid), the lipids partioned into their
43
expected monolayers while maintaining the discocytic shape of the cell. In these cases,
no phase transitions were observed and order was measured as an intermediate between
common gel or liquid crystal values. Therefore, the existence of domains as suggested by
the behavior of incorporated lipids as described in 1 and 2 is dependent on echinocytic
morphology and further speculated to be located in the protrusions of the spicules.
Influence of temperature on shape change
Erythrocytes observed at high temperatures (50 to 55 °C) turn to spherocytes, and
exhibit budding and fragmentation that has been linked to denaturation of the cytoskeletal
protein, spectrin (reviewed by Williamson et al., 1975). Alternatively, cells stored at low
temperatures develop echinocytosis over time. This has been linked to metabolic
changes due to storage, not temperature per se (Nakao et al., 1960).
Regulation of Erythrocyte Vesiculation
Physiological Importance of Microvesicles
Virtually all cells shed microvesicles (MVs), however erythrocytes have proven a
good model to study the phenomenon because of their simplicity. Microvesiculation is
the loss of membrane from the tips of the spicules of echinocytes and occurs in vivo as
well as during storage (Laczko et al., 1985). In vivo, MVs are removed from circulation
by the reticuloendothelial system. MVs shed by platelets have clinical importance
because of their ability to promote coagulation and platelet adhesion to the
subendothelium (see review by Heemskerk et al, 2002). In erythrocytes, the release of
membrane probably alleviates some of the osmotic effects due to calcium-stimulated
potassium efflux through the Gardos channel. It has also been proposed that the vesicles
44
provide a defense mechansim for erythrocytes from attack by complement (Iida et al.,
1991). Further suggestion of a protective role comes from a study in which poly
(ethylene-glycol) cholesterol induces echinocytosis and is located in the tips of the
spicules, as though the cell is trying to sequester the exogenous compound (Baba et al.,
2004). Along with membrane blebbing and PS exposure, MV release is an external
indicator of apoptosis (Chukhlovin, 1993).
Calcium-induced microvesiculation
Like the shape transition, the MVs that are released from the spicules of the
echinocyte have been well-characterized by their size and composition (Salzer et al.,
2001; 2002). Microvesicles can be produced by elevated calcium concentrations, depleted
ATP levels, or the addition of various amphipathic molecules (Allan and Thomas, 1981;
Hagelberg and Allan, 1990; Kralj-Iglic et al., 2000; Smith et al., 2001; Vest et al., 2006).
Between 1975 and 1980, a series of papers published by D. Allan and colleagues were
the first to study MV release in detail, focusing on the effect of elevated calcium
introduced by ionophore. They characterized the composition of vesicles as depleted in
PC, and cytoskeletal proteins spectrin, actin, and glycophorin while retaining band 3 and
acetylcholinesterase (see review by Greenwalt, 2005).
High calcium concentrations lead to alterations in the membrane protein patterns,
namely protein aggregation and cytoskeletal rearrangement due to proteolysis of protein
4.1 by calpain (Croall et al., 1986). Calcium also causes the breakdown of
phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate with a
simultaneous increase in 1,2-diacylglycerol and phosphatidic acid (Allan and Michell,
1975). Calcium activates scramblase and inhibits aminophospholipid translocase which
45
results in a distortion of phospholipid organization, especially PS exposure on the
extracellular surface, over the two leaflets of the bilayer (Comfurious et al, 1990).
Moreover, calcium stimulates the Gardos channel and the concomitant potassium efflux
exerts osmotic effects which may result in loss of cell water and cell shrinkage due to
blebbing (Maher and Kuchel, 2003). Recently, the GTPase Rap2 was shown to be
selectively enriched in microvesicles stimulated by calcium influx (Greco et al., 2006).
Of all the morphological or biochemical changes that calcium causes, it appears that the
activity of calpain, scramblase, and potassium efflux are required for cells to release
vesicles (Smith et al., 2001).
Bilayer and microvesicle release
Beyond the proteins that have been implicated as responsible for microvesicle
release, there exists evidence that the influence of the bilayer also plays a role. One of
which is the ability of amphipathic molecules to cause MV release (as stated above). In
addition, Salzer et al. concludes that lipid rafts are involved in the vesiculation process by
showing calcium-induced vesicles that are high in lipids and proteins from rafts,
specifically cholesterol and gangliosides, somatin, flotillin, AchE, etc. They suggest that
elevated calcium induces segregation of different rafts selectively and that membrane
fusion proteins such as synexin may also play a role (2003).
Role of cholesterol in the erythrocyte membrane
Cholesterol and membrane microscopic physical properties
Artificial membranes consisting of one species of phospholipid are characterized
by a phase transition temperature (Tm) from the gel to liquid-crystalline state. In contrast,
46
biological membranes exhibit a more complex thermal phase behavior because of the
mixture of phospholipids, sterols, and proteins with preferential interactions between
them. For example, one of the roles of cholesterol in the membrane is a fluidity buffer,
fluidizing the bilayer in the gel state and adding stability in the fluid phase by reducing
lateral mobility in between phospholipids (Wolkers et al., 2002). This effect can be seen
in a study of DPPC vesicles performed recently in our laboratory that show how
incorporation of exogenous cholesterol attenuates the temperature dependent decrease in
membrane order in a dose-dependent manner above the the phase transition of
approximately 40 °C (Harris et al., 2002). This data is in agreement with other studies in
artificial membranes (Silvius, 1992; Silvius et al., 1996).
The fluidity buffer concept has been further corroborated in biological systems.
In 2002, Wolkers et al. performed FTIR studies that show evidence that cholesterol
modulates the membrane fluidity by decreasing the lipid order at low temperatures and
by increasing the lipid order at high temperatures. His group also noted distinct phase
transition temperatures of 14 °C and 34 °C in erythrocytes, which is surprising given that
the normally high concentrations of cholesterol in the membrane usually mask distinct
phase transitions (Moore et al. saw only monotonic increases in fluidity in erythrocytes
(1999)).
Spatial removal of cholesterol
Whether cholesterol removal via MBCD occurs generally or selectively from
lateral domains or leaflets is not well understood. Cholesterol interacts preferentially
with sphingomyelin, creating a fluidity intermediate called the liquid ordered phase in
microdomains commonly known as lipid rafts (Ipsen, 1987). Rothblat et. al. observed
47
biexponential kinetics of cholesterol efflux, a fast pool (t1/2= 19 to 23 seconds) and a slow
pool (t1/2= 15 to 30 minutes) using both model membranes and a variety of cell lines (not
erythrocytes) (1999). Ilangumaran and Hoessli observered three different kinetic pools
for cholesterol in lymphocyte membranes that received the following assignments: the
fluid phase (fastest), the liquid ordered phase of the microdomains (intermediate), and a
transition zone (slowest) in between that is postulated to exist to provide “a smooth
transition in the energy states between the two phases” (1998). This indicates that MBCD
selectively removes cholesterol from outside the sphingolipid microdomains.
In contrast, Steck et. al. (2002) showed in erythrocytes that all cholesterol efflux
followed first order kinetics with a t1/2 < 1 second. While their data do not show evidence
of different kinetic pools, they do find that the transbilayer diffusion of cholesterol is fast
compared to the kinetics of extraction. They interpret this result to indicate that MBCD
removes cholesterol from both the inner and outer leaflet of the bilayer.
Cholesterol in lipid rafts
Much of the research regarding lipid rafts has been in artificial membranes,
however biological rafts have been implicated in a number of important cellular signaling
events, including apoptosis (Scheel-Toellner et al., 2002). Leidy et al. (2002) reviewed
the relationship between cholesterol and lipid raft aggregation and/or function and shares
the following relevant information. Erythrocyte membranes contain a high concentration
of cholesterol, hence, there is only a mildly cooperative phase transition at high
temperatures because cholesterol interacts with sphingomyelin within domains but also
partitions into the fluid phase. Lower concentrations of cholesterol would favor
increased domain formation (as seen in platelets & other cell types). In addition, the fact
48
that exogenous cholesterol can alter the phase boundaries in artificial cells suggests that
cholesterol is active at the domain boundary.
There are established interactions between cholesterol content and protein
function in lipid domains (Hao et al., 2001; Hoessli et al., 1998; Samuel et al., 2001;
Westover et al., 2003). This must be taken into account in the discussion of the effect of
cholesterol depletion on events such as shape change and MV release. However, the
significance of interference from protein-based signaling pathways is limited by the use
of erythrocytes as a model.
49
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LAURIE J. GONZALEZ
56 47 SW REDTOP PLACE • CORVALLIS, OREGON 973 33
PHONE (541 ) 20 7-30 34 • E-MAIL go nzal ezla urie@hot mail .com
Education
2006 M.S. Physiology and Developmental Biology, Brigham Young University
Thesis: “The influence of membrane order on cell shape and microvesiculation in
human erythrocytes” GPA: 3.8
2001 B.S. Zoology with Honors, Brigham Young University
Honors Thesis: “The effect of elevated intracellular calcium on membrane fluidity
in human erythrocytes : a fluorescent probe study” GPA: 3.5
Technical Experience
• Graduate Research Assistant, Biophysics Lab, BYU
• Graduate Research Assistant, Reproductive Endocrinology Lab, BYU
• Laboratory Technician, Huntsman Cancer Institute, SLC, Utah
Teaching Experience
• Instructor, Introduction to the Human Body and Cell Biology, Linn-Benton
Community College, Albany, Oregon, 2004-2005
• Teaching Assistant at BYU, Biopsychology, 2003; Biology 100, 2002-2003;
Human Anatomy and Physiology, 1995-1996
• Spanish Teacher, Missionary Training Center, BYU, 1999-2000
Posters
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•
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Biophysics Society Annual Meeting, 2006, Salt Lake City, UT
Society for Neuroscience Annual Meeting, 2003, New Orleans, LA
5th International Symposium of the Role of Soy in Treating Chronic Disease,
Orlando, FL
Experimental Biology Annual Meeting, 2003, San Diego, CA
Publications
Vest RS, Gonzales LJ, Permann SA, Spencer E, Hansen LD, Judd AM, Bell, JD. 2004. Divalent
cations increase lipid order in erythrocytes and susceptibility to secretory phopholipase A2.
Biophysics Journal 86(4):2251-60.
Smith SK, Farnbach AR, Harris, FM, Hawes AC, Jackson LR, Judd AM, Vest RS, Sanchez S,
Bell JD. 2001. Mechanisms by which intracellular calcium induces susceptibility to secretory
phospholipase A2 in human erythrocytes. Journal of Biological Chemistry 276 (25): 22732-41.
Skills
Fluent in spoken and written Spanish; public speaking; computer software
(proficiency in all Microsoft Office, Corel and Adobe programs with emphasis in
creating multi-media presentations); statistical analysis (Prism Graphpad, Minitab,
S-Plus, SAS); laboratory skills: in-situ hybridization, immunohistochemistry,
fluorescence spectroscopy and microscopy.
Personal
Enjoy photography, cooking, alpine skiing, triathlons, playing and coaching
women’s lacrosse
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