* Department of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan and Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Meguro-ku, Tokyo 153-0041, Japan
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Abstract |
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Mechanisms that regulate the movement of a membrane spanning protein band 3 in erythrocyte ghosts were investigated at the level of a single or small groups of molecules using single particle tracking with an enhanced time resolution (0.22 ms). Two-thirds of band 3 undergo macroscopic diffusion: a band 3 molecule is temporarily corralled in a mesh of 110 nm in diameter, and hops to an adjacent mesh an average of every 350 ms. The rest (one-third) of band 3 exhibited oscillatory motion similar to that of spectrin, suggesting that these band 3 molecules are bound to spectrin. When the membrane skeletal network was dragged and deformed/translated using optical tweezers, band 3 molecules that were undergoing hop diffusion were displaced toward the same direction as the skeleton. Mild trypsin treatment of ghosts, which cleaves off the cytoplasmic portion of band 3 without affecting spectrin, actin, and protein 4.1, increased the intercompartmental hop rate of band 3 by a factor of 6, whereas it did not change the corral size and the microscopic diffusion rate within a corral. These results indicate that the cytoplasmic portion of band 3 collides with the membrane skeleton, which causes temporal confinement of band 3 inside a mesh of the membrane skeleton.
Key words: lateral diffusion; erythrocyte membrane; membrane skeleton; single particle tracking; optical tweezers ![]() |
Introduction |
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MANY functions of the cellular plasma membrane
are based on its interaction with the membrane
skeleton. In particular, the topological and mechanical basis for the functions of the plasma membrane is
largely provided by the interaction with the membrane skeleton: i.e., such diverse functions as anchoring of specific membrane proteins, changes in the cell shape, localization of membrane proteins in polarized cells, and
assembly of specific membrane proteins in specific functional domains, such as coated pits, caveolae, and cell-cell
and cell-substrate adhesion structures, can only be
achieved by collaboration with the membrane skeleton (Carraway and Carraway, 1989; Bennett, 1990
; Bretscher,
1991
; Pumplin and Bloch, 1993
; Kusumi and Sako, 1996
).
However, our knowledge about how membrane proteins
interact with the membrane skeleton is limited.
The red blood cell membrane has long served as a paradigm for the studies of the interaction between membrane
proteins and the membrane skeleton (for reviews see Bennett, 1990; Luna and Hitt, 1992
; Bennett and Gilligan,
1993
). In particular, the involvement of the membrane
skeleton in slowing the diffusion of band 3 has been suggested in many investigations (for reviews see Golan, 1989
; Saxton, 1990
). The lateral diffusion coefficient (D)1
of band 3 was enhanced by a factor of 50-100 in mutant
erythrocytes that possess no membrane skeleton (Sheetz
et al., 1980
; Corbett et al., 1994
). Experimental modulations of the spectrin skeleton greatly affected D of band 3 (Golan and Veatch, 1980
; Schindler et al., 1980
; Smith and
Palek, 1982
; Tsuji and Ohnishi, 1986
). Rotational diffusion
measurements of band 3 provided evidence that only 20-
40% of band 3 molecules are bound to the membrane skeleton at 37°C (Nigg and Cherry, 1980
; Tsuji et al., 1988
;
Matayoshi and Jovin, 1991
; Tilley et al., 1991
; Corbett et
al., 1994
). The fraction of the rotationally immobile band 3 agrees well with that of the translationally immobile band
3 as measured by fluorescence redistribution after photobleaching (FRAP) in broad ranges of temperature and
tetramer/dimer ratios of spectrin (Tsuji et al., 1988
), suggesting that immobile band 3 in FRAP measurement reflects band 3 molecules that are bound to the membrane
skeleton.
Based on the results of FRAP and rotational diffusion
measurements of band 3 in ghosts with various ratios of
spectrin dimers versus tetramers, Tsuji et al. (1988) proposed a spectrin dimer-tetramer equilibrium gate model in
which long-range translational diffusion of band 3 molecules that are not bound to the membrane skeleton is restricted by nonspecific barriers imposed by the skeletal network and the rate of translational diffusion is regulated
by the fraction of spectrin dimers (open gate) and tetramers (closed gate). Kusumi et al. (1993)
proposed a general model to explain the slowness of lateral diffusion
of many other membrane proteins in nonerythroid cells
(membrane-skeleton fence model; see Fig. 1), in which the
cytoplasmic portion of a membrane protein collides with
the membrane skeleton, which causes temporal confinement of the membrane protein within a mesh of membrane skeletal network.
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For further understanding of the mechanism by which
the membrane skeleton regulates the lateral mobility of
band 3, we think it necessary to directly observe corralling
of band 3, collision of band 3 with the membrane skeleton,
corral size, hops from one mesh to an adjacent one, and its
frequency. Recent advent of single particle tracking (SPT)
(Wilson et al., 1996; Choquet et al., 1997
; Schütz et al.,
1997
; for review see Saxton and Jacobson, 1997
) and optical tweezers (for review see Sheetz, 1998
) would allow one
to just do these. In the present investigation, we used these
two techniques to elucidate the regulation mechanism of band 3 diffusion.
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Materials and Methods |
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Gold Probe Preparation
Antibodies specific for the extracellular domain of band 3 were provided
by Y. Takakuwa (Tokyo Women's Medical College, Tokyo, Japan). They
were raised in a rabbit against a 17 amino-acid synthetic peptide (SKLIKIFQDHPLQKTYN, residues 538-554). The specificity of the antibodies was examined by Western blotting after SDS PAGE of the erythrocyte
ghost, by the method described previously (Manno et al., 1995). The antibodies specifically recognized band 3 without any apparent binding to
other proteins. IgG was purified by column chromatography of immobilized protein A (Pierce Chemical Co., Rockford, IL) and digested with papain (immobilized papain; Pierce Chemical Co.) as described previously
(Porter, 1959
). Fab fragments were then purified on a protein A column.
Gold particles of 40 nm in diameter were prepared and "the minimal
protecting amount of protein", which is actually the minimal concentration of the protein that is needed to stabilize colloidal gold in suspension,
was determined as described previously (De Mey, 1983; Leunissen and De
Mey, 1989
). One-fiftieth of the minimal protecting amount of anti-band 3 Fab was mixed with the minimal protecting amount of rabbit Fab (Zymed
Laboratories, Inc., San Francisco, CA). The mixture was added to the suspension of colloidal gold, pH 7.4, and mixed on a slowly tumbling shaker
for 10 min at room temperature. The gold probe was further stabilized
with 0.05% Carbowax 20 M (Sigma Chemical Co., St. Louis, MO) and 1% BSA (Sigma Chemical Co.). After three washes by sedimentation and resuspension in 0.02% Carbowax 20 M/20 mM Tris/150 mM NaCl, pH 8.0, the gold probe was resuspended in 5 mM sodium phosphate buffer, pH
8.0, containing 10 mM NaCl, sterilized by filtration with a 0.22-µm filter
(Millipore Corp., Waters Chromatography, Bedford, MA), and then used
within 24 h.
Gold probe for spectrin was prepared by conjugating the minimal protecting amount of anti-human spectrin (Sigma Chemical Co.) with 40-nm colloidal gold solution, pH 9.0, followed by stabilization and washes as described above.
To probe the lipid movement, acyl-modified phosphoethanolamine (1-hexadecanoyl-2-[1-pyrenehexanoyl]-sn-glycero-3-phosphoethanolamine; Molecular Probes, Inc., Eugene, OR) was conjugated with FITC (isomer I; Molecular Probes, Inc.) in the head group (Fl-PE), and was incorporated in the erythrocyte ghost. To prepare the gold label for Fl-PE, Fab fragments of anti-fluorescein (anti-Fl) IgG (Molecular Probes, Inc.) were first prepared by papain digestion followed by protein A column chromatography. One-fiftieth of the minimal protecting amount of anti-Fl Fab was mixed with the minimal protecting amount of rabbit Fab. The mixture was added to a suspension of 40-nm colloidal gold, pH 7.0, and the gold probe was prepared as described above.
Preparation of Erythrocyte Ghost and Gold Labeling
Human erythrocyte ghosts were prepared as described by Fairbanks et al.
(1971). Human blood was obtained from M. Tomishige (Type B, Rh+).
Red blood cells were sedimented by centrifugation at 1,500 g for 10 min,
and were washed four times with 5 mM sodium phosphate buffer, pH 8.0, containing 140 mM NaCl, 0.7 mM PMSF, and 0.7 mM (p-amidinophenyl)
methanesulfonyl fluoride hydrochloride (pA-PMSF). Erythrocytes were
lysed by incubating in 5 mM phosphate buffer, pH 8.0, on ice for 20 min.
After lysis, ghosts were washed twice with 5 mM phosphate buffer, pH 8.0, containing 0.7 mM PMSF and pA-PMSF, and then washed twice more
with 5 mM phosphate buffer, pH 8.0, containing 10 mM NaCl and 0.7 mM
PMSF and pA-PMSF.
To attach the erythrocyte ghosts stably to a coverslip, coverslips were pretreated with 1 mg/ml poly-L-lysine hydrobromide (Wako, Tokyo, Japan) for 15 min at room temperature and washed with distilled water. Ghosts were incubated on coverslips for 10 min at 0°C. This procedure also greatly reduced fluctuating movements of the ghost membrane. After three washes at room temperature, a chamber was prepared by inverting the coverslip on a slide glass using strips of adhesive tape (~0.2-mm thick) as spacers, and an ~0.6 nM gold probe and 0.07 mg/ml (~ 1.2 mM) anti- band 3 Fab were added simultaneously at room temperature (Fab was required to reduce cross-linking of band 3 by the gold probes, see Results). The chamber was sealed with paraffin (Wako), and was placed on the stage of an optical microscope for immediate observation. The microscope was placed in a specially constructed chamber in which the temperature was maintained at 37 or 26°C (±1.5).
Trypsin Treatment of Ghosts
Trypsin treatment of ghosts was carried out as described previously (Tsuji
and Ohnishi, 1986). Erythrocyte ghosts (2 mg of membrane protein per
milliliter) were incubated with 0.5 µg/ml trypsin (Wako) for 40 min at 0°C
in 5 mM phosphate buffer, pH 8.0, containing 10 mM NaCl. The reaction
was stopped by adding 2.7 mM PMSF and 1.9 mM pA-PMSF (final concentrations) followed by centrifugation. The time course of protein digestion was monitored by SDS-PAGE followed by Coomassie blue staining
and Western blotting.
High-speed Video Microscopy
The movement of colloidal gold particles was observed at 37 and 26°C
with a time resolution of 33 ms using contrast-enhanced differential interference microscopy as described previously (Kusumi et al., 1993). For observation with improved temporal resolutions (2 and 0.22 ms), a charge-coupled device camera was replaced by a digital high-speed video camera
with a C-MOS sensor (model FASTCAM-ultima; Photron, Tokyo, Japan). For high-speed video microscopy, bright-field optical microscopy
rather than differential interference contrast microscopy was used to increase the light intensity on the photodetector plane, which enhanced
the signal-to-noise ratio in the image. In addition, a green interference filter was removed, and thus only UV and infrared filters were used. After
these modifications, the light intensity at the detector was increased by a
factor of 150. This compensates for the decrease in the exposure time by a
factor of 150 (from 33 to 0.22 ms). The maximal observation time, which
was limited by the present frame memory size of 1,024 frames, was 0.23 s
at a time resolution of 0.22 ms. Even at this higher light intensity, the
movement of band 3 as observed at the normal video rate before and after
the exposure of high-intensity light for at least 30 s was not affected. Images were recorded digitally on the frame memory of the camera. The sequence of images was replayed at the video rate with analogue and digital
enhancement by an image processor (model DVS-3000; Hamamatsu Photonics, Hamamatsu City, Japan), and recorded on a laser disk recorder
(model TQ-3100F; Panasonic, Kadoma, Japan).
Positions of the gold particles were determined as described previously
(Kusumi et al., 1993). The accuracy of the position measurement was estimated from the standard deviation of the coordinates of 40-nm gold particles fixed in a 10% polyacrylamide gel on a poly-L-lysine-coated coverslip, and was 5 and 17 nm at time resolutions of 2 and 0.22 ms,
respectively.
Quantitative Analysis of Band 3 Movement
Data analysis was basically the same as described previously (Kusumi et al.,
1993; Sako and Kusumi, 1994
). The microscopic diffusion coefficient Dmicro
was calculated as the slope of the mean-square displacement (MSD)-
t
plot for 0.44-0.89 ms (2-4 video frames) by least-square fitting. Higher
time resolutions were required to measure Dmicro for membrane proteins
undergoing confined diffusion in smaller domains, since the time between
successive video frames (step size) must be sufficiently short compared
with an average time for membrane proteins to collide with the fence (
tc = A/4Dmicro; i.e., 4.4 ms for Dmicro of 5.3 × 10
9 cm2/s and the domain size A
of 9,300 nm2) (Qian et al., 1991
; Saxton, 1995
). The lengths of the confinement area in the x and y directions, Lx and Ly, respectively, were estimated by fitting the MSD-
t plot from
t = 0.22 to 5 ms using equations
described previously (Kusumi et al., 1993
).
The instrumental noise determined by using gold particles fixed in a
polyacrylamide gel appeared as a sharp rise at the first step in the MSD-t
plot. The noise leveled off rapidly after the first point. The relative movement of gold particles to band 3 may also be superimposed on the translational diffusion of band 3. Such movement may involve conformational
fluctuations of proteins and rotational diffusion of band 3. However, the
rates of these movements tend to be much faster than the frame rate of
0.22 ms/frame. Therefore, these movements would be averaged over 0.22 ms, and even when these movements show up in an MSD-
t plot, they are likely to be concentrated in the first step in the MSD-
t plot. When Lx
and Ly were determined, the effects of these noise components were eliminated by fitting the MSD-
t plot after subtracting the noise amplitude
(estimated as y-intersect of the least square fitting between one and three
video frames) from the experimental MSD-
t.
Optical Tweezers Experiment
Carboxylate-modified latex beads (1 µm-diam [], 50 µl suspension, 2.5%
solid; Polysciences, Inc., Warrington, PA) were mixed with 100 µl of 1 mg/
ml anti-band 3 antibodies and 200 µl of 7 mM sodium acetate buffer, pH
6.5. After incubation for 15 min at room temperature, 10 mg of water-soluble carbodiimide (Dojindo, Kumamoto, Japan) was added (pH was adjusted to 6.5 with dilute NaOH) and mixed on a shaker for 2 h at room
temperature. The reaction was stopped by addition of glycine to a final
concentration of 100 mM and incubation of the mixture for 30 min. After
four washes by centrifugation at 250 g for 20 min and resuspension in 1%
BSA/5 mM phosphate/10 mM NaCl, pH 8.0, the pellet was resuspended in 250 µl of 1% BSA/5 mM phosphate/10 mM NaCl, pH 8.0, and stored at
4°C. Latex beads were mixed with gold probes and anti-band 3 Fab, and
the mixture was applied to the erythrocyte ghosts preadsorbed onto the
poly-L-lysine-coated coverslip.
The optical trapping system was described previously (Sako and
Kusumi, 1995; Kusumi et al., 1998
). Beads were trapped by optical tweezers and were dragged along the membrane by moving the laser beam.
Movements of the beads and gold particles were observed by a contrast-enhanced Nomarsky microscopy at 37°C. The maximal trapping force for
the latex particle with a 1-µm
in the present setup was ~20 pN.
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Results |
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Labeling of Band 3 with Colloidal Gold Particles in the Erythrocyte Membrane
Band 3 in human erythrocyte ghosts in a hypotonic medium was labeled with 40-nm- colloidal gold particles
conjugated with Fab fragments of anti-band 3 antibodies.
These antibodies specifically recognized band 3 molecules
with no apparent binding to other proteins, as found by
Western blotting of the erythrocyte ghost membrane (refer to Materials and Methods). The movement of gold particles attached to band 3 was observed by a contrast-enhanced bright-field optical microscopy at 37°C. At the
initial stage of the present study, the minimal protecting
amount (refer to Materials and Methods) of anti-band 3 Fab was used to coat colloidal gold particles. The number
of such gold probes attached to the cell surface was ~10
particles/cell on average, which was ~20-fold greater than
that of nonspecific rabbit Fab-gold probes. However, most of the gold particles did not show long-range diffusion. It is thought that, due to cross-linking of band 3 molecules by gold particles, either the cross-linked band 3 molecules are bound to the membrane skeleton because of
higher avidities, or they rarely cross the membrane skeleton corrals.
To decrease the degree of cross-linking by gold particles, we tried to decrease the number of specific Fab fragments attached on the surface of a gold particle (paucivalent gold probes; Lee et al., 1991). For this purpose,
various amounts of anti-band 3 Fab fragments were mixed
with the minimal protecting amount of rabbit nonspecific
Fab. At the mixing ratios of 1:20 and 1:50 (anti-band 3 Fab/nonspecific Fab, wt/wt), the number of gold particles attached on the cell surface decreased to ~5 per cell. However, only ~10% of gold particles exhibited long-range
diffusion.
To further reduce cross-linking by gold probes, paucivalent gold probes and free anti-band 3 Fab (which is not
bound to gold particles) were premixed and added to the
erythrocyte ghosts attached to a coverslip. The number of
bound gold particles on the cell surface further decreased
with an increase in concentration of added free Fab, indicating specific binding of paucivalent gold probes. Percentage of band 3 molecules that undergo long-range diffusion was increased with an increase of added Fab
concentration. At a Fab concentration of 0.07 mg/ml, the
number of gold particles attached on the cell surface decreased to three or four per cell, and 65% particles exhibited long-range diffusion, which is close to the mobile fraction
determined by lateral (FRAP) and rotational diffusion
measurements under the same conditions (80%, Tsuji et al., 1988; 75% on a 10-ms time scale, Blackman et al., 1996
;
two-thirds, Nigg and Cherry, 1980
; Matayoshi and Jovin,
1991
; Tilley et al., 1991
; Schofield et al., 1992
; Che et al.,
1997
). Under these conditions, Fab-gold particles occasionally came off from the membrane surface. These results suggest that, under the conditions used in the present work, most gold particles are attached to one or possibly
small groups of band 3 molecules without inducing large-scale cross-linking of band 3. As shown later, various macroscopic diffusion parameters observed here are also consistent with previous FRAP data. We believe that, at the
present moment, numerical agreements in the mobile fractions and their diffusion coefficients between SPT and
FRAP data under variety conditions are best evidences we
could collect for the lack of induction of large band 3 aggregates by the gold particles.
It should be noted that all experiments reported in this
article have been carried out at low ionic strength conditions, which is likely to promote formation of spectrin
dimers over tetramers. Tsuji and Ohnishi (1986) found
that under the same conditions used here, 67% of spectrin
molecules are dimers (the rest are tetramers), whereas under an isotonic condition 50% of spectrin molecules are
dimers. Therefore, lateral diffusion of band 3 is likely to be
enhanced under the conditions used here. These conditions were selected to simplify the comparison with most
of the previous lateral and rotational diffusion measurements of band 3 (Golan and Veatch, 1980
; Nigg and
Cherry, 1980
; Tsuji and Ohnishi, 1986
; Tsuji et al., 1988
;
Clague et al., 1989
; Matayoshi and Jovin, 1991
; Tilley et al.,
1991
; Schofield et al., 1992
; Blackman et al., 1996
; Che
et al., 1997
).
Two-thirds of Band 3 Undergoes Macroscopic Diffusion in the Erythrocyte Ghost Membrane
Approximately two-thirds of the gold particles attached to
band 3 were undergoing simple Brownian diffusion when
they were observed with a time resolution of 33 ms (video
rate) (Fig. 2 a). The remaining one-third of the particles
exhibited oscillatory movements within small regions
(~100 nm-) during the observation period of 10 s (Fig. 2
b). These particles are likely to represent band 3 molecules
that are bound to the membrane skeleton. Blackman et al.
(1996)
suggested a possibility that ~25% of band 3 molecules are aggregated to form ~5,000 mers (and each aggregate is rotationally mobile). These aggregates may also
contribute as the band 3 fraction that do not undergo long-range diffusion in the present measurement.
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The particles that were undergoing macroscopic diffusion covered all over the erythrocyte surface without exhibiting any preferred location. During an observation time of 10-30 min, they often stopped and stayed in an area for a while (several minutes), and they started traveling again (Fig. 2 c). These observations suggest that band 3 binds to and dissociates from the spectrin network in a matter of several minutes, and that the percentage of mobile particles that undergo long-range diffusion was determined by the equilibrium of band 3 binding to the spectrin network.
Band 3 Molecules That Do Not Exhibit Long-range Diffusion Are Bound to the Membrane Skeleton
In SPT observations, one-third of band 3 molecules did
not exhibit long-range diffusion at 37°C. The percentage of
such particles was close to the immobile fractions in translational (FRAP) and rotational (anisotropy decay) diffusion measurements (Tsuji and Ohnishi, 1986; Tsuji et al.,
1988
), suggesting that the immobile fractions in these experiments represent band 3 molecules bound to the membrane skeleton. The band 3 molecules that do not undergo long-range diffusion in SPT showed oscillatory movements (Fig. 2 b), which was observed even at a time resolution of 0.22 ms (Fig. 3). When such particles were dragged
along the membrane by optical tweezers at a maximum
trapping force of 0.25 pN, they could be dragged ~300 nm
until they escaped from the optical trap and returned to
the initial position (Fig. 4, a and c). Such movement and
responses to being dragged are very similar to those of
gold particles attached to spectrin (Figs. 3 and 4). Taken
together, these results indicate that band 3 molecules that
are not undergoing long-range diffusion are bound to the
spectrin network, and that the oscillatory restricted motion that these band 3 molecules exhibit would represent
thermal conformational fluctuations of the membrane
skeletal network.
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Two-thirds of Band 3 Molecules Undergo Hop Diffusion over Many Membrane Compartments
To characterize the movement of mobile band 3 molecules, short-term movements of mobile band 3 in the ghost membrane were observed using a high-speed video system (Fig. 5 a). Fig. 5, b and c show typical trajectories of band 3 recorded with time resolutions of 2 and 0.22 ms, respectively. These recording rates are greater than those of normal video by factors of 17 and 150, respectively. By following the trajectory in Fig. 5 b closely by eye, plausible compartments (domains) were found, as shown in different colors. Occasions of hops between compartments can be clearly identified as shown in the blue and green lines in Fig. 5, b and d. The adjacent compartments are closely apposed to each other, and the particles did not return to the previous compartment before the next hop occurs. When a particle did return within several seconds to the domain that it had passed through previously, the domain looked similar with regard to position and shape (Fig. 5 d). Therefore, these trajectories suggest that these band 3 molecules are temporarily confined within domains and occasionally hop to adjacent domains.
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Fig. 5 c shows a representative trajectory of band 3, with a better time resolution of 0.22 ms for 120 ms. It is likely that this particle stayed in the same domain during this period. The shape of the domain in which the particle is confined could be visualized indirectly by the trajectory of band 3, and was almost random without any characteristic shape.
Quantitative Analysis of Band 3 Diffusion
For a quantitative analysis of the movement of mobile
band 3 molecules, the MSD of the particle was plotted
against the time interval (t) (Fig. 6, a and b). Almost all
of the MSDs showed a rapid rise and leveling off in a time
window of ~10 ms (Fig. 6 a). Statistical analysis according
to the method by Kusumi et al. (1993)
indeed showed that
all of the mobile band 3 molecules undergo confined diffusion in a time window of 20 ms. The rapid rise near
t = 0 reflects fast diffusion within a compartment (the microscopic diffusion coefficient Dmicro). In a longer time window, the MSD-
t plot asymptotically approached a
straight line with a constant positive slope (Fig. 6 b). Since
the absence of hops would result in a horizontal line in a
longer time regime (Saxton, 1989
; Kusumi et al., 1993
), the
positive slope represents hop diffusion over different compartments (the macroscopic diffusion coefficient DMACRO).
Therefore, the MSD-
t plots in Fig. 6, a and b indicate that
intercompartmental hops take place on a time scale of
1,000 ms, which can be detected even in a time window of
50 ms (Fig. 6 a). These results are consistent with the impression of the trajectories shown in Fig. 5, b-d, and suggest that the macroscopic diffusion takes place as a result
of a series of intercompartmental hops.
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By analyzing the MSD-t plots in the time window of 10 ms using the method described in Kusumi et al. (1993)
,
Dmicro and the size of the confinement domain L were obtained. Distribution of Dmicro is shown in Fig. 7 a. The median value is 5.3 × 10
9 cm2/s (Table I), which is close to a
value one might expect for freely diffusing membrane proteins in the plasma membrane (Poo and Cone, 1974
; Golan et al., 1984
; Berk and Hochmuth, 1992
; Cole et al.,
1996
), suggesting that band 3 undergoes free diffusion within a compartment. Dmicro is also similar to the diffusion rate of band 3 in spectrin-deficient mouse erythrocyte
ghosts (Sheetz et al., 1980
) and in spectrin-deficient erythrocytes from patients with hereditary spherocytosis (Corbett et al., 1994
) as measured by FRAP (Table I), which
again suggests that band 3 undergoes uninhibited diffusion
within each compartment.
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Distribution of the size of the confinement domain L is
shown in Fig. 7 c. The median value for L is 110 nm (Table
II), which is by a factor of 1.6 greater than the estimated
mesh size of ~70 nm based on the number of spectrin tetramers of ~105 copies/cell and the surface area of the
erythrocyte membrane of ~135 µm2 (Steck, 1989). The
median value for confinement domain area A is 9,300 nm2
(Table II). This compartment size should be compared
with the actual mesh size of the spectrin network. The
atomic force microscope images of the cytoplasmic surface
of erythrocyte ghosts (in the low-ionic strength buffer) after rapid freezing and freeze drying showed that the median mesh area was 2,500 nm2 (Takeuchi et al., 1998
), approximately one-fourth of the compartment area for band
3 diffusion, suggesting that approximately half of the spectrin skeleton (square root of one-fourth) acts as a barrier
for the lateral diffusion of band 3. One half of spectrin molecules are likely to be located near or on the membrane surface and function like diffusion barriers. The barrier may be only transiently lowered when the spectrin
molecules undergo large conformational changes and/or
when spectrin tetramers dissociate to form dimers, which
allows band 3 molecules to hop to an adjacent compartment. In contrast, the other half of the spectrin molecules may be located far from the membrane surface and/or may
be dimers (open gates), which do not act as diffusion barriers.
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DMACRO of band 3, which describes the rate of diffusion
over many compartments were obtained in a time regime
over 500 ms (Fig. 6 b). DMACRO observed by SPT is expected to be close to the diffusion coefficient measured by
FRAP (DFRAP), because DFRAP represents the diffusion
rate in a time scale of ~10 s (time required for most of the
bleached molecules to leave the beached area of ~1-µm-). The median value for DMACRO is 6.6 × 10
11 cm2/s,
which is smaller than Dmicro by a factor of as much as 80 (Fig. 7 b and Table I). DMACRO was also measured at 26°C
to compare with that previously obtained by FRAP (Tsuji
and Ohnishi, 1986
). The SPT results showed a mobile fraction of 38% and DMACRO of 4.6 ×10
11 cm2/s, which is in
good agreement with the data (mobile fraction of 40% and
diffusion coefficient of 5.3 ×10
11 cm2/s) reported by Tsuji
and Ohnishi (1986)
(Table I).
What is the Molecular Basis for Hop Diffusion of Band 3?
These results support a model of the hop diffusion of band
3 in human erythrocyte ghosts, as shown in Fig. 1 a. The
plasma membrane is compartmentalized into microcompartments of an average of 0.01 µm2 with regard to the lateral diffusion of band 3. Band 3 molecules undergo almost
free diffusion within a domain (slowed only by the presence of other membrane proteins; Dmicro ~53 × 1010 cm2/s).
The average residency time within a compartment can be
calculated from the median values of DMACRO and the
compartment size (area/4DMACRO, Sako and Kusumi, 1994
),
which gives ~350 ms (Table II), i.e., band 3 molecules
move from one compartment to an adjacent compartment at an average frequency of ~2.8 s
1. The long-range diffusion of band 3 occurs as a the result of successive intercompartmental hops.
The next question is what constitutes the compartment boundaries for the lateral diffusion of band 3. Our working hypothesis for the compartmentalization of the membrane with regard to the lateral diffusion of band 3 involves collision of the cytoplasmic domain of band 3 with the spectrin network, as shown in Fig. 1 b. Since most of the membrane skeleton resides in close proximity to the cytoplasmic surface of the plasma membrane, the cytoplasmic domain of band 3 collides with the membrane skeleton and cannot readily move to an adjacent compartment (membrane-skeleton fence model).
Deformation of the Membrane Skeleton Using Optical Tweezers Caused Forced Movement of Band 3
To examine whether or not the cytoplasmic domain of band 3 actually collides with the membrane skeleton, we displaced the mesh of the membrane skeleton by dragging and deforming the skeletal network with optical tweezers, and investigated whether the displacement of the network causes forced movements of band 3 molecules that are not bound to the membrane skeleton (Fig. 8 a). If band 3 is confined by collision with the spectrin skeleton, even band 3 molecules that are not bound to the skeleton should be displaced by moving the membrane skeleton network.
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A 1-µm- latex particle coated with anti-band 3 IgG
molecules was attached to the center of an erythrocyte
ghost by bringing the particle and holding it there in contact with the membrane using optical tweezers. Since such
particles are bound to many band 3 molecules and one-third of these molecules in turn are bound to the membrane skeleton, the latex particles can be linked to the
spectrin network by way of band 3. When the particle that had been attached to the center of the ghost was dragged
by optical tweezers with a maximal trapping force of ~20
pN, the membrane skeleton was deformed, as observed by
the displacement of 40-nm gold particles attached to spectrin via anti-spectrin antibodies (data not shown). The
contour of the ghost was practically unaffected, because
the ghosts were stably attached to the coverslip via poly- L-lysine (Fig. 8 b). The latex bead and gold particles attached to the spectrin network were displaced as expected
for those attached to a two-dimensional elastic continuum
(our unpublished observation), i.e., the membrane skeleton was deformed rather than simply translated by such
dragging.
As the network was dragged at a rate of 1.8 µm/s, band 3 molecules that had been undergoing hop diffusion (labeled with paucivalent 40-nm gold particles; Fig. 8 c, red) were displaced in the direction of dragging (Fig. 8 c, blue, also see Fig. 8 b). When the dragging was stopped 2 µm away from its original location and the latex particle was held there for 7 s, the translated band 3 molecules were found to be still undergoing hop diffusion at new locations (Fig. 8 c, orange). When the spectrin network was returned to its original location by moving back the latex bead with optical tweezers, the band 3 molecules were displaced again. However, these molecules did not return to their original locations due to hop diffusion they underwent during these processes (Fig. 8 c, green). They continue hop diffusion at new locations (Fig. 8 c, magenta). Gold particles attached to the spectrin network (or those bound via band 3) returned to their original locations (data not shown). These results directly show that the steric hindrance imposed by the spectrin network on the diffusion of band 3 exists, which causes temporary confinement of band 3.
Forced Movements of Band 3 by Dragging of the Membrane Skeleton Is Not Due to Viscous Drag in the Membrane
Dragging of the membrane skeleton moves skeleton-bound membrane proteins and lipids, which would induce a viscous drag in the fluid membrane. To examine the extent to which such viscous drag affects the movements of free band 3 and lipids, the following two experiments were carried out. First, the scan rate of the optical trap to drag the membrane skeleton was decreased by a factor of 12 to 0.15 µm/s (Fig. 9 a). Gold particles attached to band 3 that are undergoing hop diffusion followed just as they did at a dragging rate of 1.8 µm/s (Fig. 9 b). The deviation of the trajectories of band 3 perpendicular to the direction of dragging increased as the dragging rate was decreased. This can be explained by the presence of hop diffusion of band 3, which was superimposed on the unidirectional movement due to dragging of the membrane skeleton.
|
Second, the lipid in the outer leaflet of the membrane was observed when the membrane skeleton was dragged. Fluorescein-phosphatidylethanolamine (Fl-PE) artificially incorporated into the ghost membrane was labeled with gold particles, and then the membrane skeleton was dragged. At dragging rates below 1.8 µm/s, Fl-PE was not displaced (Fig. 9 c). When the bead was dragged at a rate of as much as 18 µm/s, the lipid was only slightly moved (data not shown). Taken together, these results indicate that band 3 was moved due to the collision with the membrane skeleton rather than due to the hydrodynamic drag in the membrane.
A Decrease in the Size of the Cytoplasmic Domain of Band 3 Increases the Intercompartmental Hop Rate
According to the membrane-skeleton fence model (refer
to Fig. 1 b), if the cytoplasmic domain is made smaller,
band 3 would hop to an adjacent compartment more
readily. To test if this occurs, ghosts were briefly treated
with trypsin as described in the literature (Steck et al.,
1976; Tsuji and Ohnishi, 1986
; Clague et al., 1989
; Matayoshi and Jovin, 1991
). This treatment removes most of
the cytoplasmic domain of band 3 and cleaves ankyrin, but
leaves the spectrin network (spectrin, actin, and protein
4.1) basically intact as determined by SDS-PAGE and immunoblotting (data not shown). More extensive trypsin
treatment cleaved protein 4.1 as described previously
(Chasis and Mohandas, 1986
), but under the present conditions, cleavage of protein 4.1 was undetectable. Adducin
may be cleaved under the conditions used here (Joshi and Bennett, 1990
; Scaramuzzino and Morrow, 1993
).
After mild trypsin treatment of ghosts, the macroscopic diffusion of band 3 was enhanced. At a recording rate of every 2 ms (refer to Fig. 5 e), which is the same rate as that for Fig. 5, b and d for intact band 3, the trajectory appears to be simple Brownian motion rather than hop diffusion. However, when the recording rate was increased to every 0.22 ms (refer to Fig. 5 f), it becomes apparent that the cleaved band 3 also undergoes confined diffusion in a short time window and hop diffusion over longer time scales. The portions of the trajectory which appear to reflect hopping are shown in the blue and green lines in Fig. 5 f.
The ensemble-averaged MSD-t plot for cleaved band 3 showed a greater slope than that for intact band 3 in a time
window of 2 s (refer to Fig. 6 c). In a short time scale (refer
to Fig. 6 c, inset), and particularly within 10 ms, the difference between intact and cleaved band 3 was slight, confirming that the intracompartmental movements of intact
and cleaved band 3 are similar. Trypsin cleavage dramatically increased DMACRO by a factor of 9 from 0.66 × 10
10
cm2/s to 5.8 × 10
10 cm2/s (Fig. 7 b and Table I). Since
trypsin cleavage only slightly changed Dmicro and the size
of the confining compartment (Fig. 7, a and c), the large
increase in DMACRO must be due to the increase in the hop
frequency. The average hop rate indeed increased from once every 350 ms to once every 60 ms (Table II). Since
the viscosity in the membrane is about 100 times greater
than that in water, loss of the cytoplasmic domain of band
3 would not substantially reduce Dmicro (Saffman and Delbrück, 1975
).
Trypsin treatment, in addition to cleaving the cytoplasmic portion of band 3, digests ankyrin and also might have
cleaved other peripheral membrane proteins (e.g., adducin). However, since the compartment size does not
change greatly after trypsin treatment, the spectrin network was likely to be basically intact even after the mild
trypsin treatment. The removal of ankyrin reduces the interaction of the membrane skeleton with the membrane
directly (Jinbu et al., 1984) or indirectly by reducing the
oligomeric size of band 3 (Michaely and Bennett, 1995
;
Che et al., 1997
; Yi et al., 1997
). Therefore, it is possible
that the reduction of band 3 collision is not only due to the
reduced size of the cytoplasmic domain of band 3 but also
due to the increased fluctuation of the membrane skeleton
conformation. The results obtained here support, in either
reason, that the confinement of band 3 is due to collisions
of band 3 with the membrane skeleton.
![]() |
Discussion |
---|
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---|
Proposed Model for Regulation of Band 3 Lateral Diffusion
All of the results obtained in this research are consistent
with the membrane-skeleton fence model. About two-thirds
of band 3 molecules were mobile at 37°C. Mobile band 3 was temporarily confined within a small domain of 110 nm in
diameter, in which band 3 underwent uninhibited Brownian diffusion (Dmicro = 5.3 × 109 cm2/s). Confinement of
band 3 was caused by collisions of the cytoplasmic domain
of band 3 with the membrane skeleton, which is shown by two experimental results. (a) When the membrane skeletal
network was deformed using optical tweezers, mobile
band 3 molecules were displaced toward the same direction
as the dragging occurred. (b) The hop rate of band 3 increased by a factor of 6 after mild trypsin treatment which
cleaves the cytoplasmic portion of band 3. Band 3 molecules hop to an adjacent compartment an average of every
350 ms and undergo macroscopic diffusion by repeating hops, which is termed hop diffusion in the present paper.
A hop could occur as a result of conformational fluctuations of the spectrin molecules and/or dissociation of spectrin tetramers to dimers. We have shown that the compartment area with regard to band 3 lateral diffusion was about four times greater than the mesh area measured by anatomic force microscopy, suggesting that approximately half of the spectrin skeleton acts as barriers for the lateral diffusion of band 3. The other half of spectrin molecules do not act as barriers for band 3 diffusion, perhaps because they may be dimers and/or located far from the plasma membrane. We have been unable to differentiate these two possibilities.
Two other models for reduction of band 3 lateral diffusion have been thought of and discarded. First, a model of
band 3 hopping on spectrin molecules was considered, in
which band 3 dimers or tetramers are attached to spectrin
(which are undergoing conformational fluctuation over
the size similar to the size of the proposed compartments
[refer to Fig. 3]) via ankyrin or other peripheral membrane
proteins and hop from one spectrin molecule to another (Golan et al., 1996). However, the rotational relaxation
time of band 3 molecules (that are rotationally mobile, the
fraction equals that of band 3 that undergoes long-range
diffusion) is in the range of 1 µs to several hundreds of microseconds (Tsuji et al., 1988
), which contradicts the assumed duration of band 3 binding to the membrane skeleton of 350 ms on average. Second, the hydrodynamic
model was assumed, in which hydrodynamic interaction of
band 3 with membrane skeleton-immobilized membrane
proteins is responsible for confinement (Bussell et al.,
1995
). However, since (a) the loss of the cytoplasmic domain of band 3 causes large increases in the hop rate (Table II) and (b) PE was not moved by dragging of the membrane skeleton (Fig. 9 c), membrane proteins immobilized
on the membrane skeleton alone cannot explain hop diffusion. It follows then that the peripheral membrane proteins that are associated with integral membrane proteins
must be involved. In this case, the most likely candidate
for such a protein is indeed spectrin. Other peripheral proteins occupy limited surface area, insufficient to cause confinement for band 3.
Binding of Band 3 to the Membrane Skeleton
About 30% of band 3 molecules did not undergo long-range diffusion in a time scale of ~30 s at 37°C. Tsuji et al.
(1988) found that the immobile fraction of band 3 in
FRAP measurements are those bound to the membrane
skeleton, based on the observation that rotational diffusion measurements give the immobile fraction that is very
close to that in FRAP measurements under a variety of
conditions. These findings were further confirmed by the
present research. The immobile band 3 molecules in SPT
measurements only showed oscillatory motion (refer to
Fig. 3). Immobile band 3 could be dragged only ~300 nm
laterally using optical tweezers, and then it escaped from
the trap and returned to the initial position (refer to Fig.
4). These behavior is very similar to that of gold particles
bound to spectrin. These results indicate that immobile band 3 is tethered to the membrane skeleton that is elastic
and undergoing thermal fluctuations. Immobilization of
band 3 is thought to be caused by high-affinity binding of
band 3 via ankyrin (Bennett and Stenbuck, 1979
), since
mild trypsin treatment which also cleaved ankyrin eliminated immobile fraction of band 3.
Long-term observation of band 3 using SPT suggests
that interconversion between mobile band 3 and immobile
band 3 occurs in a time scale longer than 10 min (refer to
Fig. 2 c). Considering that all band 3 molecules are likely
to be functionally and structurally equivalent (Bennett
and Stenbuck, 1979; Hargreaves et al., 1980
), mobile/immobile fractions of band 3 may be determined by the kinetics of band 3-ankyrin association.
Membrane Skeletal Regulation of the Movements of Membrane Proteins in Nonerythroid Cells
The regulation mechanisms for band 3 lateral diffusion in
erythrocyte membranes (corralling/binding effect of membrane skeleton) are likely to be common in other cell
types, since components of the erythrocyte membrane
skeleton (i.e., spectrin, actin, ankyrin, protein 4.1, adducin,
etc.) have been found in every type of cells as ever known
(Bennett, 1985, 1990
; Bennett and Gilligan, 1993
). Corralling effect of the membrane skeleton has been found for several membrane proteins thus far (Edidin et al., 1991
,
1994
; Kusumi et al., 1993
; Sako and Kusumi, 1994
, 1995
;
Sako et al., 1998
). Sako and Kusumi (1994)
measured the
movement of transferrin receptor and
2-macroglobulin
receptor in normal rat kidney fibroblastic cells using SPT.
The majority of these receptor molecules undergo hop diffusion. Binding (tethering) effect of the membrane skeleton has also been found for many membrane proteins of
nonerythroid cells. Using optical tweezers, Sako et al.
(1998)
have shown that half of E-cadherin on the free surface of transfected L cells is tethered to an elastic structure, which is probably the membrane skeleton network.
The regulation of membrane protein diffusion would certainly involve various other mechanisms including interactions of extracellular domains of membrane proteins
(Sheetz, 1993
), entrapment in specialized lipid domains
(Sheets et al., 1997
; Simson et al., 1998
), percolation effects due to the presence of immobile and slowly diffusing
species of membrane proteins (Saxton, 1982
, 1987
, 1989
)
and so on. However, we believe that the corralling and
binding effects of membrane skeleton provide major
mechanisms for the cells to regulate the movement and localization of membrane proteins.
The present study using erythrocyte membrane would provide a basis for the studies of the mechanism by which movements of membrane proteins are regulated in nonerythroid cells. Experiments of dragging the membrane skeleton suggest that the membrane skeleton can, in addition to regulating the movement of band 3, regulate the localization of membrane proteins. By regulating the hop rate and mesh density (i.e., through modification of membrane skeletal components and the cytoplasmic domains of integral membrane proteins), and by moving the mesh toward a specific location (i.e., through ATP-driven transport), the membrane skeleton can be instrumental in controlling aggregation, assembly, and localization of membrane proteins, and perhaps the formation of specialized membrane domains in which specific membrane proteins are assembled in the plasma membrane.
![]() |
Footnotes |
---|
Received for publication 8 April 1998 and in revised form 10 July 1998.
Address all correspondence to: Akihiro Kusumi, Department of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan. Tel.: (81) 52-789-2969. Fax: (81) 52-789-2968. E-mail: akusumi{at}bio.nagoya-u.ac.jpWe thank Y. Takakuwa, S. Manno, N. Kimata, and W. Nunomura (Tokyo Women's Medical College, Tokyo, Japan) for providing anti-band 3 antibodies and for their helpful advice and discussions.
![]() |
Abbreviations used in this paper |
---|
, diameter;
D, lateral diffusion coefficient;
DMACRO, macroscopic diffusion coefficient;
Dmicro, microscopic diffusion coefficient;
Fl-PE, fluorescein-phosphatidylethanolamine;
FRAP, fluorescence redistribution after photobleaching;
MSD, mean square displacement;
pA-PMSF, (p-amidinophenyl) methanesulfonyl fluoride hydrochloride;
SPT, single particle tracking.
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