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Ultrafine Membrane Compartments for Molecular Diffusion as Revealed by Single Molecule Techniques

Kotono Murase ^*, Takahiro Fujiwara ^*, Yasuhiro Umemura , Kenichi Suzuki ^*, Ryota Iino ^*, Hidetoshi Yamashita , Mihoko Saito , Hideji Murakoshi , Ken Ritchie ^*  and Akihiro Kusumi ^* 

^* Kusumi Membrane Organizer Project, Exploratory Research for Advanced Technology Organization (ERATO/SORST), Japan Science and Technology Agency, Nagoya, Japan and Department of Biological Science and Institute for Advanced Research, Nagoya University, Nagoya, Japan

Correspondence: Address reprint requests to Akihiro Kusumi, Ph.D., Dept. of Biological Science, Nagoya University, Nagoya 464-8602, Japan. Tel.: 81-52-789-2969; Fax: 81-52-789-2968; E-mail: akusumi{at}bio.nagoya-u.ac.jp.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Plasma membrane compartments, delimited by transmembrane proteins anchored to the membrane skeleton (anchored-protein picket model), would provide the membrane with fundamental mosaicism because they would affect the movement of practically all molecules incorporated in the cell membrane. Understanding such basic compartmentalized structures of the cell membrane is critical for further studies of a variety of membrane functions. Here, using both high temporal-resolution single particle tracking and single fluorescent molecule video imaging of an unsaturated phospholipid, DOPE, we found that plasma membrane compartments generally exist in various cell types, including CHO, HEPA-OVA, PtK2, FRSK, HEK293, HeLa, T24 (ECV304), and NRK cells. The compartment size varies from 30 to 230 nm, whereas the average hop rate of DOPE crossing the boundaries between two adjacent compartments ranges between 1 and 17 ms. The probability of passing a compartment barrier when DOPE is already at the boundary is also cell-type dependent, with an overall variation by a factor of 7. These results strongly indicate the necessity for the paradigm shift of the concept on the plasma membrane: from the two-dimensional fluid continuum model to the compartmentalized membrane model in which its constituent molecules undergo hop diffusion over the compartments.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
One of the most important structural characteristics of the cell membrane is that it behaves like a two-dimensional liquid, and its constituent molecules, i.e., membrane proteins and lipids, rapidly move about in the membrane plane. However, even phospholipids do not undergo free diffusion in the cellular plasma membrane. The diffusion rates in the cell membrane are reduced by a factor of 10–100, as compared to those in artificial membranes (see Table 1). The mechanism that is responsible for such slowing has puzzled membrane biologists for the last 25 years.

View larger version (39K): [in this window] [in a new window]   FIGURE 2  Distribution of diffusion coefficients in a 100-ms time-window (D100ms). D100ms is the same as D2–4 in Kusumi et al. (1993). Arrowheads indicate the median values. (A) D100ms estimated for Cy3- (top) and Gold-DOPE observed using our standard observation protocol (bottom), which involves the observation of all of the gold particles attached to the membrane longer than 3 s, but the observation is limited for 20 min after the addition of the gold probes. (B) (Top) D100ms for Gold-DOPE estimated for particles bound to the membrane surface for shorter periods (between 3 and 150 s, the short-term reporters, solid bars) and for those bound for longer periods (5 min or longer, the long-term reporters, open bars), indicating that those bound to the membrane for short periods exhibit diffusion coefficients comparable to Cy3-DOPE. See the text for details. Note that for the determination of the long-term reporters, we only observed for 5 min, and did not examine how much longer than 5 min they stayed on the membrane surface, whereas the observation following the standard protocol would include the short-term reporters as well as the gold probes that might have stayed much longer than 20 min, and therefore, its D100ms distribution becomes broader than that for the short-term reporters and the long-term reporters combined. (Bottom) Fab fragments and the whole IgG of anti-fluorescein antibodies were labeled with Cy3, and their diffusion coefficients after binding to fluorescein-DOPE were measured. D100ms estimated for Cy3-Fab-DOPE (solid bars) and Cy3-IgG-DOPE (open bars).

The diffusion rates of lipids vary greatly from cell to cell, with an overall variation by a factor of 15 (see Table 1). FRSK cells have displayed one of the slowest diffusion rates studied thus far (because the data obtained in this research have been added to the table, the diffusion coefficient of lipid in FRSK cells does not appear to be particularly slow; however, when this work was initiated, it was one of the smallest in the literature). In this research, we examined the mechanism for slowing the lipid diffusion in FRSK cells (as well as CHO, HEPA-OVA, PtK2, HEK293, HeLa, and T24 (ECV304, a subclone of T24) cells), and investigated how this slower diffusion rate is induced in FRSK cells.

Even in FRSK cells, DOPE was found to undergo hop diffusion, in a manner dependent on the membrane skeleton, but not on the cholesterol concentration, the extracellular matrix, and the extracellular domains of membrane proteins, consistent with the anchored-protein picket model. However, compartment sizes as small as 30–40 nm were found in FRSK, CHO, HEPA-OVA, and PtK2 cells (70 nm in HEK293 and HeLa cells, 110 nm in T24 cells, and 230 nm in NRK cells), which are smaller by a factor of 7 in diameter (or a factor of 50 in area) than those previously found in NRK cells.

To obtain the correct hop rates of membrane molecules, the following method has been used extensively in this study (Fujiwara et al., 2002): the macroscopic diffusion coefficient over many compartments was obtained using SFVI, and the compartment size was obtained by high temporal/spatial-resolution single-particle tracking using 40-nm colloidal-gold probes. Since fluorescent probes, which are much smaller than colloidal-gold probes, are much less likely to interact with other molecules, and since they intrinsically represent single molecules, unlike gold probes, with which one must always carefully consider the level of crosslinking when interpreting the data, SFVI tends to provide much more reliable data on the macroscopic diffusion coefficient. However, the time resolution obtained by SFVI is limited to a millisecond at best, and this cannot be accomplished without severely sacrificing the observation durations (due to photobleaching by the employment of high excitation light intensity; Schütz et al., 2000). Our past experience indicated that to identify membrane compartments clearly by statistical analyses as well as by eye, one should observe a single molecule's trajectory for a sufficiently long period to include more than a few hops and with a time resolution high enough to have at least 40 points (on average) within a compartment. If the residency time within a compartment is short (i.e., the hop rate is high), e.g., from one to several hundred milliseconds, then such conditions cannot be fulfilled with single fluorescent molecule observations, but can be easily satisfied by high-speed SPT with gold probes (Fujiwara et al., 2002).

If the gold probe induces (even low levels of) crosslinking, then it would affect the hop rate strongly (this is already a strong indication that the plasma membrane should not be thought as a two-dimensional fluid continuum), but the observed compartment size only slightly. Therefore, the hop rate can be calculated quite accurately by using the macroscopic diffusion coefficient of a fluorescently-labeled lipid (DOPE) and the compartment size determined by SPT with gold-probe-labeled DOPE.

Hop rates measured for DOPE are as fast as once every 1–2 ms in FRSK, CHO, HEPA-OVA, and PtK2 cells (3 ms for HEK293 cells, 5 ms for HeLa cells, and 10–20 ms for T24 and NRK cells) on average. Although the residency time is very short for DOPE, it was greatly prolonged upon the crosslinking of DOPE. This suggests that when receptor molecules are liganded and clustered to form signaling complexes, they will be instantaneously arrested in the membrane skeleton mesh (oligomerization-induced trapping model; Iino et al., 2001). Further, if larger and stabilized rafts were induced upon clustering of GPI-anchored or other raft-preferring receptor molecules, the results presented here indicate they too have to be trapped in the compartment where the stabilized rafts are formed. Therefore, the "pickets" and "fences" made of the membrane skeleton and the anchored transmembrane proteins provide the cell with a mechanism for preserving the spatial information of signal transduction in the membrane.

One of the most important questions addressed in this research is: How universal is the compartmentalized structure of the membrane that even works for phospholipids? We started out from very detailed examinations of the DOPE diffusion in FRSK cells, where the DOPE diffusion is substantially slower than that reported previously (Table 1). Then we further proceeded 1), to examine whether hop diffusion is found in other cell types and 2), to obtain the compartment sizes and the hop rates if hop diffusion is found in those cells. SFVI of fluorescently-labeled DOPE and high-speed SPT of gold-particle-tagged DOPE have been carried out for all of the eight cells examined thus far, suggesting that the plasma membrane compartmentalization and the hop diffusion of membrane molecules occur universally among mammalian cells. The compartmentalization of the plasma membrane and hop diffusion of lipid molecules over these compartments were found in all of the eight cells examined here. This strongly indicates the necessity for the paradigm shift of our view on the plasma membrane, from the two-dimensional fluid continuum model to the compartmentalized membrane model in which its constituent molecules undergo hop diffusion over these compartments.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Cell culture
Fetal rat skin keratinocytes (FRSK, a cell line) were grown in Eagle's minimal essential medium (MEM) supplemented with 10% fetal bovine serum. Cells used for the experiment were cultured on 18x18-mm coverslips (IWAKI, Chiba, Japan) for high-speed video imaging and cell staining for 2 days after plating, or on 12-mm glass-bottom dishes (IWAKI) for single fluorescence molecule video imaging for one day after plating (FRSK cells grow faster in the glass-bottom dishes). All other cells were observed on the second day after inoculation in either type of dish.

Synthesis of Cy3-DOPE
DOPE (Avanti Polar Lipids, Alabaster, AL, 6 µmol), dissolved in 500 µl of CHCl₃ containing 20 µl dry (C₂H₅)₃N, was added to a solution of 3 µmol Cy3.29-succinimidyl ester (monoreactive, Amersham-Pharmacia, Tokyo, Japan) in 50 µl dry dimethylformamide. The solution was stirred under a nitrogen atmosphere at room temperature for 2 h. Next, silica-gel thin layer chromatography (TLC) (CHCl₃: MeOH: H₂O = 2:1:1 vol/vol) was used to identify the presence of a new spot (R_f = 0.6) and then to isolate the product: after the reaction mixture was evaporated to dryness, the crude product was dissolved in a mixture of CHCl₃/MeOH/H₂O (7:3:0.4 vol/vol), applied to a preparative TLC plate, and developed in the same solvent mixture, and then the Cy3-DOPE spot was scraped off from the TLC plate. For the synthesis of FITC-DOPE, 20 µmol fluorescein-5-isothiocyanate (isomer I from Molecular Probes, Eugene, OR) was added to 20 µmol of DOPE in 2 ml CHCl₃ containing 2 µl dry (C₂H₅)₃N. The solution was stirred under a nitrogen atmosphere at room temperature for 2 h. A new spot exhibiting the production of FITC-DOPE (R_f = 0.6) was identified by TLC (CHCl₃/MeOH = 4:1). FITC-DOPE was purified as follows: After the reaction mixture was evaporated to dryness, the crude product was dissolved in a mixture of CHCl₃/MeOH = 4:1 (v/v) and applied to a 10-g silica gel column C-300 HG from Wako (Osaka, Japan), and then eluted with a CHCl₃/MeOH = 4:1 (v/v) solvent mixture. The fractions with equal R_f values were combined and evaporated to dryness.

Preparation of colloidal-gold probes
The minimal protecting amount (MPA) of anti-fluorescein antibodies' Fab fragments (Molecular Probes), which is defined as the minimum concentration of the protein needed to stabilize colloidal gold in suspension, was determined to be 2.5 µg/ml, using methods described previously (De Mey, 1983; Leunissen and De Mey, 1989). Colloidal-gold probes created with attached to the MPA of Fab were prepared by mixing 50 µl of 25 µg/ml anti-fluorescein Fab in 2 mM phosphate buffer (pH 7.2) and a 500 µl suspension of colloidal gold (pH 7.4) on a slowly tumbling shaker for 1 h at room temperature. The gold probe was further stabilized with 0.05% Carbowax 20M (Sigma, St. Louis, MO). After three washes by sedimentation and resuspension in 0.05% Carbowax 20M in 20 mM phosphate buffer (pH 7.0), the gold probe was resuspended in Eagle's minimal essential medium (MEM) containing 2 mM PIPES (without NaHCO₃) and 10% fetal bovine serum, sterilized by filtration with a 0.22-µm filter (Millipore, Bedford, MA), and then used within 12 h.

Single fluorophore video imaging of Cy3-DOPE and single particle tracking of Gold-DOPE
For SFVI of Cy3-DOPE, first Cy3-DOPE in methanol (20 µg/ml) was added to HBSS buffered with 2 mM PIPES at pH 7.2 with vigorous vortexing (100 ng/ml final concentration), and then this solution was then added to the cells cultured on a glass-bottom dish at 37°C (final concentration 10 ng/ml). Individual Cy3-DOPE molecules were observed in the apical cell membrane (side of the membrane facing the medium) at the video rate, using a homebuilt objective lens-type total internal reflection fluorescence microscope (Iino et al., 2001). Briefly, a 532-nm laser beam (the second harmonic of the Nd:YAG laser beam, Model 4501-050, Uniphase, San Jose, CA) was attenuated with neutral density filters, circularly polarized, and then steered into the edge of a high numerical aperture (NA) objective lens (PlanApo100x, NA = 1.4 or 1.45, Olympus, Tokyo, Japan) with a focus at the back-focal plane of the objective lens on an Olympus inverted microscope (IX-70).

For SPT of Gold-DOPE, after FITC-DOPE was incorporated in the cell membrane by the addition of 2 µg/ml (final concentration) of FITC-DOPE, gold probes conjugated with anti-fluorescein antibodies' Fab fragments were applied to cells cultured on 18x18-mm coverslips. For observation with improved temporal resolutions, a digital high-speed camera with a C-MOS sensor was used (FASTCAM-Ultima, PHOTRON, Tokyo, Japan; Tomishige et al., 1998, Fujiwara et al., 2002). For high-speed video microscopy, bright-field optical microscopy rather than Nomarski microscopy was employed, and the green interference filter was removed to increase the light intensity on the photodetector plane, greatly enhancing the signal/noise ratio in the image. The sequence of images was replayed at the video rate with analog and digital enhancements by an image processor (DVS-3000, Hamamatsu Photonics, Hamamatsu, Japan), and recorded on a digital video tape recorder (DSR-20, Sony, Tokyo, Japan).

Positions of single gold particles or single fluorescent molecules were determined from each image, and the mean-squared displacement versus time (MSD-t) plots were obtained as described previously (Kusumi et al., 1993). From video-rate recordings, the diffusion coefficients in a 100-ms time-window (D_100ms) were obtained, which is the same as D_2–4 in Kusumi et al. (1993).

In SPT recordings at time resolutions of 110 or 25 µs, typically, recordings for 278 or 61.7 ms (2500 frames), respectively, were carried out for at least 35 particles. Almost all of the MSD-t curves showed a rapid rise followed by a leveling off. The MSD-t plot was fitted to a theoretical curve representing hop diffusion (Powles et al., 1992). For successful curve fitting, the duration for the fit must be sufficiently long to reflect the macroscopic diffusion, but should not be overly long so that the behavior near time 0 and the largest curvature in the transition region between the short-term simple Brownian diffusion and the long-range hop movements over the compartments are properly taken into account. Generally, 8.89 and 6.17 ms for 110- or 25-µs resolutions, respectively, turned out to be useful. The fit parameters included L, the compartment size, and D_MACRO, the macroscopic diffusion coefficient over the membrane compartments, and D_micro (typically D_100µs); both represent the diffusion coefficients within a compartment when the compartment size is sufficiently large, 800 nm or greater; when the compartments are smaller, these values are artificially reduced because the averaging of the image of gold particles over the frame time—even 25 µs—makes their movement appear smaller as the compartment boundaries more frequently bounce off the molecule (see Results). The residency time, , was calculated from L and D_MACRO: = L²/ 4D_MACRO. D_100µs (Table 2, Fig. 8) and D_100ms (Table 3, Table 6, and Fig. 2) were determined by fitting the MSD-t plots between the second and fourth points using a straight line (which is the same as D_2–4 in Kusumi et al., 1993) for the data obtained at 33-ms and 25-µs resolutions, respectively.

View larger version (38K): [in this window] [in a new window]   FIGURE 8  Apparent Dmicro (in a time-window of 100 µs based on 25-µs resolution observations) plotted against compartment size, as determined for each trajectory in the control and bleb membranes. With a larger compartment size, the apparent Dmicro increased, suggesting that in smaller compartments, even a 25-µs resolution is insufficient to obtain true diffusion rates. This figure is generated using the same data set used to generate Fig. 4 (at a 25-µs resolution, solid bars) and the bleb data obtained at a 25-µs resolution. The average compartment size for NRK cells is indicated (dashed blue line). The dashed red line is a visual aid.

The results of these measurements are summarized in Table 2. The mean value of the diffusion coefficients for Gold-DOPE on FRSK cells is greater than those on the coverslip or the DPPC bilayer in the gel phase by a factor of >30 in every time-window, indicating that the background noise does not make significant contributions to the diffusion coefficients of Gold-DOPE determined here. These noise factors will become important when the measured diffusion coefficients in the cell are smaller, or when the determination of the immobile fraction is desired.

Modulation of the actin cytoskeleton, partial cholesterol depletion, and membrane bleb formation
FRSK cells were incubated in the MEM medium containing either 13 µM cytochalasin D or 0.5 µM jasplakinolide (gifts from Dr. G. Marriott, University of Wisconsin-Madison) on the microscope stage at 37°C for 5 min. Microscopic observations were completed within 15 min after the addition of drugs.

For partial cholesterol removal, cells were incubated at 37°C for 20 min in the presence of 4 mM MßCD (Sigma) in MEM containing LDL-free FBS (Goldstein et al., 1983). To make the membrane blebs on the cell surface, cells were treated with 0.5 mM 2-methyl-1,4-naphthoquinone (menadione) in HBSS buffered with 2 mM PIPES (pH 7.4) at 37°C for 60 min (Malorni et al., 1991; Fujiwara et al., 2002).

Removal of the extracellular matrix and extracellular domains of membrane proteins on the cell surface
To remove the extracellular matrix and extracellular domains of membrane proteins from the surface, the FRSK cells were treated with 5, 10, or 25 µg/ml trypsin (Difco, Kansas City, MO) at 37°C for 10 min. To monitor the extent of cleavage, the extracellular surface proteins were first tagged with sulfosuccinimidyl biotin (Sigma), and were visualized by fluorescein-streptavidin (Molecular Probes) before and after trypsin treatment. Chondroitin sulfate glycosaminoglycan was detected by an indirect immunofluorescence method, using mouse anti-chondroitin sulfate IgM (Seikagaku, Tokyo, Japan) and TRITC-conjugated goat anti-mouse IgM (ICN, Costa Mesa, CA). Epifluorescence images of cells were quantitated by the MetaView software (Universal Imaging, Downingtown, PA).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
DOPE undergoes slow, apparently simple Brownian diffusion in the plasma membrane of FRSK cells at a time resolution of 33 ms
All experiments were carried out at 37°C unless otherwise specified. To track the movement of individual phospholipid molecules in the cell membrane, DOPE molecules tagged with Cy3 in the headgroup region were incorporated in the cell membrane of FRSK cells, and then individual Cy3-DOPE molecules were observed under an objective-lens-type total internal reflection fluorescence microscope at video rate (33-ms resolution). The Cy3-DOPE thus observed was confirmed to be individual molecules by the single-step photobleaching and the single-peak distribution of its fluorescence intensity (data not shown), as described previously (Iino et al., 2001; Fujiwara et al., 2002). Therefore, this method is referred to as SFVI (see Introduction). A typical trajectory of a Cy3-DOPE molecule, recorded at a 33-ms resolution for a period of 3.3 s, is shown in Fig. 1 a.

View larger version (29K): [in this window] [in a new window]   FIGURE 1  Typical images and trajectories of Cy3- and Gold-DOPE recorded at a 33-ms resolution (video rate) for 3.3 s (100 frames). (a) SFVI of Cy3-DOPE. (b) SPT of Gold-DOPE (gold probes coated with anti-fluorescein antibody Fab fragments bound to fluorescein-DOPE, which was preincorporated in the cell membrane). The colors (purple, blue, green, orange, and red) represent the trajectories over time periods of 20 steps (every 660 ms). The color sequence is consistent throughout this article. The actual video images are also shown.

Crosslinking effects of gold probes and optimization of gold-particle labeling
Such a great reduction in the diffusion coefficient in the cell membrane from the artificial membranes suggests the presence of mechanisms for restricting lipid movement in the FRSK cell membrane that are not resolvable at a 33-ms resolution. Since achieving higher time resolutions in single fluorophore observations is difficult due to the problem of low signal/noise ratios, we employed single particle tracking (SPT) by binding 40 nm- colloidal-gold probes to fluorescein-DOPE molecules preincorporated in the cell membrane. Note that the fluorescein moiety is not used as a fluorescent probe, but as a tag to anchor the gold probes on DOPE via the anti-fluorescein Fab.

Conditions for labeling DOPE with gold probes were optimized by adjusting the concentration of fluorescein-DOPE preincubated with the cells and the amount of anti-fluorescein Fab fragments conjugated with the gold probes (see Materials and Methods). Thus, the effect of crosslinking by the gold probes was minimized, while sufficient specificity for their binding to the cell surface was maintained. Reducing the Fab concentration mixed with gold probes increased the diffusion rate of the gold-DOPE complex determined at video rate (D_100ms), which reached a plateau value of 0.044 µm²/s (median) at 2.5 µg/ml of Fab. At this concentration, the ratio of particles bound specifically versus nonspecifically (using 40 nm- colloidal-gold probes not coated with Fab fragments) was 5:1.

In all of the measurements using colloidal-gold probes in this research (unless otherwise stated), they were made within 20 min after the addition of gold probes: sufficient concentrations of gold particles are always present in the observation medium, and they are in dynamic equilibrium with those bound to the cell surface (standard observation protocol). Fig. 1 b shows a typical trajectory of a Gold-DOPE complex recorded at video rate (33-ms resolution). A statistical analysis (Kusumi et al., 1993) classified it into the simple Brownian diffusion mode. The mean values of D_100ms and D_3s were 0.044 µm²/s (Fig. 2 A, bottom) and 0.028 µm²/s (median), respectively, which were smaller by a factor of 5 than those of Cy3-DOPE. The distributions of D_100ms for Cy3-DOPE and Gold-DOPE are shown in Fig. 2 A.

These results suggest that the diffusion of Gold-DOPE may be slowed, due to steric hindrance and/or the crosslinking effect of gold probes attached to DOPE. Previously, using the same Cy3-DOPE and Gold-DOPE, Fujiwara et al. (2002) found that these probes gave the same diffusion coefficients in the NRK cell membrane, as long as the time-window for evaluating the diffusion coefficient was <100 ms. This was not the case in FRSK cells. This is probably due to the very small compartment size in the FRSK cell membrane (40 nm vs. 230 nm in NRK cells), as described below. These probe molecules collide with the compartment boundaries 30 times ([230/40]²) more often in FRSK cells than in NRK cells, which is likely to make the D_100ms of Gold-DOPE very sensitive to low levels of gold-induced crosslinking of DOPE.

Evaluation of the level of crosslinking by gold probes
To evaluate the degree of crosslinking by gold probes, we initially tried to directly measure the number of fluorescein-DOPE molecules bound to the gold probe on the cell surface by carrying out SFVI of fluorescein-DOPE. However, this turned out to be impossible due to the strong signal from the gold particle even in the absence of fluorescein-DOPE (the nature of the light scattered or emitted from the gold particle could not be determined).

To examine the relationship between the crosslinking level of fluorescein-DOPE by our gold probes (multiple binding of a gold particle to the cell membrane via several fluorescein-DOPE molecules) and the diffusion coefficient of the gold-DOPE complex, we first examined the binding duration of each gold particle on the membrane surface, and obtained the distributions of the diffusion coefficients separately for the gold particles that stayed on the membrane surface between 3 and 150 s (the short-term reporters), and for those that stayed >5 min (the long-term reporters, Fig. 2 B). Those that stayed <3 s were not counted because differentiating such probes from those that nonspecifically stayed near the membrane surface is difficult.

Only 5% of the gold probes were short-term reporters, but their D_100ms exhibited a distribution with the median value (0.17 µm²/s, mean = 0.23 µm²/s; Fig. 2 B, top, solid bars) comparable to those for Cy3-DOPE (0.19 µm²/s, mean = 0.27 µm²/s; Fig. 2 A, top). This result suggests that the short-term reporters are likely bound to single molecules of DOPE. The long-term reporters exhibited diffusion coefficients (median = 0.066 µm²/s, mean = 0.074 µm²/s) smaller than those for the short-term reporters by a factor of 2.5–3. These results strongly indicate that the slowed diffusion of gold probes compared with that of Cy3 probes can largely be explained by the crosslinking effect of gold probes (further examination on this point is described in the text corresponding to Figs. 5–9).

View larger version (49K): [in this window] [in a new window]   FIGURE 5  Models for the mechanisms that could be responsible for the temporal corralling and hop diffusion of DOPE.

View larger version (31K): [in this window] [in a new window]   FIGURE 9  Plots of log(MSD/time) against log(time) provide useful information on the changes of the diffusion characteristics that depend on the observation time intervals. MSD of the trajectories was estimated using data obtained at time resolutions of 25 µs (5000 frames long), 110 µs (5000 frames long), and 33 ms (500 frames long), for the time-windows where the theoretically expected statistical errors in MSD are <40% (Qian et al., 1991). Then, the mean log(MSD/time) values (•) and their standard deviations (blue and yellow vertical bars, which may look like a band due to the high density of the data points here) were plotted as a function of log(time). Normal (simple Brownian) and anomalous diffusion can be distinguished in this display as lacking time-dependence (slope 0) and having negative slopes, respectively (Saxton, 1994; Feder et al., 1996). The best fit for the data obtained for the cell membrane using the three linear segments, depicted by blue lines, with -values of 0.97 (50 µs 0.13 ms), 0.53 (1 10 ms), and 0.94 (300 ms 2 s) gives transitions at 0.1 ms (or less) and between 10 and 100 ms. (Fitted regions are shown in solid lines. To help the eye, they are extended, which are shown in broken lines. The fit between 1 and 10 ms appears bad, but in fact there are many more points in the lower black sequence of •; i.e., the fit was done correctly.) For comparison, the plots for the trajectories in bleb membranes ( = 1000 frames; n = 24) and the best regression result (orange line, = 1.0) are also shown.

The distributions of their D_100ms values are shown in Fig. 2 B (bottom). First, we compare the distribution of D_100ms values for Cy3-Fab-DOPE (Fig. 2 B, bottom) with that for Cy3-DOPE (Fig. 2 A, top). The median values of D_100ms for Cy3-Fab-DOPE and Cy3-DOPE were 0.13 and 0.19 µm²/s, with mean values of 0.26 and 0.27 µm²/s, respectively. Namely, the distribution is largely similar (P = 0.095 in the U-test of Mann-Whitney), but 20% of Cy3-Fab-DOPE exhibited D_100ms smaller than any Cy3-DOPE molecules. This result suggests that some of the Cy3-Fab fragments attached to DOPE might slightly interact with surrounding molecules, increasing the drag very slightly, but that, practically, the effect of Fab-binding to the lipid headgroup on the lipid movement is negligible.

Next, we compare the histogram of D_100ms for Cy3-Fab-DOPE with that for Cy3-IgG-DOPE, both shown in Fig. 2 B (bottom). The average D_100ms for Cy3-IgG-DOPE is smaller than that for Cy3-Fab-DOPE by a factor of 2, with median values of 0.075 and 0.13 µm²/s, and mean values of 0.12 and 0.26 µm²/s, respectively. Assuming that the fraction of Cy3-IgG-DOPE exhibiting D_100ms is smaller than either 0.046 (based on the comparison with the distribution for Cy3-Fab-DOPE in Fig. 2 B, bottom) or 0.1 µm²/s (based on the comparison with the distribution for Cy3-DOPE in Fig. 2 A, top), representing those bound to two DOPE molecules, the amount of Cy3-IgG molecules bound to two molecules of DOPE can be estimated very roughly as 36 or 61%, in the respective evaluations, whereas the remaining 64 or 39% of Cy3-IgG molecules were bound to single molecules of DOPE. Given the level of approximation for such estimates, it can be said that approximately one-half ±15% of Cy3-IgG molecules were bound to either one or two molecules of DOPE.

The observation that even such a low level of clustering (mixtures of monomers and dimers) induces the twofold reduction in the diffusion coefficient on average (Fig. 2 B, bottom), itself indicates that the two-dimensional fluid continuum model for the membrane is not applicable. The Saffman-Delbrück equation that assumes the two-dimensional fluid continuum predicts that dimerization of a diffusant will have an almost negligible effect on the diffusion coefficient. Therefore, such a large reduction upon partial dimerization of the diffusant is consistent with compartmentalization of the cell membrane (also see Table 6).

Third, we compare the D_100ms distribution for the long-term reporters of Gold-DOPE (Fig. 2 B, top, open bars) with that for Cy3-IgG-DOPE (Fig. 2 B, bottom, open bars). The distribution for the long-term reporters overlaps with 80% of that for Cy3-IgG-DOPE (Fig. 2 B, bottom, open bars), suggesting that the majority of gold probes may bind to one or two molecules of DOPE. Comparing these two distributions, particularly at their low ends, and assuming that the fraction of the long-term reporters that exhibited D_100ms <0.022 µm²/s represents those bound to more than two DOPE molecules, we suggest that 20% of the long-term reporters may be bound to three or more DOPE molecules.

Fourth, finally, we compare these distributions to that for Gold-DOPE obtained following our standard observation protocol (shown in Fig. 2 A, bottom; observing all of the gold particles attached to the membrane longer than 3 s, but the observation is limited to 20 min after the addition of the gold probes, as described above. Note that for the determination of the long-term reporters in Fig. 2 B (top), we only observed for 5 min, and did not examine how much longer than 5 min they stayed on the membrane surface, whereas the observation following the standard protocol would include the short-term reporters as well as the gold probes that might have stayed much longer than 20 min, and therefore, the D_100ms distribution becomes broader than that for the short-term reporters and the long-term reporters combined). Approximately 40 and 50% of the distribution of D_100ms for Gold-DOPE observed in our standard protocol exhibit overlaps with those for Cy3-DOPE (Fig. 2 A, top) and Cy3-Fab-DOPE (Fig. 2 B, bottom), respectively. Meanwhile, comparing this distribution obtained under standard conditions with the histograms for the long-term reporters (Fig. 2 B, top, open bars) and Cy3-IgG-DOPE (Fig. 2 B, bottom, open bars), particularly at the low ends of the distributions, and assuming that the fraction of Cy3-IgG-DOPE that exhibited D_100ms <0.01 or 0.022 µm²/s (following similar arguments as described above), we suggest that 15–30% of Gold-DOPE in our standard protocol may be bound to three or more DOPE molecules. Taken together, under our standard conditions, Gold-DOPE may very roughly represent 40, 40, and 20% (±10% each) of monomers, crosslinked dimers, and crosslinked oligomers greater than dimers (due to the variations of individual probes in terms of the number of active Fab molecules and their geometrical distributions on the gold surface, any theoretical prediction for such a binding is difficult to make).

This complicates the measurements of the hop parameters for DOPE diffusion using gold probes. However, although crosslinking would affect the frequency of hops across the compartment boundaries, it would not affect the measured compartment size. Therefore, in this research, we determine the macroscopic diffusion coefficient using the video rate observations of Cy3-DOPE (at this observation rate, the hop movements over compartment boundaries cannot be seen, but the rate of macroscopic diffusion over many compartments could be obtained), and the compartment size by the high-speed SPT of gold particles.

Cy3-DOPE is an intrinsically better probe in the sense that it is free from the effect of crosslinking and the probe moiety is much smaller than the gold probe, reducing the chances of nonspecific interactions with cellular structures. Nevertheless, to achieve higher time resolutions for investigating the mechanism for the 50-fold reduction in the phospholipid diffusion rate in the cell membrane (compared with that in liposomes and reconstituted membranes), we had to use Gold-DOPE, because the poor signal/noise ratio of single Cy3-DOPE imaging hindered observations with high spatial and temporal resolutions (making direct observations of hop movements impossible). We then tried to reach a cohesive conclusion by combining the high-speed, high-resolution SPT data (the compartment size, using Gold-DOPE) with the SFVI observations (macroscopic diffusion rate, using Cy3-DOPE; see the following sections).

Gold-DOPE shows hop diffusion at 25- and 110-µs resolutions
The movement of Gold-DOPE complexes was examined at time resolutions of up to 25 µs, an enhancement by a factor of 1350 from the normal video rate (once every 33 ms). Their typical trajectories recorded at 33-ms and 110-µs resolutions are shown in Fig. 3. At a 110-µs resolution, qualitatively, all trajectories exhibited temporal confinement (Fig. 3 b), with occasional hops to adjacent compartments, a process we call hop diffusion (Kusumi et al., 1993; Sako and Kusumi, 1994; Kusumi and Sako, 1996; Tomishige et al., 1998; Fujiwara et al., 2002). Each trajectory underwent a quantitative analysis, based on the mean square displacement plotted against time (MSD-t plot), and a statistical classification into the simple Brownian diffusion mode or the hop plus confined diffusion mode, as described previously (Powles et al., 1992; Kusumi et al., 1993; Fujiwara et al., 2002; see Materials and Methods for further details).

View larger version (56K): [in this window] [in a new window]   FIGURE 3  Gold-DOPE observed at a 110-µs resolution exhibited hop diffusion. What appears to be simple Brownian diffusion at a 33-ms resolution (a, video rate) is actually fast hop diffusion, as visible in recordings at a 110-µs resolution (b, 300-fold faster than the video rate). (a) Each color represents 60 step periods (every 2 s). (b) Each color indicates plausible confinement within a compartment, and black indicates intercompartmental hops. The residency time for each compartment is indicated. These compartments were detected by computer software we developed (Fujiwara et al., 2002) as well as by eye.

View larger version (24K): [in this window] [in a new window]   FIGURE 4  Distributions of the compartment size (left) and the residency time (right) for Gold-DOPE indicate the presence of 41-nm compartments (median observed at a 25-µs resolution) and 15-ms residency time (median) in a compartment. Open bars and solid bars indicate the distributions observed at 110-µs and 25-µs resolutions, respectively. Arrowheads indicate the median values. The inset in the histogram for the compartment size shows more detailed distributions (25-µs resolution data).

The extracellular matrix, extracellular domains of membrane proteins, and rafts are not involved in DOPE hop diffusion
Fig. 5 shows a model for the mechanisms by which the movement of DOPE on FRSK cells could be confined in 40-nm compartments.

1. The involvement of the extracellular matrix and/or extracellular domains of membrane-associated proteins, due to interactions with Gold-DOPE.
   
2. Rafts acting as diffusion obstacles for unsaturated phospholipids such as DOPE, which is excluded from rafts.
   
3. The effect of transmembrane proteins anchored to the actin-based membrane skeleton, due to circumferential slowing effects and steric hindrance (anchored membrane-protein picket model).
   

We examined each model, as described below. All examinations were performed at a 110-µs resolution.

To partially remove the extracellular matrix and extracellular domains of membrane proteins, the cells were mildly treated with trypsin. To monitor the extent of the extracellular protein removal, sulfosuccinimidyl biotin was attached to the surface proteins, and was visualized by fluorescein-streptavidin (Fig. 6 A, a and b). A quantitative analysis of the fluorescence intensity indicated that the amount of fluorescein-streptavidin bound to the cell surface decreased to 45% after a 10 µg/ml trypsin treatment for 10 min (Fig. 6 B, left). To further examine the density of the extracellular matrix in particular, the chondroitin sulfate on the cell surface was stained by an immunofluorescence method (Fig. 6 A, c and d). The signal from chondroitin sulfate glycosaminoglycan decreased to 40% under the same trypsinization conditions (Fig. 6 B, right).

View larger version (60K): [in this window] [in a new window]   FIGURE 6  The extracellular matrices and the extracellular domains of the membrane proteins are partially cleaved by treatment of cells with trypsin. (A) (a, b) After the amino groups of the cell surface proteins were first tagged with biotin, the cells were incubated with 10 µg/ml trypsin (or trypsin-free medium) for 10 min and then visualized with FITC-streptavidin (biotin labeling after trypsin treatment gave nearly the same results): a, no treatment; b, after trypsin treatment. The focus of the microscope is on the lamellipodia, where most SPT experiments were carried out. (c, d) The amount of remaining chondroitin sulfate glycosaminoglycan was quantitated by immunofluorescence staining before (c) and after (d) trypsin treatment. (B) Fluorescence intensity due to the remaining cell surface proteins and chondroitin sulfate after trypsin treatment (10 min), plotted as a function of trypsin concentration. The fluorescence intensity (70 x 70 pixels 8 x 8 µm) was normalized to that before trypsin treatment. The background was determined as the intensity in the area on the cover glass where no cell was attached, and was subtracted from the measured intensity in each area on the cell. A total of 40 cells were used for the measurements, with a total measured area of 3000 µm2.

View larger version (32K): [in this window] [in a new window]   FIGURE 7  Histograms showing the distributions of the compartment size (left) and the residency time (right) after various treatments (solid bars). Observations were carried out at a 110-µs resolution for a period of 278 ms for each trajectory. The compartment size and the residency time were determined as described in the legend to Table 4. From top to bottom, trypsin treatment (10 µg/ml, 10 min, 68 particles), partial depletion of cholesterol by MßCD (4 mM, 20 min, 51 particles), partial depolymerization of f-actin by cytochalasin D (13 µM, 5–15 min, 68 particles), and stabilization of f-actin by jasplakinolide (0.5 µM, 5–15 min, 45 particles). In the bottom left corner, the distribution of the compartment size after cytochalasin D treatment is shown in a broader range. Arrowheads indicate median values. Open bars indicate the distributions before treatment (control, same as those in Fig. 4), and reflect the collective control results (before treatment) for all treatments.

The actin-based membrane skeleton is responsible for cell membrane compartmentalization
To examine the effects of the actin-based membrane skeleton, cytochalasin D (final 13 µM) was added to the cultured cells on the microscope stage. To obtain a cytochalasin D effect comparable to that with fibroblasts, a high concentration (increased by a factor of >10) must be used with cells of an epidermal origin, like FRSK (or F cells; see Kusumi et al., 1993). Observations of the DOPE movement were completed within 15 min, to observe the effect of slight actin depolymerization. Under these conditions, larger compartments appeared, with the median diameter increased by a factor of 2, or the area by a factor of 4, but the mode of diffusion did not change (91% hop plus confined, Table 5). Without cytochalasin D treatment, no compartments >120-nm diameter were found, whereas after the treatment, 40% of the compartments found were >120 nm (see the bottom left box in Fig. 7).

Treatment with 0.5 µM jasplakinolide changed the movement of DOPE dramatically (Fig. 7 and Table 5). After the jasplakinolide treatment, approximately the same fraction of DOPE showed the hop plus confined diffusion mode (93% vs. 92%), but 40% became confined within a single compartment (no hop movement) on a timescale of 278 ms (observation time), up from 0% for total confinement without treatment. Jasplakinolide treatment increased the median residency time by a factor of 7, to 156 ms. It also increased the compartment size by a factor of 1.6. Both can be explained well by the dual effects of jasplakinolide. It first increases filamentous actin and actin bundles, with a concomitant decrease in monomeric G-actin, which then induces depolymerization of thin actin filaments, resulting in a coarser but stronger actin meshwork (Bubb et al., 2000).

The movement of DOPE molecules in membrane blebs, in which the membrane skeleton had been partially depleted, was also observed. Approximately 70% of the DOPE (as compared to 10% in the control cell membrane) showed simple Brownian diffusion at a 110-µs resolution (Table 5).

Taken together, these results indicate that the actin-based membrane skeleton is responsible for restricting the diffusion of DOPE in untreated cells, and are consistent with the anchored-protein picket model.

The diffusion coefficient of DOPE within a small compartment is as large as that in artificial membranes
The microscopic diffusion coefficient, D_micro, determined by the membrane viscosity and the temperature, within the 41-nm compartments on FRSK cells, was estimated. The D_micro determined for Gold-DOPE in liposomes, as well as in the bleb membranes of NRK cells after latrunculin A treatment (Fujiwara et al., 2002), over 100 µs, was 10 µm²/s (trajectories at least 100-µs long are needed to determine D_2–4 or D_100µs at a 25-µs resolution). If we assume that D_micro for DOPE in FRSK cells is also 10 µm²/s, then the DOPE would cover an area of 60 nm across during 100 µs ([4 x 10 µm²/s x 10^–4 s]^1/2). This is greater than the compartment size (41 nm), and therefore D_100µs cannot be the correct diffusion coefficient within 41-nm compartments, but rather is an operationally defined rate, which we call the apparent D_micro here.

The relationship between the apparent D_micro and the compartment size, observed in control cells and bleb membranes in FRSK cells, is shown in Fig. 8 (both observed at a 25-µs resolution). The apparent D_micro monotonically increases with an increase in the compartment size, and asymptotically approaches 8 µm²/s. These results indicate that the true D_micro in the FRSK cell membrane is likely to be comparable to that in artificial membranes or in the NRK cell membrane, suggesting that the true D_micro in 41-nm compartments is 8 µm²/s.

The diffusion rate within 200-nm compartments is 5 µm²/s, which is nearly as fast as that within 230-nm compartments in NRK cells (the dashed blue line). This indicates again 1), that the microscopic diffusion rate within a compartment in the cell membrane may appear small due to the lack of sufficient time resolutions (with the reduction of the compartment size, more enhanced time resolution is required); 2), that it may be basically very similar in various cell types; and 3), that the (macroscopic) diffusion in the cell membrane is reduced not because diffusion per se is slow, but because the cell membrane is compartmentalized with regard to lateral diffusion of phospholipids.

Compartmentalization is confirmed by quantitative anomaly analysis
To further confirm the compartmentalization of the FRSK cell membrane, we carried out a quantitative analysis using the mean square displacement (MSD), i.e., the mean log(MSD/time) for Gold-DOPE is plotted as a function of log(time), based on the data obtained at time resolutions of 25 µs, 110 µs, and 33 ms (Fig. 9). The slope in this display is sensitive to diffusion anomalies. Due to the relationship log(MSD/time) = (–1) log(time) (0 1), parameterizes the level of anomaly (Saxton, 1994, 1996; Feder et al., 1996). In the case of simple Brownian diffusion, the plot becomes flat (the slope –1 = 0), and = 1 (Fujiwara et al., 2002). When diffusion is anomalous, becomes <1, giving a negative slope (–1 < 0) to the plot. For DOPE molecules undergoing free Brownian diffusion in membrane blebs (Fig. 9), the plot is almost flat between 50 µs and 12 ms when observed at a 25-µs resolution. The best fit for the plot yields an –1 of 0.012 ( 1), which indicates that DOPE in membrane blebs undergoes simple Brownian diffusion. On the other hand, the plot for the normal cell membrane exhibits multiple negative slopes, depending on the observation time intervals. The plot can be fitted with three lines, with -values of 0.97 (50 µs 0.13 ms), 0.53 (110 ms), and 0.94 (300 ms 2 s). Two transitions were found, one at 0.1 ms (or less), and the other between 10 ms and 100 ms. The former is likely to represent the collision of DOPE with compartment boundaries, and the latter is comparable to the residency times within 40-nm (15-ms) compartments. We interpret this to mean that these transitions occur at the interfaces of the following three time zones:

1. The short time zone in which Gold-DOPE corralled inside 40-nm compartments does not sense the presence of compartment boundaries (flat left end).
   
2. The medium time zone in which Gold-DOPE undergoes microscopic diffusion inside the compartment on short timescales, although occasionally hopping to adjacent compartments (the steep slope in Fig. 9).
   
3. The long time interval in which Gold-DOPE appears to undergo simple Brownian diffusion by hopping over many compartments (trajectories in Figs. 1 b and 3 a, flattened end on the right in Fig. 9).
   

Although anomalous diffusion in the cell membrane could be caused by various kinds of obstacles and binding sites (Saxton, 1994, 1996), the good agreement of these transition times in Fig. 9 with the periods for free diffusion within a compartment and the residency time (15 ms) supports compartmentalization of the plasma membrane with respect to DOPE diffusion. Furthermore, the difference in these plots between intact and bleb membranes indicates the involvement of the membrane skeleton in the hop diffusion of DOPE.

Estimation of the correct hop rate
D_100ms and D_3s for Gold-DOPE are smaller by a factor of 5 than those for Cy3-DOPE (Table 3). This difference is probably due to the crosslinking effect of gold probes, rather than the interaction of gold probes with the extracellular matrix and extracellular domains of membrane proteins, as trypsin treatment did not affect the movement of Gold-DOPE. Cy3-DOPE would give more correct macroscopic diffusion rates over many compartments, and thus a more correct hop rate, whereas the low time resolution of SFVI does not allow direct observation of the hop events and compartment size. In contrast, SPT of Gold-DOPE would provide the correct compartment size (41 nm), although it cannot give the correct hop rate because of the crosslinking of DOPE molecules, which would enhance interactions with the anchored protein pickets. Therefore, the correct hop rate can be estimated using the D_100ms of Cy3-DOPE (median = 0.19 µm²/s, Table 3) and the 41-nm compartment size obtained by using Gold-DOPE, thus yielding an average of once every 2.3 ms ([0.041 µm]²/ 4 x 0.19 µm²/s).

Monte Carlo simulations of the point diffusant in the presence of anchored-protein picket lattices
A series of Monte Carlo simulations of phospholipids diffusion, including the effects of steric hindrance and circumferential slowing caused by proteins anchored to the membrane skeleton (Sperotto and Mouritsen, 1991; Almeida et al., 1992), were carried out to examine if the anchored protein-picket model could be applicable to the case of an extremely small compartment size (41 nm on average) and a short residency time (2.3 ms). The residency time within a compartment, as determined from the Monte Carlo simulations, is plotted as a function of barrier coverage, with the dashed red line showing the experimentally determined 2.3-ms residency time for 40-nm compartments (Fig. 10). Approximately 17, 30, or 38% coverage of each side of a square compartment, corresponding to seven 1-nm proteins, six 2-nm proteins, or five 3-nm proteins, respectively, were needed to reproduce the experimental residency time of 2.3 ms, supporting the anchored-protein picket model.

View larger version (25K): [in this window] [in a new window]   FIGURE 10  Two-dimensional, Brownian dynamics/Monte Carlo simulations indicate that 17% coverage of the boundary with 1-nm- transmembrane anchored proteins is sufficient to reproduce the experimentally determined hop rate of 2.3 ms on average. The average residency time of Monte Carlo particles in a 40-nm compartment is plotted against percent of coverage of each side of the square compartment. The red broken line indicates the experimentally determined residency time (2.3 ms). The results indicate that seven 1-nm proteins (17% coverage), six 2-nm proteins (30% coverage), or five 3-nm proteins (38% coverage), located along each side of the square compartment, were needed to reproduce the experimental residency time values of 2.3 ms.

View larger version (22K): [in this window] [in a new window]   FIGURE 11  Summary of the hop diffusion parameters observed in various cell types.

Probability of passing a barrier when the lipid is already at the boundary varies a factor of 7 over all of the cell types

The overall variation in PP among various cell types is a factor of 7 (Table 6, Fig. 11). PP would depend on the number of transmembrane proteins anchored to the membrane skeleton, the structural stability of the membrane skeleton and the membrane, and the dissociation kinetics of the actin filaments that form the membrane skeleton.

The overall variations in the residency time (a function of both PP and the compartment size) and the compartment size are factors of 17 and 3 (Table 6, Fig. 11), respectively (note that NRK cells are excluded from this discussion, because double compartmentalization makes the interpretation complex).

Oligomerization of DOPE greatly enhances the corralling effect
In FRSK, CHO-B1, HEK293, HeLa, HEPA-OVA, and PtK2 cells, D_100ms and PP for small oligomers of DOPE, induced by Fab-gold probes, were reduced by a factor of 1.5–4 from those for the monomeric DOPE (Cy3-DOPE).

The reduction of diffusion rates upon oligomerization is at marked variance with the general understanding of the translational diffusion rate of membrane-constituent molecules in a pure lipid bilayer; translational diffusion is rather insensitive to variations in the size of the diffusing unit, if the membrane is a simple two-dimensional continuum (Saffman and Delbrück, 1975). Therefore, the results obtained with small oligomers of DOPE, as described above (Table 6), clearly show that the cell membrane cannot be regarded as a continuous fluid. It is likely that monomers hop easily from one compartment to an adjacent one, but that, upon oligomerization, the corralling effects are enhanced—greatly decreasing the hop rate, and thereby D_MACRO, for oligomers (Kusumi and Sako, 1996; Fujiwara et al., 2002).

Based on the SFVI observations of E-cadherin-GFP, which showed various levels of oligomerization, Iino et al. (2001) proposed that large decreases in translational mobility with the formation of oligomers can be explained by an oligomerization-induced trapping model, in which, upon oligomer formation, E-cadherin-GFP was trapped in place due to the greatly enhanced tethering and corralling effects of the membrane skeleton on oligomers. We think that the oligomerization-induced trapping model can be applied to phospholipids as well as lipid-anchored extracellular proteins, like GPI-anchored proteins. These molecules are confined by picket lines, consisting of various transmembrane proteins anchored to the actin-based membrane skeleton. When they are oligomerized by a gold particle or by liganding, they have a much lower chance of crossing a picket line, due to the increased interactions with the anchored membrane-protein pickets. Such a corralling effect would be further enhanced if liganding were to induce stable rafts (Simons and Toomre, 2000; Sheets et al., 1999).

Previously, Fujiwara et al. (2002) found that Gold-DOPE and Cy3-DOPE exhibited similar diffusion rates, as long as the time-window was kept shorter than 100 ms. Similar observations were made in this study for T24 cells (110-nm compartment size). However, since compartment sizes and residency times are much smaller in FRSK, CHO-B1, PtK2, HEPA-OVA, HEK293, and HeLa cells than in NRK cells, the effects of DOPE crosslinking were probably greater in these cells in the 100-ms window.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
DOPE diffusion in the cell membrane is regulated by rows of anchored membrane-protein pickets
By employing SPT at a high time resolution and SFVI, in all of the cultured cells examined, we found that an unsaturated phospholipid diffuses macroscopically, by being temporarily confined in small compartments of 30–230 nm and then hopping among adjacent compartments once every 1–17 ms, on average. The existence of such small domains, 30–40-nm compartments in particular, was unexpected.

Such compartmentalization depends on the actin-based membrane skeleton (although the phospholipid molecules we obs