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GPI-anchored receptor clusters transiently recruit Lyn and Gα for temporary cluster immobilization and Lyn activation: single-molecule tracking study 1

The signaling mechanisms for glycosylphosphatidylinositol-anchored receptors (GPI-ARs) have been investigated by tracking single molecules in living cells. Upon the engagement or colloidal gold–induced cross-linking of CD59 (and other GPI-ARs) at physiological levels, CD59 clusters containing three to nine CD59 molecules were formed, and single molecules of Gαi2 or Lyn (GFP conjugates) exhibited the frequent but transient (133 and 200 ms, respectively) recruitment to CD59 clusters, via both protein–protein and lipid–lipid (raft) interactions. Each CD59 cluster undergoes alternating periods of actin-dependent temporary immobilization (0.57-s lifetime; stimulation-induced temporary arrest of lateral diffusion [STALL], inducing IP3 production) and slow diffusion (1.2 s). STALL of a CD59 cluster was induced right after the recruitment of Gαi2. Because both Gαi2 and Lyn are required for the STALL, and because Lyn is constitutively recruited to CD59 clusters, the STALL of CD59 clusters is likely induced by the Gαi2 binding to, and its subsequent activation of, Lyn within the same CD59 cluster.

Transient confinement zones (TCZs), a term coined by Simson et al. (1995) and Sheets et al. (1997), are the membrane domains where the diffusion of a membrane molecule is substantially slowed. It is defined as a domain where the observed molecule (normally tagged with a colloidal gold particle) stays much longer than statistically expected from the average diffusion coefficient of the molecule (Simson et al., 1995). Note that this is an operational definition, because it assumes that the observation is carried out at slow rates, like normal video rate, at which individual membrane compartments are usually smeared out because of time averaging on (low time resolution of) the camera. Therefore, the observed molecules are undergoing apparent simple Brownian diffusion with a macroscopic diffusion rate determined by the hop movements over many compartments, outside the TCZs.
These TCZs have appeared on the central stage of the raft research when Sheets et al. (1997) showed that (1) slightly crosslinked, putative raft-associating molecules exhibit TCZs much more often than (cross-linked) putative non-raft-associating molecules and (2) the occurrence of TCZ is formed in a cholesterol-dependent manner (Dietrich et al., 2002). In the present paper and in Suzuki et al. (2007), we showed that temporary immobilization of GPI-anchored proteins, such as CD59, DAF, and PLAP, takes place much more often when they form clusters, and such temporary immobilization is likely due to the binding of the clusters to actin filaments. Because the term zone suggests the presence of preformed microdomains in the membrane, TCZ would not be a proper word for describing the actin binding-induced temporary cessation of diffusion of the clustered GPI-anchored molecules. Therefore, here, we use the term stimulation-induced temporary arrest of lateral diffusion (STALL).

The difference between the membrane compartments (corrals) and STALL
The membrane compartments (corrals) and the domains where CD59 clusters exhibit STALL might be confusing. CD59 clusters are immobilized for periods much longer than expected from the average diffusion coefficient, but because of the measurement noise, and perhaps because of the oscillative motion of actin filaments that the CD59 clusters bind to, the binding spot on an actin filament would give an appearance of a domain. As shown in Suzuki et al. (2007), the Gaussian fit of such a domain gave a diameter of 48 nm. Because the Gaussian fitting for the noise (determined for the gold particles attached to a coverslip) gave the standard deviation of 3 nm (giving a diameter of 6 nm), the actual domain size due to the motion of actin filament would be 42 nm. In the following, we try to clarify the difference between the membrane compartments and STALL domains.
The membrane compartments cover the entire cell membrane, except for other membrane structures, like clathrin-coated pits, caveolae, and cell-to-substrate and cell-to-cell adhesion structures. Contiguity of the compartments with each other is one of the important characteristics of the compartmentalized structure, as this is seen as close apposition of compartments in the trajectories of membrane molecules observed at sufficiently high time resolutions (for an example, see Fig. S4). The fence and picket models are consistent with these observations, because they insist that the compartments are separated by narrow quasilinear fences or by the fence posts that keep these fences near the bilayer, so there is not any significant diffusion distance between adjacent compartments. The actin-based fences are thought to be quasilinear at around the 1 μm level, because the persistent length of the actin filament was found to be between 2 and 15 μm (Yanagida et al., 1984;Gittes et al., 1993;Steinmetz et al., 1997;Liu and Pollack, 2002).
In contrast, contiguity of STALL domains with each other is not generally seen (although STALL domains might be concentrated in a small region). Namely, STALL domains are mostly separated from each other, often a micrometer or farther away. It's not proper to call the inter-STALL times "hops," as they can be quite long, both spatially and temporally. The residency time within the membrane compartment is 1-25 ms on average (200 ms for CD59 clusters), whereas a STALL period is on average on the order of half a second. The diffusion coefficient within a compartment is comparable to those found in artificial membranes (5-10 μm 2 /s), whereas the movement during the STALL period might be determined by the fluctuating movement of the actin filament.
How do the partitioned membrane compartments and STALL sites appear when the movement of CD59 clusters is observed at high frame rate, such as once every 0.11 ms?
What will we see if CD59 clusters are tracked at a high time resolution? Both membrane compartments and STALL domains are likely to be observed as distinct domains. STALL domains (48 nm as determined at video rate) are likely to be generally smaller than the size of the corralled compartments (110 nm on average), and the average residency time in a STALL domain is 0.58 s, whereas the residency time in a membrane compartment will be generally shorter, i.e., 0.2 s on average. Because this is a subject matter entirely different from those of this paper and Suzuki et al. (2007), and will be published elsewhere, we give a glimpse of these results, using a representative trajectory observed at 0.11-ms resolution in Fig. S4. Figure S1. Oligomerization-induced trapping of membrane molecules within membrane compartments. (a) Paradigm shift of the plasma membrane structure, from the simple fluid-mosaic model to the partitioned fluid model. The diffusion coefficients of membrane molecules in the cell membrane are reduced by a factor of 5-50 compared to those in artificial membranes. The reduction mechanism has puzzled membrane biologists for >30 yr. Single-molecule tracking of membrane molecules at an imaging frame rate as high as 40 kHz or more (a time resolution of 25 μs or shorter) for >1 s (40,000 frames or longer) has shown that the major mechanism for such reduction is that the entire plasma membrane, except for specialized domains such as clathrin-coated pits, caveolae, and cell-adhesion structures, is parceled up into apposed domains of a size between 30 and 250 nm (called compartments) and that both membrane lipids and integral proteins undergo short-term confined diffusion within the membrane compartments and long-term hop diffusion between the compartments. Namely, diffusion in the plasma membrane is slow not because diffusion per se is slow, but because molecules are temporarily confined in these compartments and it takes some time to hop from a compartment to an adjacent one. As it turned out, the residency time within a compartment is as short as 1-25 ms for phospholipids and 3 ms to 1 s for transmembrane proteins (depending on the cell type and molecule; Kusumi and Sako, 1996;Fujiwara et al., 2002;Murase et al., 2004;Kusumi et al., 2005). Furthermore, generally, at least 40 measured points within a compartment were required to distinctively detect a compartment as such, and thus the time resolution of 25 μs was required in many cases (1 ms devided by 40). In video-rate observations, a membrane molecule undergoing hop diffusion looks as if it were undergoing slow simple Brownian diffusion (apparent or effective simple Brownian diffusion). See Fig.  S4 for an example. (b) The membrane-skeleton fence model (left) and anchored transmembrane-protein picket model (right). The compartment boundaries are likely to be made of the actinbased membrane skeleton (the cytoplasmic domains of transmembrane proteins directly collide with the membrane skeleton; the membrane-skeleton fence model), and rows of transmembrane-protein pickets anchored to the membrane skeleton (the anchored-protein picket model). Therefore, for transmembrane proteins, both the fence and pickets work together to suppress their diffusion in the membrane. In understanding the picket model, it is important to realize that these picket proteins exert the effects of enhanced packing or hydrodynamic friction on surrounding lipids, as well as the steric hindrance effect by their own volume on diffusing molecules in the membrane (Almeida et al., 1992;Bussell et al., 1995;Dodd et al., 1995). Therefore, the movement of lipids is slowed up to 2-3 nm away from the picket proteins. This, coupled with the alignment of these picket proteins along the actin membrane-skeleton fence, forms effective picket lines that induce temporary confinement of lipids and proteins in the membrane-skeleton mesh . Actin-based corralling has further been supported by a recent finding, using electron tomography of the cytoplasmic surface structures of the plasma membrane (Morone et al., 2006). In this work, the distribution of the mesh size of the actin filaments right on the cytoplasmic surface of the plasma membrane was deduced from the three-dimensional structure of the membrane skeleton. The mesh size distribution obtained from electron tomography agreed well with the distribution of the compartment size determined from the single-molecule tracking of diffusing phospholipid in the plasma membrane. (c) Oligomerization-induced trapping model. Using E-cadherin as a paradigm, Iino et al. (2001) previously showed that, when E-cadherin-GFP molecules are clustered, their diffusion coefficient was dramatically decreased, even when the cluster was as small as a trimer. Such a reduction of the diffusion coefficient would not occur in the absence of fences and pickets (left). In the presence of pickets and fences (right), even if monomers of a membrane molecule undergo rapid hop movement across the compartment boundaries, when it forms oligomers, the hop rate would be substantially decreased because much greater space between the membrane and the membrane skeleton or much greater opening between the dissociated membrane-skeletal filaments are necessary to allow the passage of oligomers than monomers. Furthermore, the oligomers of membrane molecules tend to bind to the membrane-skeletal filaments much more readily than monomers, because of the avidity effect (see the right figure; MSK tether). In either case, oligomerization of membrane molecules would greatly prolong their stay within a compartment. The results shown in Fig. S2 suggest that the pentamers of anti-CD59 IgG, which are likely to bind to 5-10 molecules of CD59 in the plasma membrane, are basically immobile as a result of the effect of oligomerization-induced trapping of membrane molecules.

STALL detection
The detection of STALL was carried out by using a plot of a TCZ probability level L versus time, as developed by Simson et al. (1995). L is a convenient yardstick, showing the likelihood with which a particle is confined at the given time, based on the average diffusion coefficient over a single trajectory (Fig. S3 b). Three parameters were used to calculate L and determine TCZs: the window size to calculate L (Sm); the critical threshold value of L (Lc), which is used to select the TCZ candidates; and the critical threshold duration (tc), which determines the minimal duration for the TCZ candidates to last to be real TCZs. They generally recommended using the following criteria: Sm = 17 steps, Lc = 3.16, and tc = 8 steps (Simson et al., 1995), and these values were used here. The method by Meilhac et al. (2006) was not useful for detecting STALL, as expected from the theory.
The term STALL was used instead of TCZ in the present papers, to avoid confusion, because we were unable to rule out the possibility that the temporary immobilization of CD59 clusters is induced by their binding to the actin-based membrane skeleton, rather than by being confined within preexisting zones.
The length of the trajectory used for the analysis had no influence on the estimated parameters, as long as it was >5 s. The size of the area covered by a CD59 cluster during STALL was estimated by the 2D Gaussian fitting of the determined coordinates of the CD59 cluster during the STALL period (see Fig. S3 a). This evaluation method (definition) of the STALL domain size is different from what was described in by Simson et al. (1995), where the maximal expected domain size was evaluated. As our results suggest that STALL is induced by the binding of a CD59 cluster to an actin filament, we estimated the STALL size from the spread of the measured points during STALL by a 2D Gaussian fitting. This measures the jittering motion (convoluted with the spread as a result of the measurement error) of the CD59 cluster bound to the actin filament.
STALL is conceptually different from the phenomena related to compartments (STALL is probably induced by the binding of CD59 clusters to actin filaments); in addition, STALL cannot be seen without inducing clustering of GPI-ARs. Figure S2. The diffusion coefficients for the spots containing five anti-CD59 IgG molecules, showing that they are practically immobile. (a) The distribution of the fluorescence signal intensities of the individual spots in the image of anti-CD59 IgG tagged by Cy3 (dye/protein ratio = 0.9) attached to the T24 cell surface, before (top) and after (bottom) the addition of the secondary antibodies (goat anti-mouse IgG). To avoid the effect of photobleaching, the fluorescence intensity was measured right after illumination is started in the new view-field, by averaging the signal intensities of a spot in the first five video frames after illumination was initiated. Even in cells without cross-linking with secondary antibodies (top), a substantial fraction of the spots existed in a peak with the double signal intensity (green curves representing Gaussian fitting), probably representing IgG bound by two Cy3 molecules, as expected from the Poisson distribution with an average of 0.9. After anti-CD59 IgG was clustered by the secondary antibodies (bottom), the distribution of the signal intensities of the individual fluorescent spots broadened substantially. Note that the concentration of the secondary antibodies used here is 10-fold less than that used for the experiments shown in Fig. 6, inducing much lower levels of CD59 clustering. Gaussian fitting was done with free parameters, with initial parameters given by an operator (peaks and widths read out from the figure). From this fitting result, the fluorescent spots that exhibited the fluorescence intensity of five Cy3 molecules were selected (yellow bar). These clusters on average contain five anti-CD59 IgG molecules, which in turn represent clusters of 5-10 molecules of CD59. (b) The distributions of the effective (or apparent) diffusion coefficients for IgG-gold-induced CD59 clusters (shaded bars), for the clusters containing an average of five Cy3-conjugated anti-CD59 IgG molecules (the histogram with thick, black outlines; using the points included in the yellow band in panel a), and for anti-CD59 IgG molecules conjugated with an average of five Cy3 molecules, attached to the coverslip (histograms with red outlines; because the noise level determines the apparent diffusion coefficient of immobile molecules, giving the lower limit of the method to determine the diffusion coefficient, the number of Cy3 molecules in a spot had to be matched to five in the latter two cases). By comparing the histograms outlined by black and red lines, it is concluded that the fluorescent spot containing five anti-CD59 IgG molecules (in turn, containing 5-10 CD59 molecules) are practically immobile. Meanwhile, the majority of the IgG-gold-induced CD59 clusters are mobile (see the shaded histogram in panel b). Therefore, it was concluded that each IgG-gold-induced CD59 cluster contains <10 CD59 molecules beneath the gold particle (otherwise, the IgG-gold-induced CD59 clusters would be immobile like the anti-CD59 IgG molecules bound to the coverslip). This can be understood naturally or, more specifically, by the anchored transmembraneprotein picket model and oligomerization-induced trapping we proposed previously (Iino et al., 2001;Kusumi et al., 2005; see Fig. S1 for further explanation of these models; the greater the oligomer grows, the slower it diffuses, until it finally stops diffusion). One cannot use GFP for the experiments to determine the number of molecules in a cluster, because one generally does not know the fraction of GFP that actually fluoresces. Figure S3. The definition and the detection protocol for STALL. (a) A representative trajectory of a CD59 cluster recorded at video rate (33 ms). Four STALL regions were detected, using a display shown in panel b. The numbers shown here corresponds to those in panel b. (b) The detection of STALL was carried out by using a plot of a TCZ probability level L versus time, as developed by Simson et al. (1995) (see supplementary Materials and methods). In the case of the trajectory shown in panel a, five STALL candidates were selected from Lc (1-4 + asterisk), and four of them satisfied tc requirement (1-4), but one peak showed the duration shorter than the critical duration of eight steps (with asterisk), being ruled out from STALL. The plot of L versus t for a typical simulated simple Brownian trajectory is shown in the bottom box, where L was found to be always smaller than Lc. After the submission of this paper, a somewhat related paper by Chen et al. (2006) was published. They observed that gold particles that can highly cross-link Thy-1 or 5'-NT sometimes exhibited much less mobility during STALL, which tends to last much longer (>10 s). We did not find such events, perhaps because the level of cross-linking we are using (five times the minimal protecting amounts of IgG for gold particles) is between those for gold particles that exhibited transient confinement and transient anchorage, described in their paper. (c) The plot of the probability level L versus time for the trajectory shown in Fig. S5. Figure S4 (facing page). Detection of membrane compartments and STALL in the trajectories observed by high-speed singleparticle tracking. (a and b) Typical trajectories of CD59 clusters simultaneously recorded at normal video rate (33 ms/frame; panel a) and at a frame rate of 0.11 ms/frame (300 times faster than normal video rate; panel b). The domain-detection software  was applied to the 0.11-ms/frame trajectory shown in panel b, and the compartments and STALL sites were detected, as shown by different colors. The color is changed following the sequence of compartment appearance, in the order of purple, blue, green, yellow, red, and back to purple again. The compartments are shown in dashed lines, with their locations and shapes approximated by ellipsoids, determined from the x (long axis) and y coordinates for individual compartments. (c) By definition, the occurrence of STALL domains must first be identified in the corresponding 33-ms trajectory (a), using the STALL-detection software and the display in panel c, as described in the caption to Fig. S3. See the STALL periods 1 and 2, delimited by vertical lines. The STALL domains in the high-speed trajectory in panel b were found from the determined compartments, based on the match of the time periods for the STALLs in panel a trajectory and those for compartments in panel b trajectory (see the periods for staying in compartments 5 and 7, as shown by the horizontal thick lines on the x axis of panel c). The STALL compartment is shown by a blue ellipsoid in the panel b trajectory (also see panel d). In the trajectories shown in panels b and d, note that the two STALLs occurred in the same place, in the compartment (5) and (7) (see below). In addition, note that the compartment (5 and 7) is located at the border of compartments (3) and (4), as well as at the border of compartments (3 [green]) and (6 [orange]), consistent with the concept of STALL induction by the binding of CD59 clusters to the actin filaments. (d) To clarify the occurrence of each compartment, the trajectory shown in panel b is broken into six separate sequences. Each time the CD59 cluster enters a new compartment, a new number is given to that compartment. However, the CD59 cluster revisited some of the compartments more than once. When this occurred, the sequential compartment number is followed by a parenthesis with two hyphenated numbers. The first number indicates the sequential compartment number when the CD59 cluster first entered the same compartment, and the second number shows whether it was the first or the second visit there (in this particular example, we did not see the return of the CD59 cluster three times or more to the same compartment). In this example, the compartments marked by the sequential numbers (3 and 10), (5 and 7 [STALL 1 and 2]), (6 and 8), and (14 and 16) are the same compartments. In the case of the (3 and 10) compartment, the compartment slightly shifted toward the direction of 7 o'clock (see the green [3] and red [10] parts of the trajectory). In the case of the (6 and 8) compartment, the compartment slightly shifted toward the direction of 2 o'clock. The median residency time of CD59 clusters in a compartment (and not a STALL) was 200 ms, much shorter than the STALL lifetime of 570 ms. All of such paired trajectories (trajectories simultaneously obtained at rates of 33 and 0.11 ms/frame) exhibited virtually the same characteristics as described above. The median size of the STALL sites in the 0.11-ms resolution trajectories detected this way was 64 nm, which is in reasonable agreement with the 48-nm diameter determined by the Gaussian fitting for the STALL sites in the 33-ms trajectory. This value is much smaller than the normal compartments (median = 110 nm), presumably corralled by actin filaments (the size distributions for normal compartments and STALL domains as determined as described above were statistically different; P < 0.01 by U-test). Further explanations on the difference between the membrane compartments (corrals) and STALL are given in the supplemental Materials and methods. See Figs. S1 and S4 for further explanations about the difference between these two concepts.
Furthermore, STALL was basically abolished after the MβCD pretreatment (4 mM, 30 min, 37°C) of cells. Meanwhile, such MβCD treatment did not affect the compartment sizes significantly. The median compartment sizes changed from 260 to 280 nm in NRK cells, and from 110 to 100 nm in T24 cells (statistically insignificant difference). Figure S5. STALL sites are not caveolae. A typical trajectory of a CD59 cluster (white, 3-min-long, observation initiated 1 min after the addition of IgG-gold), with 41 STALL sites indicated by magenta circles (with a 48-nm median diameter for all of the STALL sites; see Suzuki et al., 2007), is superimposed on the image of GFP-caveolin 1 (green spots). None of the STALL sites were colocalized with caveolae, under the experimental conditions used here (only the initial events, generally within 5 min after IgG-gold addition were observed). About 3% (20%) of the IgG-gold became colocalized with caveolae after a 5-min (30min) incubation (not depicted). However, when CD59 was extensively cross-linked by the primary and secondary antibodies, CD59 quickly became trapped in caveolae as they form large aggregates, in agreement with Chen et al. [2006]). In addition, IgG-gold rarely colocalized with clathrin-coated pits, as marked by GFP-AP2α (not depicted). These results indicate that the temporary immobilization of CD59 clusters at the earlier stages of signaling is not caused by their interactions with either caveolae or clathrin-coated pits.