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Article |
Correspondence to Hidekazu Tanaka: htanaka{at}pharma1.med.osaka-u.ac.jp
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Abstract |
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Abbreviations used in this paper: CCD, charge-coupled imaging device; DIV, day in vitro; SCCL, spine cotyloid curve length; W2A-cadherin, W2A mutant of N-cadherin.
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Introduction |
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The CNS synapse is an adhesive junction comprising pre- and postsynaptic membranes, which are accompanied by the machinery for intercellular communication. Among certain families of adhesion proteins that have been suggested to be responsible for this adhesion, cadherins are likely to provide the adhesive force to maintain synaptic membranes in apposition (Yamagata et al., 1995; Fannon and Colman, 1996; Uchida et al., 1996; Benson and Tanaka, 1998; Inoue et al., 1998; Miskevich et al., 1998). Recent studies have shown that synaptic cadherins are involved in synaptic plasticity (Yamagata et al., 1999; Manabe et al., 2000). In particular, N-cadherin, which is enriched in hippocampal synapses (Benson and Tanaka, 1998), is required for the establishment of long-term potentiation (Tang et al., 1998; Bozdagi et al., 2000). Furthermore, N-cadherin rapidly redistributes after vigorous depolarization and acquires pronounced trypsin resistance, a property of stable cadherins engaged in adhesive interactions (Tanaka et al., 2000). The enhanced adhesiveness of N-cadherin is assisted by the recruitment of ß-catenin to the activated synapse (Murase et al., 2002). The modification of N-cadherin with ß-catenin is not dependent on new protein synthesis, probably providing the structural framework responsible for a rapid phase synaptic plasticity.
N-cadherin is a member of the classical cadherin family, homophilic adhesion molecules with five extracellular subdomains separated from the cytoplasmic domain by a single transmembrane segment (Takeichi, 1990). The coupling of the cytoplasmic domain with the actin-cytoskeleton through catenins seems to be essential for full adhesive activity (Gumbiner and McCrea, 1993). Hence, cadherins provide the framework that links the cellcell contact on the membrane surface to the cytoskeleton. However, it has not been directly studied whether cadherins are involved in the actin-mediated remodeling of synapses.
Here, we set out to the time-lapse analysis of GFP-visualized spines in order to understand the relationship between the morphological plasticity and the adhesive machinery linked to the actin-cytoskeleton. Activation of AMPA-glutamate receptor induces the lateral expansion of the spine surface that apposes the presynaptic membrane. The overexpression of a dominant negative form of N-cadherin abolishes the lateral spine expansion. Inhibition of actin-polymerization with cytochalasin D also blocks the spine expansion. This work suggests that the cooperation of the cadherin-actin complex is required for rearrangement of the adhesive surface of the synaptic junction, which could be at least in part responsible for the rapid phase of synaptic plasticity.
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Results |
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To quantify this spine enlargement, we took advantage of a confocal microscope (Fig. 2). The three dimensional information of the spine of interest was analyzed by optical section (z-series) images collected at 0.2 µm focus intervals. The profile of the z-stack images was sufficient to obtain an unambiguous spine profile (Fig. 2 A). The width of spine heads was measured on the spine profile being thresholded at half maximal fluorescence intensity (Fig. 2 B). The maximum spine width was determined by laying the axis as to run across the widest point of the spine head (Fig. 2 B, gray line). The spine width changed from 1.18 ± 0.0490 µm to 1.36 ± 0.0620 µm (Fig. 2 C; mean ± SEM).
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Although the confocal technology enables us to avoid out-of-focus image blurring, it takes at least tens of seconds to collect data of the area that includes several spines, while the spine changes its shape within several seconds (Fischer et al., 1998). To obtain time-lapse images rapidly enough, we collected only a single focus image at each time point with 6-s intervals by the use of a CCD; these experiments confirmed the rapid spine motility (Fig. 3 A). We measured the dimensions of spines from these single-focus images. Measurements of the (a) width, (b) length, and (c) curve of the apex of the spine (spine cotyloid curve length [SCCL]) were performed on thresholded images at half maximal fluorescence intensity (Fig. 3 C). A possible drawback of this procedure, however, is a limited resolution. Therefore, we obtained optical section images of the GFP-filled spines in fixed neurons, and compared undeconvoluted images with deconvoluted ones (Fig. 3 B). In spite of the relatively low resolution of the undeconvoluted images, the dimensions measured on these undeconvoluted images were very well correlated with those measured on the deconvoluted images (Fig. 3 D). For further accuracy, we averaged the values obtained from 21 time series with 6-s intervals for each time window of 2 min.
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In addition to the spines with laterally broad heads, there were spines with their apexes curved deeply (Fig. 3 F). This type of spine did not show lateral expansion, but extended the both ends of the cotyloid curves longitudinally. However, this type of spine also appeared to broaden the adhesive contact surface, where the spine apex is apposed to the presynaptic membrane (Fig. 1 C). Therefore, it seems reasonable to measure the SCCL in order to include the remodeling of the deep cotyloid spines (Fig. 3 C). As expected, the change in SCCL was found to be more prominent than one in simple spine width (Fig. 3 G). The mean SCCL changed from 2.80 ± 0.136 to 3.38 ± 0.168 µm. The mean SCCL at 30 min after the depolarization became 1.22 ± 0.0527 times larger than the resting state (Fig. 3 H). The temporal profiles of the SCCL of individual spines differ with each other, but the enlargement usually peaked at 15 or 30 min after the stimulation (Fig. 3 I). The data support the hypothesis that strong synaptic inputs accelerate synaptic efficacy, which may accompany the expanded area size for the trans-synaptic communication (Buchs and Muller, 1996; Tanaka et al., 2000; Colicos et al., 2001).
The AMPA receptormediated pathway is responsible for the spine broadening
Dendritic spines comprise an excitatory postsynaptic structure equipped with glutamate receptors. To see if the lateral spine enlargement is coupled with excitatory synaptic functions, we tried to identify the pathway responsible for the phenomenon. In the presence of an AMPA receptor antagonist CNQX (100 µM), neither spine shrinkage during depolarization, nor spine enlargement after recovery occurred (Fig. 4). The mean SCCL being 3.41 ± 0.175 µm at rest remained 3.38 ± 0.160 µm in 30 min of recovery. In contrast, an NMDA receptor antagonist APV (100 µM) did not strongly affect these responses of the spines (Fig. 4). The mean SCCL changed from 2.63 ± 0.103 µm to 3.08 ± 0.112 µm in 30 min of recovery. The data suggest that the broadening of spine head is under the influence of AMPA-type glutamate receptor activation. The observation is consistent with the hypothesis that the rapid rearrangement of the synaptic adhesion zone might be related to the rapid phase of synaptic plasticity.
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To gain insight into the involvement of N-cadherin in the activity-induced enlargement of the spine head, we pursued the redistribution of N-cadherin by expressing recombinant N-cadherin fused with the Venus fluorescent protein, a variant of GFP (Fig. 5 A; Nagai et al., 2002). Although Venus was ligated adjacent to the catenin-binding domain of N-cadherin, N-cadherin-venus normally bound to ß-catenin and exhibited homophilic adhesive activity on transfected L929 cells (unpublished data). N-cadherin-venus was distributed widely throughout the spine head, whereas PSD-95, a postsynaptic density marker protein, was often restricted to the center of the spine head (Fig. 5 B). The distribution of N-cadherin-venus well fitted in with the distribution of intrinsic N-cadherin as examined by immunostaining (Fig. 5 C). N-cadherin-venus was redistributed along the laterally moving spine head in resting state (Fig. 5 D). Upon depolarization, N-cadherin-venus showed lateral dispersion along the expanding spine head (Fig. 5, E and F). The lateral extent of spinal N-cadherin changed from 3.23 ± 0.222 µm to 3.60 ± 0.274 µm in 30 min of recovery. Thus, N-cadherin seems to be redistributed in accordance with the broadening of the synaptic apposition zone.
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To rule out the possible artifact by the overexpression of N-cadherin in the experiments above, we examined the effect of overexpression of N-cadherin on the change in spine shape. We cotransfected N-cadherin and gfp cDNAs into cultured neurons, and confirmed that the green neurons coexpressed abundant recombinant N-cadherin by immunostaining retrospectively (Fig. 6 A). 57% of the dendritic protrusions displayed typical cotyloid spine, whereas only 16% were filopodia (Table I). The data suggest that overexpression of N-cadherin increases the ratio of mature spine and decrease the ratio of filopodia. However, the total number, and the mean length of the protrusions were not changed by the overexpression of N-cadherin (Fig. 7 D; Table II). The morphological responses to membrane depolarization were essentially the same as in the control neurons; the spines rounded up and halted during the depolarization, and became enlarged after the repolarization (Fig. 6 B). The mean SCCL changed from 3.08 ± 0.0975 µm to 4.00 ± 0.134 µm in 30 min of recovery (Fig. 6 C). The extent of the spine enlargement was similar to that in control neurons (1.32x enlargement of SCCL, SEM = 0.0359; Fig. 7 J).
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We then focused our analyses on the normal shape (C-1) and flat apex (C-2) spines, and pursued the morphological plasticity during and after neural stimulation. Depolarization of the W2A-cadherin transfected neurons resulted in rounding up and freezing as observed in control neurons (Fig. 7 F). However, there was no lateral enlargement observed during the recovery phase (Fig. 7 F). The lateral size of spines became rather smaller than in the resting state. The spine width changed from 1.17 ± 0.0374 µm to 1.02 ± 0.0377 µm in 30 min of recovery (Fig. 7 G). The observation was confirmed by the analysis using a confocal microscope; the spine width changed from 1.36 ± 0.076 µm to 1.12 ± 0.049 µm in 30 min of recovery (Fig. 7, H and I). Moreover, rapid frame time-lapse analyses enabled us to distinguish the core portion of C-2 type spines from the additional protrusions that move very quickly and randomly and that disappear shortly (see Fig. 8 D). Thus, we ruled out transient protrusions, and could determine the spine apex curve for the measurement. The SCCL changed from 3.16 ± 0.146 µm to 2.52 ± 0.136 µm in 30 min of recovery (Fig. 7, G and J). All these observations were confirmed by using another dominant negative cadherin construct, NcadE (Fig. 7, D and J; Tables I and II). The data suggest that cadherin activity is necessary for the lateral expansion of spine in response to synaptic activity as well as for the maintenance of the spine shape.
By the overexpression of W2A-cadherin, the distribution of AMPA receptor as marked by GluR2/3 immunoreactivity remained normal accumulating on spines (Fig. 7 K). Therefore, the synaptic input and the subsequent signal transduction through the AMPA receptor may not be disturbed by W2A-cadherin. Together, the abolishment of the activity-induced spine expansion with W2A-cadherin is unlikely to be a secondary event due to the abrogation of AMPA receptors.
Activity-induced spine head expansion is dependent on actin polymerization
The rapid motility of spines is dependent on actin polymerization (Fischer et al., 1998). In addition, cadherin-based adhesion is dependent on linkage to the actin-cytoskeleton, bridged by ß- and -catenins (Gumbiner and McCrea, 1993). Therefore, we examined if the cadherin-dependent spine remodeling is associated with the actin polymerization. Low concentration of cytochalasin D arrests spine motility without significant depolymerization of preexisting actin fibers (Fischer et al., 1998). In the presence of 40 nM cytochalasin D, spines showed rounding up during depolarization, suggesting that the phenomenon is not dependent on actin polymerization (Fig. 8 A). In contrast, no enlargement of the spines was observed during the recovery phase in the presence of cytochalasin D (Fig. 8, AC). The mean SCCL being 3.14 ± 0.102 µm at rest remained at 3.33 ± 0.137 µm in 30 min of recovery in the presence of cytochalasin D. The data indicate that the activity-induced expansion of spines is dependent on the remodeling of the actin-cytoskeleton, as well as its surface partner, the cadherins.
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Therefore, we asked whether the spine shape is dependent on F-actin itself. To address this, F-actin was depolymerized using the G-actin binding drug latrunculin B, and its effect on spines was assessed. Addition of 5 µM latrunculin B, which results in a progressive loss of F-actin (Zhang and Benson, 2001), resulted in a rough spine head with random protrusions (Fig. 8 E, arrowheads). Together, the data suggest that synaptic stimulation drives the polymerization of actin and the remodeling of cytoskeleton, which in turn results in the random and transient protrusion of filopodia-like structures, as the actin-cytoskeleton is disconnected from the cadherin-based adhesion apparatus. On the other hand, this driving force might result in the normal expansion of the synaptic adhesion zone, if there are enough cadherin molecules to link with the activity-driven actin-cytoskeleton in synaptic membranes.
Change in diffusion rate of GFP does not affect spine profile
There is possibility that the change in shape of GFP-filled spine is interfered by the alteration of the diffusion rate of soluble GFP. The change in molecular diffusion rate in response to synaptic activity has been observed in the case with the membrane bound form of the GFP (Richards et al., 2004). To assess the extent of the possible influence due to the diffusion rates of GFP, we have performed FRAP experiments in GFP-filled spines under various conditions (Fig. 9). In either condition, the diffusion of soluble GFP was so rapid that we observed subsecond recovery curves as reported previously (Majewska et al., 2000). There was no detectable difference in the FRAP of cadherin-inactivated neurons (Fig. 9 B). Although there was some delay in recovery with the addition of cytochalasin D, submaximal recovery was achieved within 1 s (Fig. 9 C). Therefore, the change in spine size may not be interfered by the diffusion rate of GFP. Moreover, we observed rapid spine motility both before and after depolarization. This suggests that the GFP diffusion was rapid enough to fill the moving spine in this time course. Therefore, the shape change of GFP-filled spine in control, N-cadherin inactivated, and cytochalasin Dtreated neurons likely reflect the actual change in spine shape.
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Discussion |
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In this work, insight is provided into the mechanism of how excitatory synapses consisting of glutamatergic terminals and dendritic spines undergo enlargement of the synaptic apposition zone. We propose a model comprising several steps. First, synaptic stimulation that involves AMPA receptormediated signaling pathways activates the vigorous rearrangement of actin-cytoskeleton, the putative key regulator of spine morphology (Fischer et al., 2000; Colicos et al., 2001; Furuyashiki et al., 2002). Second, the enriched actin-cytoskeleton may link with its surface counterpart, the cadherins, and become stabilized. Consistent with this idea, there is evidence that ß-catenin, a molecular bridge between the actin-cytoskeleton and cadherin, is recruited to the activated spines and coupled with synaptic N-cadherin (Murase et al., 2002), which also becomes stabilized (Tanaka et al., 2000). Third, the synaptic cadherins recruited at the tip of the newly assembled cytoskeleton establish stable cellcell contacts, if the cadherins find their homophilic partner across the synaptic cleft.
The overexpression of adhesion-defective cadherin mutants, W2A-cadherin and NcadE, on the plasma membrane disconnects the linkage between cadherin and the actin-cytoskeleton. In this condition, the actin under remodeling loses its anchor to the cell surface, and in turn, to the adhesive interactions with another cell. Thus, the actin-cytoskeleton under vigorous rearrangement loses its control, protruding random-shaped structures ad libitum (Fig. 8 D), and yet the changes do not persist without the storage of a "structural memory" in the synaptic adhesive structure.
The activity-induced protrusion on the top of the spines of W2A-cadherin overexpressing neurons (Fig. 8) is reminiscent of the activity-induced development of filopodia (Maletic-Savatic et al., 1999; Ostroff et al., 2002). A small subpopulation of these activity-induced dendritic filopodia establish synaptic contacts with axon terminals and develop mature spines with synaptic markers, such as PSD-95 (Prange and Murphy, 2001). However, many of those which do not establish contact with the presynaptic membranes are likely to be unstable and will retract (Ostroff et al., 2002). Together, with our experiments with W2A-cadherin, both the remodeling of existing synapses and synaptogenesis stimulated by activity persists when these structures establish adhesive contact with the presynaptic membrane. The adhesion molecules on such potential synaptic contacts might be sensors that transmit the extracellular cue to the intracellular machinery that shape synaptic structure.
Long-term effect of the breakage of cadherin-actin linkage
Cultured hippocampal neurons exhibit spontaneous activity (Bekkers and Stevens, 1989). The spontaneous synaptic activity may repetitively stimulate each synaptic junction to enlarge its adhesive area. The disconnection of cadherin-based adhesion from the actin-cytoskeleton should also inhibit these spontaneous forces for synapses to expand the adhesive area (Fig. 7). Probably because of the chronic effect of W2A-cadherin overexpression, the width of each spine becomes smaller. As a result, neurons transfected with W2A-cadherin or NcadE sprouted less numbers of normal spines but more numbers of filopodia, whereas the total numbers of the dendritic protrusions remained the same (Tables I and II). Consistent with this speculation, Togashi and colleagues (Togashi et al., 2002) have demonstrated that a dominant negative cadherin, which has a similar function to W2A-cadherin and Ncad
E, yields narrow-headed, filopodia-like spines. Thus, the apparent developmental effect derived from the chronic manipulation of the cadherin-actin complex seems to be the summation of the results of repetitive synaptic events.
Possible involvement of multiple classical cadherins in the remodeling of synaptic junctions
Various classical cadherin members, such as N- and R-cadherins, cadherin-6, cadherin-8, and cadherin-11 are expressed in CNS (Benson and Tanaka, 1998; Manabe et al., 2000; Tanaka et al., 2000). At least some of them are localized in synaptic membranes. There is the observation that there are some synapses which express ß-catenin, but without N-cadherin; it is possible that these synapses may express other classical cadherins (Benson and Tanaka, 1998). Certain classical cadherins may label a certain population of N-cadherin-negative synapses, and some other classical cadherin members may colabel a subpopulation of N-cadherinlabeled synapses in combination.
Expression of the W2A-cadherin or NcadE probably modulates cadherin-based synaptic adhesion by competing with the intrinsic cadherincatenin interaction. Also, it is probable that in some synapses W2A-cadherin may inhibit the adhesiveness of other classical cadherins, because these classical cadherins share common cytoplasmic binding sites for catenins, and in turn, the actin-cytoskeleton. Therefore, W2A-cadherin overexpression may yield an overall suppression of the sum of the adhesiveness by various classical cadherins. However, concerning the excitatory synapses that are established on the top of the dendritic spines of hippocampal neurons, all the synapses are highly positive for N-cadherin (Benson and Tanaka, 1998). Moreover, N-cadherin, but not R-cadherin, cadherin-6, or cadherin-8, exhibits activity-induced dimerization and the acquirement of trypsin-resistance (Tanaka et al., 2000). Therefore, it is likely that the phenomena resulting from W2A-cadherin overexpression may largely reflect the dysfunction of N-cadherin.
Conclusion
This work shows a significance of the linkage of the cell adhesion molecules resides in the pre- and postsynaptic membranes with actin-cytoskeleton during the remodeling process of the synaptic junction. This work, as well as previous studies (Fischer et al., 2000; Colicos et al., 2001), indicates that synaptic activity triggers active remodeling of the synaptic cytoskeleton that involves actin-polymerization. This driving force for the cyto-architectural rearrangement is organized and stabilized by its linkage to cadherins, which recognizes the partner cell membrane with which to form synapse. Synaptic cadherin might be a sensor that transmits the environmental information across the synaptic cleft to the cytoskeleton under active rearrangement in synaptic cytoplasm.
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Materials and methods |
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Time-lapse imaging
Neurons transfected with gfp or N-cadherin-venus cDNAs were cultured until 18 DIV. After changing the culture medium to Tyrode solution (25 mM Hepes-NaOH, 119 mM NaCl, 2.5 mM KCl, 2.0 mM CaCl2, 2.0 mM MgCl2, pH 7.4), the culture dish was mounted in a chamber at 37°C for live recording. For high K+-stimulation, 1.5 ml of Tyrode-HK solution (25 mM Hepes-NaOH, 71.5 mM NaCl, 50 mM KCl, 2.0 mM CaCl2, 2.0 mM MgCl2, pH 7.4) was added to the culture dish filled with 1 ml of normal Tyrode making the final [K+] at 31 mM.
Microscopes and measurements
CCD images were obtained on a microscope (model Eclipse TE100; Nikon) equipped with GFP-optimized filter set (model MBE34931;Nikon) and CCD camera (model C4742-95; Hamamatsu). A 60x 1.40 NA oil-immersion objective (PlanApo; Nikon) was used to project images to the camera without an intermediate projection lens. Luminavision software (Mitani) was used to control mechanical shutters and filter wheels. Measurements of the projected images were performed with MacSCOPE software (Mitani). Optical section images of sixty focal planes 0.2 µm apart were collected by the use of a DeltaVision deconvolution microscope system (Applied Precision, Inc.) as described previously (Haraguchi et al., 1999). Images of spines resulted in a nominal spatial resolution of 0.11 µm/pixel by the use of a 60x 1.40 NA oil immersion objective (PlanApo; Olympus). Deconvolution of the collected data were performed as described previously (Agard et al., 1989). Confocal time-lapse imaging was performed by the use of a heating chamber (IN-ONI-F2; Tokai Hit) combined with a confocal microscope system (Radiance 2100; Bio-Rad Laboratories) assisted by LaserSharp software (Bio-Rad Laboratories). A 60x 1.40 NA oil immersion objective was used (PlanApo; Nikon). Images of spines were acquired at the 3x digital zoom, resulting in a nominal spatial resolution of 0.13 µm/pixel and in a time resolution of 39 µs/point in frame-scan mode. For each imaged spine, a z-stack at intervals of 0.2 µm was taken to allow the reconstruction of spine morphology. Measurements of signal intensity and dimensions were performed by using LaserPix software (Bio-Rad Laboratories).
The thickest main dendrite in each neuron was subjected to the analyses. For the measurement of the dimensions of spines, the spines on the 10-µm-long dendritic segment between 30 and 40 µm from the proximal origin were analyzed. About eight to nine protrusions, including spines and nonspine protrusions, were found on the 10-µm-long segment. Among these protrusions, data were collected from all the spines visualized in the same focal plane. For the counting of the type of the dendritic protrusions, and in the analyses with confocal microscopy, the dendritic segment between 20 and 40 µm from the proximal origin was analyzed. At least four independent experiments were performed to obtain more than four different neurons subjected to data collection. Capturing image and measuring spine dimension were performed blind by independent researchers. Values are shown as mean ± SEM.
FRAP experiments
FRAP experiments were performed by the use of a confocal microscope (model LSM510; Carl Zeiss MicroImaging, Inc.) equipped with a 63x, 1.40 NA oil immersion objective (Plan-Apochromat; Carl Zeiss MicroImaging, Inc.) as described previously (Richards et al., 2004). The whole area of the spine of interest was defined as the region of interest, and bleached with 100% laser intensity for 1.5 s. Recovery was measured by rapid imaging of a single image plane of 256 x 256 pixels2 size, with the 3x digital zoom, resulting in a nominal spatial resolution of 0.19 µm/pixel and in a time resolution of 1.76 µs/point in frame-scan mode. Two trials were performed per spine and averaged.
Quantification of the size of synaptic puncta
Each coverslip was scanned from one end to another, and every neuron was serially photographed with a 40x objective. The first five neurons from one end of the coverslip were counted. Each photograph was taken by 1,000 ms of exposure, and the labeled area was extracted with a threshold of signal intensity at the level of 100 among 0255. Puncta on the proximal 50 µm segment of the thickest dendrite of each neuron were measured. Huge areas >3 µm2 were excluded from the measurement because those were supposed to be the fused area consisted of multiple puncta. The counting was performed with four coverslips from independent preparations. Photographing and counting were performed blind by independent investigators. The data were compared by unpaired t test.
Construction of plasmid vectors
Expression vectors for N-cadherin mutants were constructed by modifying pCXN2-Ncad-myc, in which N-cadherin fused with 6xmyc-tag is ligated downstream of ß-actin promoter (a gift from W. Shan, Montreal Neurological Institute; Shan et al., 2000). The NH2-terminal region of N-cadherin between EcoRI and KpnI sites was replaced with the corresponding fragment derived from the W2A-cadherin (a gift from K. Tamura, Hyogo Medical School, Kobe, Japan; Tamura et al., 1998). N-cadherin-venus was constructed by replacing the 6xmyc region (between XhoI and BglII sites) of pCXN2-Ncad-myc with venus (a gift from A. Miyawaki, RIKEN, Wako, Japan; Nagai et al., 2002) amplified by PCR. The integrity of the vectors was verified by DNA sequencing.
Cell aggregation assay
N-cadherin-myc was stably transfected to L929 cells by Neomycin selection. The double transfectant cells were obtained by transfecting W2A-cadherin-flag vector possessing Zeocin resistant cassette to N-cadherin-myc stable transfectant and selected with 100 µg/ml Zeocin. A Zeocin-resistant W2A-cadherin-flag expression vector was constructed by ligating a blunt-ended ZEO-cassette (pCMV/Zeo, cut with EcoRV and PvuII; Invitrogen) into the StuI site, a W2A-cadherin between the EcoRI and XhoI sites and a flag between XhoI and BglII sites of pCXN2, respectively. Cell aggregation assay was performed as described previously (Takeichi, 1977).
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Acknowledgments |
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This work was supported by Grant-in-Aid for Scientific Research on Priority Area [C]-Advanced Brain Science Project from Ministry of Education, Culture, Sports, Science and Technology, Japan (to H. Tanaka), and NS20147 from National Institutes of Health (to D.R. Colman). K. Okamura was supported by Iwadare Association for Dental Graduate Students.
Submitted: 4 June 2004
Accepted: 28 October 2004
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