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First published online November 18, 2003
doi: 10.1242/10.1242/jcs.00822

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Active surface transport of metabotropic glutamate receptors through binding to microtubules and actin flow

Arnauld Sergé, Lawrence Fourgeaud, Agnès Hémar and Daniel Choquet^*

Physiologie Cellulaire de la Synapse, CNRS UMR 5091, Institut François Magendie, rue Camille saint Saëns, 33077 Bordeaux, Cedex, France

View larger version (40K): [in a new window]   Fig. 1. mGluR5 displays anchorage through its C-terminus. Typical trajectories (red lines) on a hippocampal neuron growth cone (A) and on fibroblast lamellipodium (B,C) of latex beads coupled to mGluR5-myc (A,B) or a C-terminal deletion mutant (C) superimposed on a DIC image of the cell. Trajectories in A and B present anchorage as an irreversible transition between Brownian diffusion and linear retrograde movement. The point of the trajectory corresponding to the transition is depicted by a yellow cross. Recording time, 125-200 seconds; sampling time, 25 images/second. (D-F) Plots of diffusion versus time corresponding to the trajectories in (A-C). For each timepoint, the diffusion coefficient was computed from the slope of the MSD calculated for 40 points. Note the sudden drop in diffusion values in D and E, depicted by arrows and corresponding to the yellow crosses in A and B. (G-I) Plots of the anchorage coefficient versus time corresponding to the trajectories in (A-C). For each timepoint, the anchorage coefficient was computed as the ratio between the mean diffusion during the 20 seconds before and after that timepoint. The maximum of this coefficient (black arrows) thus corresponds to the drop in diffusion values depicted by the corresponding arrows in D and E, whereas the coefficient remains low during all the recording time for mGluR5-DelC (F). (J,K) Plots of the MSD versus time for the control trajectory recorded on fibroblast depicted in B, computed before and after anchorage. Note the linearity of the MSD before anchorage, and the positive curvature displayed after.

View larger version (13K): [in a new window]   Fig. 5. (A,B) Histogram of the mean diffusion and mean relative deviation from Brownian diffusion in control conditions (ctl), for the C-terminal deletion variant (delC) or in the presence of drugs affecting the cytoskeleton. Note that diffusion is increased in the presence of cytochalasin (cyt), demecolcine (dem) or nocodazol (noc) (*P<0.05) and relative deviation is decreased for the C-terminal deletion variant or in the presence of cytochalasin or demecolcine (**P<0.01).

View larger version (20K): [in a new window]   Fig. 2. Anchoring is not induced by cross-linking of receptors by the bead. (A) Plot of the percentage of beads efficiently attached to transfected cells. In all graphs of this figure, the anti-myc antibody coupled to the bead was diluted with another anti-HA antibody, unable to bind to the tagged receptors, in the given proportions (percentage of anti-myc relative to total amount of antibodies coupled to the bead, as depicted on the x-axis). (B) Plot of the percentage of trajectories displaying anchorage for various dilutions of the anti-myc antibody. (C) Plot of the mean ratio of the diffusion coefficients computed before and after anchorage, for various dilutions of the anti-myc antibody. In all figures, values are given with s.e.m. (D) Plot of the time for anchorage for various dilutions of the anti-myc antibody. Note that only the percentage of beads efficiently coupled to transfected cells is diminishing with dilution of the anti-myc antibody, whereas the percentage of trajectories displaying anchorage, the mean ratio of the diffusion coefficients computed before and after anchorage, and the time of anchorage remain almost constant. This shows that anchorage does not rely on aggregation of receptors by the beads.

View larger version (23K): [in a new window]   Fig. 3. Spatial aspect of anchorage. (A) Plot of the spatial position of the anchorage points (diamonds), corresponding to the maximum of the anchorage coefficient function and depicted by the yellow cross on trajectories, as illustrated in Fig. 1, for control trajectories recorded on fibroblasts. Trajectories were aligned by rotation with the cell body along the positive x-axis (as measured by the direction of the linear movement after anchorage), with the initial point of the trajectory assigned to the origin of the plot. Note that the mean position (square, indicated by the arrow) is already situated along the positive abscises. Trajectories anchored from the beginning were discarded for this plot. (B,C) Histograms of the abscises and ordinates of the receptors positions during the first, diffusive phase. Note that the mean abscise is positive whereas the ordinates are centered on 0 (arrows).

View larger version (57K): [in a new window]   Fig. 4. Pharmacological characterization of the anchoring structure. (A-D) Typical trajectories (red) on fibroblast lamellipodium of latex beads coupled to mGluR5-myc superimposed on a DIC picture of the cell, in the presence of drugs that affect the cytoskeleton. (A) Trajectory in the presence of 1 µM cytochalasin, which depolymerizes actin. (B,C) Trajectories in the presence of 10 µM demecolcine or 1 µM nocodazol, which depolymerize microtubules. (D) Trajectory in the presence of 10 µM taxol, which stabilizes microtubules. Anchorage points are depicted by yellow crosses (A,D). Note that anchorage remains when actin is depolymerized (A) or when microtubule polymerization is favored (D), and is abolished when microtubules are depolymerized (B,C). (E-H) Corresponding diffusion versus time plots. Note the sudden drop in diffusion values in E and the low values recorded from the beginning in H (arrows), corresponding to the yellow crosses in A and D, respectively. (I-J) plots of the mean MSD versus time for control trajectories recorded on fibroblasts in control conditions and in the presence of drugs that affect actin (I) or microtubules (J). Note that the positive curvature of the mean MSD in the presence of taxol (tax) is even higher than in control (ctl), whereas the mean MSD is lower and linear with time in the presence of cytochalasin (cyt) and nocodazol (noc) (P<0.01 and P<0.05, respectively). Insets: blowup of the MSD versus time plots around the origin; J shows the increase in diffusion under nocodazol.

View larger version (32K): [in a new window]   Fig. 6. mGluR5 is immunoprecipitated with ß-tubulin in rat hippocampal lysates. Hippocampal lysates were immunoprecipitated with anti-mGluR5 (lane a) or anti-ß-tubulin (lanes b and c) as indicated. Immunoprecipitates and lysate (lane d) were immunoblotted as indicated for mGluR5 (lanes a and b) or transferrin receptor proteins (TfR) (lanes c and d, negative control). IP, immunoprecipitate; WB, western blot.

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