Intracellular Association of Glycine Receptor with Gephyrin Increases Its Plasma Membrane Accumulation Rate

Gephyrin expression increases the amount of GlyR at the cell surface in COS cells. Cells cotransfected with GlyR α₁ or GlyR α₁βgb subunits with a thrombin cleavable myc-tag, and gephyrin::GFP (Ge::GFP). Accumulation of GlyR at the surface of COS-7 cells monitored after a temperature-induced block of the TGN exit (see Materials and Methods for details). A, Temporal sequence of the temperature-induced block of TGN exit, thrombin cleavage, and surface recovery of GlyR. Eighteen hours after transfection, cells were maintained for 1 hr at 19.5°C (t₀ –60 min) in the presence or absence of thrombin to remove cell-surface myc-tags. Cells were then washed, returned to 37°C (t₀ of the restoration phase), and processed for immunolabeling of cell-surface myc-tags (t₀ + 30 and t₀ + 90 min). Some cells directly fixed after the thrombin treatment (t₀) were permeabilized before selective immunolabeling of intracellular GlyR. Fluorescence associated with GlyR was then quantified at the indicated time points. B, GlyR-associated immunofluorescence at the cell surface at various points of the restoration phase (B1, B5, 0 min; B3, B7, 30 min; B4, B8, 90 min). B1–B4, Cells cotransfected with GlyR α₁ and Ge::GFP; B5–B8, cells cotransfected with GlyR α₁βgb and Ge::GFP. Note the absence of myc immunoreactivity at the cell surface immediately after exposure to thrombin (B1, B5) in cells displaying GFP fluorescence (B2, B6, same field as B1, B5, respectively). C, Surface GlyR expression in transfected cells quantified in parallel by Western blot and immunofluorescence analysis. Left, Immunoblot of anti-myc antibody: lanes 1–4: 0, 0.125, 0.25, and 0.5 ng, respectively, of pure anti-myc antibody for calibration; lanes 5–14: 15 μl (lanes 5–9) or 30 μl (lanes 10–14) of cell lysates obtained 0 (lanes 6 and 11), 4 (lanes 7 and 12), 14 (lanes 8 and 13), and 25 hr (lanes 9 and 14) after transfection; lanes 5 and 10: lysates obtained 14 hr after transfection and not exposed to anti-myc antibody. Molecular size markers are in kilodaltons. Right, Plot of surface myc-GlyR-associated fluorescence intensity as a function of surface anti-myc amount as quantified by Western blotting [mean mass ± SEM expressed as nanograms in 15 μl; means were derived from n = 2 pictures of the same blot and n = 12–18 fields (25× objective lens) for mean fluorescence intensity] at the corresponding time of expression (r = 0.98; p < 0.01; regression analysis). D, Fluorescence associated with GlyR α₁ or GlyR α₁βgb-bound anti-myc antibody immunoreactivity. Measurement over the whole field (25× objective lens) normalized by the number of transfected cells (mean ± SEM). At steady states (18 hr after transfection), note the equivalent immunoreactivity of intracellular GlyR α₁ and GlyR α₁βgb in cells cotransfected with Ge::GFP and the increased GlyR α₁βgb amount at the cell surface (n = 18–20 fields from two experiments analyzed for each condition). During the restoration phase, note the increased expression of GlyR α₁βgb in the presence of Ge::GFP after 90 min, but not in the presence of GFP, or in the absence of the βgb domain (n = 6–9 fields analyzed for each condition after 90 min of restoration). E, Quantification of cell-surface GlyR-associated fluorescence during the restoration phase (n = 14–20 fields analyzed for each condition). Inset, Quantification analyzing the surface fluorescence density in transfected cells (see Materials and Methods) during the restoration phase (mean ± SEM; n = 13–19 fields containing 20–30 transfected cells from two experiments analyzed for each time point and each transfection condition). In both cases, the GlyR α₁βgb-associated fluorescence increased more rapidly than that of GlyR α₁. Note that values of fluorescence associated with cell-surface and intracellular GlyR at t₀ (D) and cell-surface GlyR during the restoration phase (D, E) cannot be directly compared because different exposure times were used for the acquisition of fluorescence pictures. *p < 0.05; **p < 0.01; ***p < 10^–4; Student's t test.

Gephyrin expression increases the amount of GlyR at the cell surface of cultured neurons. Spinal cord neurons (3 DIV) were treated as described in Figure 5. Fluorescence quantifications on confocal sections are shown (see Materials and Methods). A, Projections of confocal sections of neurons cotransfected with GlyR α₁ (A1–A4) or GlyR α₁βgb (A5–A8) and gephyrin::GFP (Ge::GFP), obtained at 0 (A1, A5), 15 (A2, A6), 30 (A3, A7), and 90 (A4, A7) min of the restoration phase. Cytoplasm of transfected cells was outlined (red line) using Ge::GFP and MAP2 immunoreactivity-associated fluorescence. Note that the number of GlyR-IR clusters increased with time. Nontransfected cell is indicated by the asterisk in A3 and A7. At t₀ + 15 min, surface GlyR clusters were concentrated over the soma (arrowheads) and proximal parts of neurites (arrows). At t₀ + 30 and t₀ + 90 min, GlyR clusters were detected more distally on neurites (arrows). B, Fluorescence recovery measured at somata plus neurites (B1), somata only (B2), and neurites only (B3) (mean ± SEM cell fluorescence; n = 8–23 cells from three experiments analyzed for each time point and for each transfection condition). As observed for COS-7 cells, the GlyR-associated fluorescence increased with time more rapidly with GlyR α₁βgb than with GlyR α₁. Note that the overall fluorescence recovery was accounted for by a biphasic time course for the soma, with twice as much fluorescence with GlyR α₁βgb as with GlyR α₁, at 15 min. C, GlyR immunoreactivity clusters 15 min after the temperature shift for GlyR α₁ (C1) and GlyR α₁βgb (C2) in cells cotransfected with Ge::GFP (confocal sections, pseudo-color codes for intensity levels). Note the higher fluorescence and larger size of clusters for GlyR α₁βgb compared with GlyR α₁. D, Quantification of GlyR cluster number per cell (D1), cluster surface area (D2), and fluorescence intensity (D3) (means ± SEM). Note the higher number of clusters per cell, and the increased surface area and fluorescence intensity of individual clusters for GlyR α₁βgb compared with that of GlyR α₁; 1502 clusters in 15 cells and 771 clusters in 12 cells from three experiments were analyzed for GlyR α₁βgb and GlyR α₁, respectively. Scalebars: A, 10; C, 1 μm. *p < 0.05; **p < 0.01; ***p < 10^–4; Student's t test.

GlyR–gephyrin interaction affects gephyrin::GFP intracellular movements in COS cells. A, B, Temporal sequences derived from time lapses of GFP fluorescence in cells cotransfected with Ge::GFP and GlyR α₁βgb (A) or GlyR α₁ (B) analyzed by videomicroscopy. Note in A2 that intracellular fluorescent puncta (arrow and crossed arrow) move rapidly compared with peripheral ones (arrowheads), and in B2 that large puncta display only little displacements. Time is indicated in min:sec. C, Examples of trajectories of Ge::GFP puncta formed with GlyR α₁βgb (C1) and GlyR α₁ (C2), respectively. D, Diffusion coefficients of Ge::GFP when transfected alone (Ge::GFP), with GlyR α₁ (α₁ + Ge::GFP), or with GlyR α₁βgb (α₁βgb + Ge::GFP) (mean ± SEM; for n see Results). The latter is significantly (p < 10^–4; Student's t test) higher than the two former ones.E, Examples of MSD variation as a function of time, for Ge::GFP puncta in the presence (line) or absence (dashed line) of interaction with GlyR. The straight dotted lines represent the corresponding slopes at the origin. Scale bars: A1, B1, 10 μm; A2, B2, 5 μm.

Association of GlyR–gephyrin::GFP with microtubules. A, B, Confocal sections of two COS-7 cells cotransfected with gephyrin::GFP and GlyR α₁βgb at low-power (A1, B1) and high-power (A2, B2) magnifications. Microtubule immunoreactivity and Ge::GFP fluorescences are in red and green, respectively. Intracellular Ge::GFP puncta are associated with microtubules (A2, B2, arrows). Note that clusters at the cell periphery (A2, arrowheads) are not associated with microtubules. Scale bars: A1, B1, 10 μm; A2, B2, 1 μm.

Influence of microtubule depolymerization on GlyR–gephyrin::GFP movements in COS cells. A, Tubulin immunoreactivity (projection of confocal section stacks) before (A1) and after (A2) nocodazole treatment demonstrating the depolymerization of the microtubules. B, “Subtractive projections” of Ge::GFP fluorescence time-lapse sequences (see text for details) of a cell transfected with GlyR α₁βgb recorded for 3 min before (B1) and during (B2) nocodazole treatment, at different magnifications. The signal (gray) corresponds to object displacements between two successive frames (the contrast has been inverted for a better visualization). C, Ratio of subtractive surface after (No) to subtractive surface before (Ctrl) nocodazole treatment for individual cells (mean ± SEM, the movements were analyzed in three and seven cells for GlyR α₁ and GlyR α₁βgb, respectively). Although the movements of Ge::GFP puncta formed with GlyR α₁ were not affected (C), the movements of Ge::GFP puncta formed with GlyR α₁βgb decreased significantly (p < 0.01; Student's t test) with nocodazole treatment.