Multiple association states between glycine receptors and gephyrin identified by SPT analysis

Abstract

The scaffolding protein gephyrin is known to anchor glycine receptors (GlyR) at synapses and to participate in the dynamic equilibrium between synaptic and extrasynaptic GlyR in the neuronal membrane. Here we investigated the properties of this interaction in cells cotransfected with YFP-tagged gephyrin and GlyR subunits possessing an extracellular myc-tag. In HeLa cells and young neurons, single particle tracking was used to follow in real time individual GlyR, labeled with quantum dots, traveling into and out of gephyrin clusters. Analysis of the diffusion properties of two GlyR subunit types--able or unable to bind gephyrin--gave access to the association states of GlyR with its scaffolding protein. Our results indicated that an important portion of GlyR could be linked to a few molecules of gephyrin outside gephyrin clusters. This emphasizes the role of scaffolding proteins in the extrasynaptic membrane and supports the implication of gephyrin-gephyrin interactions in the stabilization of GlyR at synapses. The kinetic parameters controlling the equilibrium between GlyR inside and outside clusters were also characterized. Within clusters, we identified two subpopulations of GlyR with distinct degrees of stabilization between receptors and scaffolding proteins.

FIGURE 1

Examples of VeGe(2) and GlyR α₁βgb distribution patterns and trajectories in cotransfected HeLa cells and 3–4 DIV neurons. (A) Gephyrin clusters (YFP fluorescence, green) and GlyR α1βgb immunopositive clusters (red, cell surface labeling: labeling at 4°C before fixation) colocalize at the cell surface (yellow puncta) in HeLa cells (A1–4) and in 3 DIV neurons (A5–8). A2 is a zoom of a characteristic large flat area of HeLa cells (white box in A1). Ve∷Ge and GlyR α₁βgb channels are shown separately (A3–4) and superimposed (A2). (A6–8) zooms of areas on a distal and on a proximal part of dendrite and on the soma, respectively (white boxes in A5). (B) Examples of MSD as a function of time for a simple Brownian diffusion (blue dots) and a restricted diffusion (green dots). (Inset) The corresponding trajectories are outside (blue) or inside (green) gephyrin clusters (red), respectively. Scale bar: 1 μm. (full lines) theoretical curves for the type of motion on a long timescale. (Green dotted line) linear fit on a short timescale for the MSD corresponding to a restricted diffusion.

FIGURE 2

Comparison of GlyR α₁ and α₁βgb dynamics. (A) Cumulative distribution of the initial diffusion coefficients. (B) Confinement diameter calculated on trajectories showing a restricted motion (mean ± SE). Experiments were performed in HeLa cells (A1, B1) and neurons (A2, B2). Comparison of the values obtained with GlyR α₁ (dark gray, n = 313, 213, 85, and 76 for A1, A2, B1, and B2, respectively), GlyR α₁βgb inside (gray, n = 59, 91, 33, and 55 for A1, A2, B1, and B2, respectively) and outside (black, n = 279, 332, 125, and 156 for A1, A2, B1, and B2, respectively) gephyrin clusters. Kolmogorov-Smirnov test (A) or Mann Whitney test (B) between indicated distribution and the values obtains for GlyR α1βgb outside gephyrin clusters: **p < 10⁻², ***p < 10⁻³, ****p < 10⁻⁴. (C) In neurons, cumulative distribution of the initial diffusion coefficients for all GlyR α₁βgb outside VeGe(2) clusters (dark diamond, n = 332), for GlyR α₁βgb not cotransfected with gephyrin (dark square) and for GlyR α₁βgb inside gephyrin clusters (gray circle). Estimation of the cumulative distribution of GlyR α1βgb outside VeGe(2) clusters affected by the presence of gephyrin (N1 values) for different value of N1/N: 0.1–0.4 (gray curves) and 0.5–0.9 (black curves). The calculated curves are bounded by 1 only when N1/N > 0.4.

FIGURE 3

Effects of latrunculin and nocodazole on the initial diffusion coefficient of GlyR in 3–4 DIV neurons. Comparison of the cumulative distributions obtained in control conditions (Ctr, full lines) and (A) after latrunculin treatment (lat, dotted lines), (B) after nocodazole treatment (NZ, dotted lines). Values obtained for GlyR α₁ (dark gray, n = 213, 198, and 130 for Ctr, Lat, and NZ, respectively), GlyR α₁βgb inside (gray, n = 91, 62, and 60 for Ctr, Lat, and NZ, respectively) and outside (black, n = 332, 218, and 209 for Ctr, Lat, and NZ, respectively) gephyrin clusters. All cells were cotransfected with VeGe(2). Kolmogorov-Smirnov test p-values are >0.05 except for the comparison of the distributions of GlyR α₁βgb outside VeGe(2) clusters in Ctr and NZ conditions (p-value < 0.01). (B, inset) Highlighting of the median values for GlyR α₁βgb outside VeGe(2) clusters. Wilcoxon rank sum test for equal medians, *p-value < 0.05.

FIGURE 4

Identification of two GlyR subpopulations within a gephyrin cluster. (A) Examples of trajectories for a “stable” (upper trajectory, circle) and a “swapping” GlyR (bottom trajectory, triangle); (gray) parts inside VeGe(2) clusters (dark gray). (B, C) Cumulative probability of diffusion coefficients inside VeGe(2) clusters for “stable” (circle) and “swapping” (triangle) GlyR in HeLa cells (B) and neurons (C). “Swapping” GlyRs diffuse faster than “stable” GlyRs (Kolmogorov-Smirnov test: HeLa cells, p-value < 0.01; neurons, p-value < 0.05). (inset) Vertical bars give the confinement diameter (d_conf, mean ± SE) for trajectories showing a restricted motion.

FIGURE 5

Properties of the equilibrium between GlyR inside and outside clusters. (A) Simple scheme of the dynamic equilibrium (top panel). (Bottom panel) Example of a trajectory (in neurons) alternating between the inside (gray) and the outside (black) gephyrin clusters (dark gray) compartments. Scale bar: 5 μm. (B) Examples of localization (IN and OUT) as a function of time. The upper plot corresponds to the trajectory in (A). (C) Comparison between the cumulative probability of in (gray circle) and out (black square) dwell times in HeLa cells (left panel) and neurons (right panel). (Full lines) Biexponential fit of the data. Mean dwell times inside (〈t_IN〉) and outside (〈t_OUT〉) VeGe(2) clusters. (D) Proportion of QDots within gephyrin clusters as a function of time, averaged over all films (n =39, 46 for HeLa cells (left panel) and neurons (right panel), respectively, computed for “swapping” GlyR.

FIGURE 6

Schematic view of the different paths leading to stabilization of GlyR by gephyrin clusters. Receptor (R) and its scaffolding protein gephyrin (S) may be preassembled before to be inserted in the cell membrane (black arrow) or they may reach the membrane separately (gray arrow). Receptor-scaffold complexes may be formed outside (equilibrium 1) or inside (equilibrium 4) gephyrin clusters. Both exchanges of receptors (equilibrium 3) and of receptor-scaffold assemblies (equilibrium 2) may occur between the inside (suffix I) and the outside (suffix O) gephyrin clusters' compartments. Once within clusters, receptor-scaffold complexes may reach a higher level of stabilization (equilibrium 5, *).