Collybistin activation by GTP-TC10 enhances postsynaptic gephyrin clustering and hippocampal GABAergic neurotransmission

Abstract

In many brain regions, gephyrin and GABAA receptor clustering at developing inhibitory synapses depends on the guanine nucleotide exchange factor collybistin (Cb). The vast majority of Cb splice variants contain an autoinhibitory src homology 3 domain, and several synaptic proteins are known to bind to this SH3 domain and to thereby activate gephyrin clustering. However, many functional GABAergic synapses form independently of the known Cb-activating proteins, indicating that additional Cb activators must exist. Here we show that the small Rho-like GTPase TC10 stimulates Cb-dependent gephyrin clustering by binding in its active, GTP-bound state to the pleckstrin homology domain of Cb. Overexpression of a constitutively active TC10 variant in neurons causes an increase in the density of synaptic gephyrin clusters and mean miniature inhibitory postsynaptic current amplitudes, whereas a dominant negative TC10 variant has opposite effects. The enhancement of Cb-induced gephyrin clustering by GTP-TC10 does not depend on the guanine nucleotide exchange activity of Cb but involves an interaction that resembles reported interactions of other small GTPases with their effectors. Our data indicate that GTP-TC10 activates the major src homology 3 domain-containing Cb variants by relieving autoinhibition and thus define an alternative GTPase-driven signaling pathway in the genesis of inhibitory synapses.

Keywords: Cdc42; RhoQ; neuroligin; postsynaptic scaffold; synaptogenesis.

Fig. 1.

TC10 stimulates Cb-dependent gephyrin microcluster formation in COS-7 cells. (A1–A5) Images of COS-7 cells transfected as indicated. GFP-gephyrin accumulates in large cytoplasmic aggregates when expressed alone (A1) or together with Myc-SH3(+)CbII (A3) or HA-TC10 (A4). In the presence of Myc-ΔSH3CbII (A2), GFP-gephyrin forms microclusters at the plasma membrane. Similarly, HA-TC10 and Myc-SH3(+)CbII jointly trigger GFP-gephyrin microcluster formation (A5). (Scale bar, 10 µm.) (B) Schematic representation of the Cb splice variants and mutants, as well as of the TC10 WT and mutants, used in this study. (C1) Percentages of GFP-gephyrin (co)-transfected cells classified as displaying GFP-gephyrin microclusters (>50 puncta per cell; n = 3 independent transfections, n = 321–428 cells). Significance levels compared with cells transfected with GFP-gephyrin only (gray bar) are shown within the bars. (C2) Total numbers of GFP-gephyrin clusters and aggregates from images of transfected COS-7 cells (n = 14–34 transfected cells per transfection condition). Significance indicated as in C1. (C3) Gephyrin puncta counted in C2 were binned according to their size (n = 14–34 cells). (Insets) Relative fractions of small microclusters (0.05–0.2 µm²; Left) and aggregates (>1 µm²; Right). (D) Percentages of microclusters (0.04–0.4 µm²) per cell in COS-7 cells transfected as indicated (n = 6–34 cells). Statistical significance was tested between the conditions without coexpression of HA-TC10 (first four columns) and those in the presence of TC10 (WT, CA, or DN).

Fig. 2.

ΔSH3CbII and SH3(+)CbII differentially activate TC10 in nonneuronal cells. (A1–A3) HEK 293 cells were transfected with Myc-TC10 either alone (-) or together with the indicated HA- (A1) or Myc-tagged (A2 and A3) Cb constructs in the absence (A1 and A2) or presence (A3, Top, last 5 lanes) of GFP-gephyrin. Cell lysates were used for cosedimentation with immobilized GST-PAK1-PBD. GTP-bound TC10 was detected by Western blotting with an anti-Myc antibody. MemCode staining (Bottom) was used to confirm that equal amounts of GST-PAK1-PBD had been added to each lysate. (B) Relative band intensities of TC10 bound to GST-PAK1-PBD (n = 3–4 independent experiments). Statistical significance was compared with Myc-TC10 expressed alone (first bar).

Fig. 3.

GTP-TC10 interacts with the PH domain of Cb. (A) Purified His-TC10, either nf or preloaded with GDP or GTPγS, was incubated with the indicated recombinant proteins bound to glutathione-Sepharose beads. Bound His-TC10 was detected by Western blotting. Note that both GST-ΔSH3CbII WT and the RR/AA mutant interacted preferentially with GTPγS-preloaded TC10, as did GST-PAK1-PBD but not GST (Top). (Middle) Similar amounts of His-TC10 were included in all incubations; (Bottom) respective MemCode stainings indicating comparable amounts of the GST-tagged bait proteins used. (B) His-TC10 was incubated with the indicated recombinant proteins bound to glutathione-Sepharose beads. Western blot analysis of the proteins bound revealed a faint interaction of SH3(+)CbII with TC10 that was similar for GDP, GTPγS, or nf conditions (Top). Deletion of the PH domain [GST-SH3(+)CbIIΔPH] strongly reduced the interaction with GTPγS-TC10, whereas the isolated PH domain (GST-PH) displayed preferential binding to TC10 in its GTP-bound state, as did GST-PAK1-PBD but not GST (Top). Asterisks in A and B indicate nonspecifically stained bands with a different migration pattern.

Fig. 4.

TC10 activity enhances SH3(+)CbII-mediated clustering of gephyrin in cultured hippocampal neurons. (A1–A5) Cultured rat hippocampal neurons were cotransfected at DIV 4 with the empty pcDNA3 vector and either the Myc-ΔSH3CbII (A2) or Myc-SH3(+)CbII (A3) cDNAs, or cotransfected with Myc-SH3(+)CbII and HA-TC10 CA (A4) or HA-TC10 DN (A5), respectively; untransfected cultures (A1) served as control. At DIV 14, the cells were fixed and immunostained for gephyrin, VIAAT, HA, and Myc. (Upper) Endogenous gephyrin immunoreactivity; (Lower) corresponding costainings. Note the increase in punctate gephyrin immunoreactivity in neurons cotransfected with TC10 CA (A4) and reduced gephyrin clustering in neurons coexpressing TC10 DN (A5) compared with neurons expressing Myc-SH3(+)CbII alone (A3). (Scale bar, 10 µm for A1–A5.) Dotted lines in A1–A5 indicate the borders of transfected cells. (B1–C2) Bar diagrams of (B1) perisomatic gephyrin cluster densities per 100 µm² surface area and (B2) average sizes of perisomatic gephyrin clusters (n = 258–1,344 clusters analyzed), (C1) gephyrin immunoreactive clusters per 40 µm dendrite length and (C2) average sizes of dendritic gephyrin clusters (n = 179–590 clusters analyzed). Bars correspond to values obtained from the perisomatic surface area and one randomly selected second-order dendrite (60–100 µm distal to the soma) per neuron, respectively (n = 10–28 cells, n = 3 independent experiments).

Fig. 5.

TC10 activity enhances GABAergic mIPSCs in cultured hippocampal neurons. (A1) Representative traces of mIPSCs recorded from neurons coexpressing GFP and Myc-SH3(+)CbII without (green) or together with HA-TC10 CA (purple) or HA-TC10 DN (blue), respectively. (A2 and A3) mIPSC mean amplitudes (A2) and frequencies (A3) of neurons transfected as described in A1. Note significant increase in mIPSC amplitude in neurons coexpressing TC10 CA compared with control cells. In contrast, coexpression of TC10 DN led to a significant decrease in the mean mIPSC frequency. (B1 and B2) Representative recordings (B1) and mean amplitudes (B2) of high [K⁺] eIPSCs from neurons transfected as described in A1. Data in A2 and A3 were obtained from n = 96–135 neurons, and in B2 from n = 41–55 neurons. n = 3–4 independent experiments.