Gephyrin interacts with Dynein light chains 1 and 2, components of motor protein complexes - PubMed (original) (raw)

Gephyrin interacts with Dynein light chains 1 and 2, components of motor protein complexes

Jens C Fuhrmann et al. J Neurosci. 2002.

Abstract

The clustering of glycine receptors and major subtypes of GABA(A) receptors at inhibitory synapses is mediated by the tubulin-binding protein gephyrin. In an attempt to identify additional components of inhibitory postsynaptic specializations, we performed a yeast two-hybrid screen using gephyrin as bait. Multiple positive clones encoded either the dynein light chain-1 (Dlc-1), also known as dynein LC8 and protein inhibitor of neuronal nitric oxide synthase, or its homolog Dlc-2. Dlc-1 protein bound efficiently to gephyrin in in vitro binding assays and colocalized with gephyrin during coexpression in HEK293 cells. The binding site for Dlc was mapped to a fragment of 63 amino acids within the central linker domain of gephyrin. In hippocampal neurons, endogenous Dlc protein was enriched at synaptic sites identified by synaptophysin and gephyrin immunostaining. Immunoelectron microscopy in spinal cord sections revealed Dlc immunoreactivity at the edges of postsynaptic differentiations, in close contact with cytoskeletal structures and at the periphery of the Golgi apparatus. Because Dlc-1 and Dlc-2 have been described as stoichiometric components of cytoplasmic dynein and myosin-Va complexes, our results suggest that motor proteins are involved in the subcellular localization of gephyrin.

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Figures

Fig. 1.

Fig. 1.

Interaction between gephyrin and Dlc-1/2 in the yeast two-hybrid system. A, Amino acid sequence of rat Dlc-1 and Dlc-2. Identity is indicated by a vertical bar. B, Schematic diagram showing the results from yeast two-hybrid analysis of the interaction between gephyrin and Dlc-1/Dlc-2. Interaction was assayed by lacZ/LEU2 reporter gene induction on agar plates and scored as + (blue color visible after 24 hr; this is comparable with gephyrin interacting with the cytoplasmic loop of the GlyR β subunit or with itself; data not shown) and − (no blue color after 4 d). Gephyrin deletion constructs are depicted schematically, and numbers refer to amino acid positions in the gephyrin splice variant p1 (Prior et al., 1992). Identical results were obtained for Dlc-1 and Dlc-2.

Fig. 2.

Fig. 2.

Binding between gephyrin and Dlc-1/2_in vitro_. A, Dlc-1 tagged with a myc epitope coimmunoprecipitates with gephyrin from transfected HEK293 cells. Myc-Dlc-1 and gephyrin were expressed alone or in combination, and the presence of the recombinant proteins, as well as lower amounts of endogenous proteins, were confirmed in the input lanes. Complexes between myc-Dlc-1 and gephyrin were precipitated by either myc- or gephyrin-specific antibodies immobilized on protein G-Sepharose. Both endogenous and recombinant gephyrin were specifically coimmunoprecipitated with myc-Dlc-1, and antibodies against gephyrin precipitated myc-Dlc-1 when gephyrin was overexpressed.IP, Immunoprecipitate; G, gephyrin; HC, antibody heavy chains; LC, antibody light chains; Dlc, Dlc-1/2. B, GST-Dlc-1 pulls down gephyrin from rat brain. Glutathione Sepharose beads were loaded with either GST-Dlc-1 or a truncated control protein. Beads containing GST-Dlc-1, but not the control protein, retained gephyrin from the brain lysate, as demonstrated by immunoblotting with a gephyrin-specific antibody. C, GST-gephyrin specifically enriches Dlc-1/2 from rat brain. The experiment was performed as in B using GST-gephyrin and GST as the negative control. Detection with an antibody recognizing Dlc-1 and Dlc-2 revealed specific binding to GST-gephyrin. D, Amino acids 181–243 of gephyrin are required for interaction with Dlc-1. Wild-type and mutant gephyrin-GFP were expressed in HEK293 cells and tested for binding to GST-Dlc-1. Gephyrin-GFP (top band) and endogenous gephyrin (bottom band) were specifically retained by GST-Dlc-1, whereas gephyrin(Δ181–243)-GFP (middle band) did not bind. No significant binding was detected when a truncated control protein was used instead of GST-Dlc-1 (data not shown).

Fig. 3.

Fig. 3.

Recombinant and endogenous Dlc-1/2 colocalize with gephyrin in transfected HEK293 cells. A, Gephyrin-GFP overexpression leads to the formation of characteristic aggregates in HEK293 cells (arrow), whereas myc-Dlc-1 transfected alone displays a diffuse cytoplasmic distribution (B). Endogenous Dlc protein is localized in the cytoplasm with no obvious compartmentalization (C). Both endogenous and recombinant Dlc proteins also show nuclear localization to a variable degree. D, Double transfection of gephyrin-GFP (green) and myc-Dlc-1 (red) leads to recruitment of Dlc-1 to gephyrin aggregates (arrows), indicating an interaction between both proteins. Overlap of both signals is indicated_yellow_ in the merged picture. E, In cells transfected with cDNA for gephyrin-GFP alone, endogenous Dlc protein (red) colocalizes with aggregates of gephyrin (green, arrows). Merge is shown in_yellow_. Note the homogenous distribution of Dlc in the untransfected cell to the left. F, Gephyrin-GFP(Δ181–243) aggregates do not recruit myc-Dlc-1 in transfected cells. HEK293 cells were cotransfected with cDNAs encoding GFP-gephyrin(Δ181–243) and myc-Dlc-1 and stained for the myc epitope tag. GFP autofluorescence (green) shows the presence of gephyrin aggregates similar to the wild-type situation, whereas Dlc-1 (red) is not enriched in these structures. Scale bars, 10 μm.

Fig. 4.

Fig. 4.

Gephyrin and Dlc colocalize at inhibitory synapses in cultured hippocampal neurons. Hippocampal neurons were cultured for 20 DIV and stained for the indicated proteins. A, Colocalization of Dlc and synaptophysin at synaptic sites. The merged image shows synaptophysin immunoreactivity in red and Dlc staining in green; overlapping signals are_yellow_. Dlc is not exclusively synaptic but displays prominent cytosolic staining in addition. Insets, 2× magnification. B, Colocalization of Dlc and gephyrin at inhibitory synapses. Many, but not all, Dlc-positive punctae stain positive for gephyrin. Display as in A.C–E, Dependence of the degree of membrane-associated Dlc immunoreactivity on culture density. Hippocampal neurons were plated at densities of 30,000 (C), 40,000 (D), or 60,000 (E) per 14 mm coverslip and allowed to differentiate for 20 DIV. Dlc staining reveals variable ratios of cytosolic and submembranous Dlc signal intensity. Note the high number of fine neurites, likely to represent both axons and dendrites, in the high-density culture. Scale bars, 10 μm.

Fig. 5.

Fig. 5.

Ultrastructural localization of Dlc immunoreactivity in neurons. A1–A2, Detection of Dlc immunoperoxidase associated electron-dense deposits (arrows) at postsynaptic differentiations adjacent to synaptic boutons containing pleiomorphic populations of synaptic vesicles. Note that not all postsynaptic differentiations associated with a synaptic bouton contain Dlc-associated immunoreactivity (asterisk in A2). B, Detection of Dlc-associated electron-dense deposits (arrow) in front of a synaptic bouton containing spherical synaptic vesicles. C, Detection of Dlc-IR at the membrane of the Golgi apparatus (Go) with gold-toned silver-intensified nanogold particles (arrows).D, Two examples of simultaneous detection of gephyrin-associated electron-dense immunoperoxidase deposits and Dlc-associated gold-toned silver-intensified nanogold particles (arrows). Note that the Dlc immunoreactivity predominates at the periphery of the postsynaptic differentiations. Scale bar: A1, 0.50 μm; A2, 0.85 μm;B, 0.30 μm; C, 0.44 μm;D1, 0.40 μm; D2, 0.60 μm.

Fig. 6.

Fig. 6.

Gephyrin targeting to inhibitory synapses in transfected hippocampal neurons does not require the Dlc-binding domain. A, GFP-gephyrin accumulates at inhibitory synapses. Hippocampal neurons cultured for 12 DIV were transfected with pEGFP-C2-gephyrin. After 20 hr, cells were fixed and stained for VIAAT (red) to indicate inhibitory terminals. GFP autofluorescence is shown in green, and merge between red and green channels is shown in yellow. GFP-gephyrin forms numerous hotspots near the plasma membrane of cell body and dendrites, the large majority of which colocalize with VIAAT immunoreactivity. B, GFP-gephyrin(Δ181–243) does not display a localization defect when compared with wild-type GFP-gephyrin. The experiment was performed as in A, except that neurons were transfected with pEGFP-C2-gephyrin(Δ181–243). Most of the submembranous clusters of GFP-gephyrin(Δ181–243) colocalize with the VIAAT signal, indicating undisturbed synaptic targeting. Scale bar, 10 μm.

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