Helbig I , Sammler E , Eliava M , Bolshakov AP , Rozov A , Bruzzone R , Monyer H , Hormuzdi SG .

Wellcome Trust

	Supplementary Fig. 1. Comparable levels of Cx36-EGFP-expressing cells are present in transgenic mice regardless of genomic Cx36 expression. Immunohistochemistry of brain slices with anti-GFP antibody processed with the Vectastain ABC kit (Vector Laboratories) confirmed that numbers of Cx36-EGFP cells are comparable between homozygous wildtype (left; Cx36+/+-TgCx36-EGFP) and knockout (right; Cx36−/−-TgCx36-EGFP) Cx36 mice expressing the transgene indicating that the protein is synthesized in both genotypes. A portion of the hippocampus demonstrating the presence of the fusion protein in interneurons in the two genotypes is shown. Scale = 200 μM
	Supplementary Fig. 2. Formation of intercellular clusters by Cx36 variants. A. Schematics illustrating the wildtype Cx36 and two variants of Cx36-EGFP (Cx36-EGFPg and Cx36-EGFPint). The protein coding fragments of Cx36 and EGFP (gray and clear rectangles respectively) along with amino acid residues flanking the latter and location of the translation termination codon (asterisk) are indicated. The ability of the two EGFP fusion constructs to form intercellular clusters typically seen with Cx36 (a, a′) is illustrated in B. The images were obtained after immunocytochemical visualization with an anti-Cx36 (Chemicon) antibody and represent maximum projections of optical sections acquired on a Lecia SP5 confocal microscope. The results show that Cx36-EGFPint frequently forms large aggregates of the protein at intercellular domains between adjacent Cx36-EGFPint-expressing cells (b, b′), whereas Cx36-EGFPg forms smaller dense clusters within the cytoplasm (c, c′) and only occasionally forms intercellular aggregates reminiscent of those formed by Cx36 (arrowhead in c′). Scale bar, upper panels = 50 μM; lower panels = 20 μM.
	Fig. 1. Cx36-EGFP protein is assembled into intercellular channels at the electrical synapse. GFP immunoreactivity (b,d) in combination with antibodies for calretinin (a) and calbindin (c) highlights the prominent distribution of Cx36-EGFP puncta within olfactory bulb glomeruli (b, dashed ovals) and in the molecular layer (ML) of the cerebellum (d, PCL = Purkinje cell layer indicated by arrowheads, GCL = granule cell layer). Immunoelectron microscopy with anti-GFP antibodies revealed the presence of EGFP-containing molecules at gap junctions (indicated by arrows; magnified f,h) between dendrites (D1 and D2) in the olfactory bulb (e,f) and cerebellum (g,h). Scale: a,b,c,d = 100 μM; e,g = 200 nM; f,h = 100 nM.
	Fig. 2. Cx36-EGFP puncta reflect the prevalence and distribution of Cx36-containing electrical synapses. A. A section of the Cx36-EGFP transgenic cerebellum demonstrating the colocalization of Cx36 puncta (red), detected with an anti-Cx36 antibody, with Cx36-EGFP fluorescent clusters (green). Only a small number of Cx36 immunopositive puncta were found to not colocalize with Cx36-EGFP fluorescent clusters. B. Measurement of Cx36-EGFP puncta quantity in the anterior and posterior (A and P respectively) striatum from 3 mice demonstrates a 2.4-fold average increase in prevalence in the latter. C.i. A portion of the transgenic hippocampus subjected to immunohistochemistry with anti-parvalbumin antibody (parv) illustrates the numerous parvalbumin-positive interneurons in the CA1 stratum pyramidale (SP) that also express the transgene (EGFP, arrows indicate examples of coexpressing interneurons). ii. Two images derived from the transgenic CA1 hippocampal region containing Cx36-EGFP-expressing interneurons (arrows) demonstrate the specific concentration of fluorescent puncta in the stratum oriens (SO) towards the alveus (A), and in the stratum radiatum (SR) near the stratum lacunosum-moleculare (SL-M). Scale: A = 25 μM, C = 50 μM.
	Fig. 3. Cx36 function is retained after addition of the EGFP molecule to the carboxy-terminus of the protein. A. Immunohistochemistry of the neocortex with anti-GFP (ia) or with anti-parvalbumin (Parv) and anti-GFP (GFP) antibodies (ib,c) demonstrating the presence of puncta and of coexpression of Cx36-EGFP and parvalbumin indicating that parvalbuminergic interneurons in the neocortex express the transgene (arrows 1b; arrowheads in 1c demonstrate the presence of the fusion protein in parvalbumin-negative neurons). Electrophysiological experiments indicated that the transgene is unable to provide electrical coupling to FS neurons in the Cx36 knockout. Representative traces (postsynaptic response above, presynaptic below) obtained from a pair exhibiting a CC of 0.0014 demonstrate our ability to detect low levels of gap junction-mediated communication in such experiments (ii). B. The junctional conductance (Gj, mean ± SEM) measured from oocytes injected with RNAs encoding either of the two Cx36 variants was similar and significantly higher than those measured from oocytes injected solely with the oligonucleotide controls (Bi). The relationship of Vj to steady-state junctional conductance (Gjss), measured at the end of the Vj step and normalized to the values recorded at ± 20 mV, is shown as the mean ± SEM of 4 pairs fit to a Boltzmann equation (dashed lines, Bii). The relationship demonstrates that the voltage-gating properties of Cx36 () or Cx36-EGFP (■) were almost identical with the exception of a slight shift of the transjunctional voltage required to elicit a conductance midway between Gjmax and Gjmin (Cx36 = 85 mV, Cx36-EGFP = 95 mV), consistent with the observation that changing the length of the carboxy-terminal tail alters the kinetics of Vj gating (Revilla et al., 1999). Scale = 50 μM.
	Fig. 4. Cx36-EGFP puncta do not form in the absence of genomic Cx36 expression. The cerebellum (a, ML = molecular layer) and olfactory bulb glomeruli (c,e) of Cx36−/−-TgCx36-EGFP backcrossed progeny are devoid of GFP-containing puncta unlike the corresponding structures (b, cerebellum; d,f, olfactory bulb glomeruli) of their Cx36+/+-TgCx36-EGFP littermates. GFP puncta were observed both by epifluorescence (a,b,c,d) and immunohistochemical analysis (e,f) with anti-gfp and anti-calretinin antibodies (green and red respectively). The presence of puncta within glomeruli, demarcated by calretinin-positive cells, in Cx36+/+ mice (f) and absence in homozygous knockout littermates (e) is clearly noticeable at higher magnification. Scale: a,b,c,d = 100 μM, e,f = 25 μM.
	Fig. 5. An obligatory requirement for Cx36 in the formation of Cx36-EGFP-containing junctional clusters in HeLa cells. A. The presence of connexin-containing clusters at the plasma membrane of HeLa cells expressing Cx36 (a,a′), or Cx36-EGFP (b,b′), or coexpressing the two (c,c′) was determined by immunocytochemistry with an anti-Cx36 antibody (a,a′) or by GFP epifluorescence (b,b′,c,c′). Arrowheads indicate the presence of large, clearly visible junctional clusters that are much more frequent in cells containing Cx36 than in cells expressing Cx36-EGFP alone. B. The percent contribution of individual connexin size classes formed within HeLa cells expressing Cx36 (■) or Cx36-EGFP () or Cx36 and Cx36-EGFP () to the total voxel count highlights the requirement of wildtype Cx36 for the formation of clusters in the largest size category. C. HeLa cells transfected with pRK5 (lanes 1,5), Cx36 (lanes 2,6), Cx36-EGFP (lanes 3,7), and co-transfected with Cx36 and Cx36-EGFP (lanes 4,8) were immunoprecipitated with an anti-EGFP antibody (lanes 1–4), blotted, and probed with an anti-Cx36 antibody. Arrows indicate the positions of the Cx36 and Cx36-EGFP proteins. Scale: a,b,c = 20 μM; a′,b′,c′ = 10 μM.
	Fig. 6. A carboxyl-terminal four amino acid motif is necessary for targeting Cx36 chimeras to the plasma membrane. A. Schematics depicting the unaltered and fluorescent protein-tagged variants of Cx36 transfected in HeLa cells. The Cx36 (gray box) and EGFP/ECFP (clear box) protein segments along with novel amino acid residues flanking the fluorescent protein segment inserted as a result of cloning are indicated. Also shown are the Cx36 residues retained in the construct after deleting specific carboxy terminal segments as well as the location of the translation termination codon (*). B. Cx36-ECFP and its derivatives bearing specific mutations at the carboxyl terminus were co-transfected into HeLa cells with either a cytoplasmic (DsRed) or membrane-bound (MbCherry) fluorescent reporter protein as indicated. The ability of the cyan fluorescent protein-tagged Cx36 variants to form protein complexes at the plasma membrane was determined by visual examination. Cx36-ECFP and Cx36-ECFP[− 10/+ 4] but not Cx36-ECFP[− 4] and Cx36-ECFP[− 10] were able to form large clusters at the membrane. C. The percentage of adjacent cell pairs expressing the specified Cx36 construct that contain intercellular clusters is plotted. The data highlight the significant handicap in the ability of Cx36-EGFP, Cx36-ECFP[− 4], and Cx36-ECFP[− 10] to form such aggregates, which, in the case of the first two, could be demonstrated to be overcome by coexpression with Cx36. D. The requirement of the terminal four amino acid residues for intercellular cluster formation is evident in cells expressing Cx36[− 4]. Such clusters (arrows indicate examples) are shorter (E, left) and less voluminous (E, right) than those formed by Cx36. Scale: B = 10 μM, D = 30 μM.

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