The Journal of Neuroscience, April 26, 2006, ():
Activity-Dependent Movements of Postsynaptic Scaffolds at Inhibitory Synapses
J. Neurosci. Hanus et al.
26: 4586
Supplemental data
Files in this Data Supplement:
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Supplemental material
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Figure S1 Primary structure of Venus::gephyrin
The Venus-tag (239 amino-acids) was fused to gephyrin (clone p1, Prior et al., 1992) N-terminus via a 5 amino-acids linker. The letters G, L and E refer to gephyrin MoG-A-homology, Linker and MoE-A-homology domains, respectively (Sola et al., 2001, 2004).
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Figure S2 Gephyrin redistribution upon interaction with the glycine receptor (GlyR) in heterologous cells.
Confocal sections of COS7 cells co-transfected with untagged- (A-B) or Venus-gephyrin (C-D) and either GlyR standard α1 (A and C) or modified α1 subunits containing the gephyrin binding (βgb) domain of the β-subunit (α1βgb, B and D), at low (A1-2, B1-2, C1-3, D1-3) or high magnification (A3, B3, C4 and D4). Red rectangles indicate the position of the fields showed in A3, B3, C4 and D4. The distribution of GlyR was assessed by immuno-fluorescence (IF) labeling (myc-IF, A2, B2, C3, D3, red in A3, B3, C4 and D4). The distribution of gephyrin was assayed by IF labeling (A1, B1, C2, D2, green in A3, B3, C4 and D4) and imaging Venus intrinsic fluorescence (Venus, C1 and D1).
Note : 1) the comparable distribution patterns of untagged- and Venus-gephyrin, in the presence or absence of the βgb domain; 2) that the presence of the βgb domain induces a redistribution of gephyrin (compare A1-3 to B1-3, C1-4 to D1-4, and the two cells expressing both Venus::gephyrin and GlyR with the one expressing only gephyrin and labeled by an asterisk in D1-3). Co-localization of gephyrin is obtained with cell-surface (arrows in B3 and D4) and intracellular receptors (crossed-arrows in B3 and D4).
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Figure S3 Resolution limit of the quantification assay
Comparison of diffusion coefficients (Dini) derived in control cells (ctrl, black), in cells treated with 3 μM latrunculin (lat, red), 10 μM nocodazole (NZ, green), or 30 μM BAPTA-AM (blue) and in fixed cells (pink).
A. Dini distributions (630, 331, 551, 373 and 56 clusters were analyzed for ctrl, lat, NZ, bapta-am, and fixed cells, respectively).
B. Mean diffusion coefficients median in individual cells (meanąsem; 31, 16, 21, 22 and 2 cells were analyzed for ctrl, lat, NZ, bapta-am, and fixed cells, respectively).
Note that Dini values with latrunculin or BAPPTA-AM treatment are well above values in fixed cells
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Figure S4 Calcium spikes in a spinal cord neuron (10 DIV) transfected with mRFP
A. mRFP fluoresence in a dendrite
B. Fluo4 fluorescence in the field shown in A.
C. Variations of Fluo4 fluorescence in the different area drown in B.
Note the calcium spikes in transfected (#1) and non-transfected neurons (#5,6 and 8; #3 is baseline).
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Figure S5 The gephyrin-cytoskeleton machine
In addition to inhibitory receptors, gephyrin interacts with different molecules (ref. in Moss and Smart 2001) such as the Rapamycin/FKBP Target Protein (RAFT1), dynein light chain subunits 1 and 2 (Dlc1/2), the Cdc42 GTP/GDP exchange factor (Cdc42GEF) protein collybistin, the GABAA Receptor-Associated Protein (GABARAP), profilin and Mena/VASP (Gieseman et al., 2003). Most of these molecules are functionally connected to the dynamics of F-actin (molecules shown in blue) or microtubules (molecules shown in green) or both (molecules shown in purple). Notably, Dlc1/2 could connect gephyrin to myosin V and then kinesins (Langford, 2002) and to cytoplasmic dynein. Calcium regulation of the cytoskeleton (red stars) could arise from changes in profilin activity (Oertner and Matus 2005), or that of myosin V (Krementsov et al., 2004) or Cdc42 downstream pathways (Watanabe et al., 2005, Briggs and Sacks 2003).
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Movie 1. Rapid lateral motion of postsynaptic scaffolds at inhibitory synapses (movie corresponding to figure 2A.
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Movie 2. Rapid motion of postsynaptic structures is not restricted to dendritic spines : raw data (movie corresponding to figure 3A)
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Movie 3. Rapid lateral motion of postsynaptic scaffolds at inhibitory synapses: filtered data (movie corresponding to figure 3D).
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Movie 4. Effects of 50 μM 4-AP and 1 μM TTX on neuron electrical activity
Calcium concentration oscillations in spinal cord neurons loaded with Fluo4, in basal conditions (panel 1), after the addition of 50 μM 4-AP (panel 1, same field as in panel1; and panel 3, in another field) and then 1 μM TTX (panel4, same field as in panel 3). Note movements of mitochondria in panel 4.
