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Fig. S1. Endogenous GAD65-GFP colocalizes with wt GFP-Rab5a in Golgi membranes and in axonal but not somatodendritic puncta. Confocal images of a hippocampal neuron transfected with a plasmid encoding GFP-tagged wt Rab5a. Neurons were double immunolabeled for endogenous GAD65 and GFP. Arrows: dendrites; arrowheads: axon. Endogenous GAD65 in an inhibitory neuron colocalizes with wt Rab5a in Golgi membranes (enlarged frame a) and in axonal puncta (arrowheads, enlarged frame b) but not in somatodendritic puncta (enlarged frame a; arrows in enlarged frame b). The GFP-Rab5a(wt)-positive puncta in enlarged frame b, which are not labeled with arrows/arrowheads, represent an axon of a transfected non-GABA-ergic neuron. Scale bars, 10 mm.
Fig. S2. Endogenous GAD65 is targeted to GFP-Rab5a(Q79L)-positive axonal but not somatodendritic endosomes. Confocal images of hippocampal neurons transfected with GFP-Rab5a(Q79L). Neurons were double immunolabeled for endogenous GAD65 and GFP. Endogenous GAD65 colocalizes with GFP-Rab5a(Q79L) in axonal endosomes (arrowheads, enlarged frames a,b) but not in dendritic endosomes (arrows, enlarged frame a). A view of the soma (enlarged image c), shows endogenous GAD65 in Golgi membranes but not in giant endosomes containing GFP-Rab5a(Q79L). GAD65-positive puncta in enlarged frame c, represent axons of neighboring non-transfected GABA-ergic neurons. Scale bars, 10 mm.
Fig. S3. Colocalization of VGAT-HA and wt GFP-Rab5a. Confocal images of a hippocampal neuron transfected with plasmids encoding VGAT-HA and GFP-tagged wt Rab5a. Neurons were double immunolabeled for HA and GFP. GFP-Rab5a(wt) is expressed in axons (arrowheads) and dendrites (arrows). VGAT-HA colocalizes with GFP-Rab5a(wt) in presynaptic clusters in the axon (enlarged frame a) but not in dendritic puncta representing somatodendritic early endosomes (enlarged frame b). Scale bars, 10 mm.
Fig. S4. VGAT and VAMP2 colocalize with Rab5a(Q79L) in both axonal and somatodendritic giant endosomes. Confocal images of hippocampal neurons co-transfected with either VGAT-HA (i,ii) or VAMP2-GFP (iii) together with GFP-Rab5a(Q79L). Neurons were either double immunolabeled for HA and GFP (i) or triple immunolabeled for GFP, HA, and the Golgi marker GM130 (ii, iii). (i) VGAT-HA colocalizes with GFP-Rab5a(Q79L) in giant endosomes in the cell body (enlarged frame a), proximal dendrites (arrows) and along the axon (arrowheads, enlarged frames b) and c). (ii) A view of the soma of a neuron expressing VGAT-HA and GFP-Rab5a(Q79L) shows localization of VGAT-HA in the Golgi compartment as well as in the large somatodendritic endosomes. (iii) VAMP2-GFP colocalizes with HA-Rab5a(Q79L) in giant endosomes in the cell body (enlarged frame a), proximal dendrites (arrows) and along the axon (arrowheads, enlarged frame b). VAMP2-GFP is relatively absent from the Golgi compartment (enlarged frame a). Scale bars, 10 mm.
Fig. S5. Rab5a(S34N) and endogenous GAD65 in Golgi membranes. Confocal images of hippocampal neurons 24 (i) and 72 (ii) hours after transfection with a plasmid encoding GFP-tagged Rab5a(S34N). (i) Neurons were double immunolabeled for GFP and the Golgi marker GM130. Rab5a(S34N) colocalizes with GM130 in Golgi membranes. (ii) Neurons were double immunolabeled for endogenous GAD65 and GFP. Endogenous GAD65 colocalizes with Rab5a(S34N) in Golgi membranes. GAD65-positive puncta (red) traversing the image, represent endogenous GAD65 in presynaptic clusters in axons of surrounding non-transfected GABA-ergic neurons. Scale bars, 10 mm.
Fig. S6. Accumulation of wt but not palmitoylation-deficient GAD65 in the cell periphery in COS-7 cells in the presence of the Rab5(S34N) mutant. Confocal images of COS-7 cells transfected with either wt (i-iii) or palmitoylation-deficient GAD65-GFP (iv-v) together with the dominant negative mutant HA-Rab5a(S34N) (ii-v). Cells were double labeled for GFP and either the Golgi marker GM130 or the HA-tag. (i) In the absence of Rab5a(S34N), wt GAD65-GFP is detected almost exclusively in Golgi membranes. (ii) In the presence of Rab5a(S34N), GAD65 is still detected in Golgi membranes but also in patches in the cell periphery (arrowheads). (iii) Rab5a(S34N) colocalizes with GAD65 in Golgi membranes and is also detected with GAD65 in the peripheral patches (arrowheads), suggesting that they are fusion incompetent early endosomes. (iv) The palmitoylation-deficient GAD65-GFP remains in the Golgi compartment in the presence of Rab5a(S34N). v. Palmitoylation-deficient GAD65 is absent in endosomal patches in the cell periphery containing Rab5a(S34N) (arrowheads). Scale bar, 10 mm.
Fig. S7. Coexpression of wt GAD65-GFP with wt Rab5a or Rab5a(S34N) for 24 hours. Confocal images of hippocampal neurons 24 hours after transfection with plasmids encoding wt GAD65-GFP and either HA-tagged Rab5a(S34N) (i) or wt Rab5a (ii, iii). Neurons were double immunolabeled for GFP and either endogenous EEA1 or HA. (i) In co-expression experiments with Rab5a(S34N), wt GAD65 accumulates in the tip of dendrites (arrows). EEA1 is below detection limit in cells expressing Rab5a(S34N). (ii) A view of the soma and proximal neurites of a neuron co-expressing wt GAD65 and wt Rab5a reveals the polarized targeting of wt GAD65 to presynaptic clusters in the axon (arrowhead), where it colocalizes with wt Rab5a, and its relative absence from dendrites expressing wt Rab5a (enlarged frame a). In these conditions neither GAD65 nor Rab5a accumulate at the tip of the dendrites (arrows). (iii) A view of the distal axon of the neuron shown in ii shows wt GAD65 and wt Rab5a in presynaptic clusters (enlarged frame b). Scale bars, 10 mm.
Fig. S8. The Rab5a(S34N) dominant negative mutant inhibits axonal trafficking and presynaptic clustering of endogenous GAD65. Confocal images of hippocampal neurons 72 hours after transfection with a plasmid encoding GFP-tagged Rab5a(S34N). Neurons were double immunolabeled for endogenous GAD65 and GFP. A view of a transfected GABA-ergic neuron reveals that it is surrounded by axons of non-transfected inhibitory neurons with abundant GAD65-positive presynaptic clusters. In the transfected GABA-ergic neuron, endogenous GAD65 and Rab5a(S34N) are primarily located in Golgi membranes in the soma. In the enlarged frames showing GFP-Rab5a(S34N), the green color has been enhanced to reveal the axon of the tranfected GABA-ergic neuron. Endogenous GAD65 (red) is abundant in puncta in surrounding non-transfected axons, but largely absent in the axon of the transfected neuron expressing Rab5a(S34N). Scale bars, 10 mm.
Fig. S9. The Rab5a-dependent pathway is not involved in trafficking of PSD-95. Confocal images of hippocampal neurons expressing PSD-95-GFP in the presence and absence of HA-tagged Rab5a mutants. Cells were fixed at either 24 hours (i, ii) or 72 hours (iii-iv) after transfection and double immunolabeled for GFP and HA. (i) PSD-95-GFP does not colocalize with wt HA-Rab5a in somatodendritic puncta in 24 hour co-expression experiments. (ii) PSD-95 traffics to somatodendritic puncta in 24 hour co-expression experiments with Rab5a(S34N). (iii) PSD-95-GFP is absent from the giant somatodendritic puncta containing Rab5a(Q79L) in 72 hour co-expression experiments. (iv) PSD-95-GFP traffics to somatodendritic puncta in the presence of Rab5a(S34N) in 72 hour co-expression experiments. (v) A view of a neuron expressing PSD-95-GFP in the absence of Rab5a(S34N), 72 hours after transfection, is shown for comparison. Scale bars, 10 mm.
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