The Journal of Neuroscience, November 1, 2006, ():
The Dynamic Distribution of TrkB Receptors before, during, and after Synapse Formation between Cortical Neurons
J. Neurosci. Gomes et al.
26: 11487
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplemental Figure 1. Live-surface staining does not induce clustering of TrkB. A, The pattern of TrkB labeling obtained after light fixation with 2% paraformaldehyde does not differ from the live-surface staining that we used for all of our experiments. Scale bar, 5µm. B, Both staining methods for sTrkB reveal a similar number of puncta per area. Thus, our live-surface staining protocol is not confounded by antibody-induced clustering of TrkB.
- supplemental material
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Supplemental Figure 2. TrkB-EGFP-transfected neurons overexpress TrkB but maintain the same density of TrkB puncta as nontransfected cells. Transfected and non-transfected cells were stained for TrkB (TrkBec (Up)). A, TrkBec puncta in transfected neurons are significantly higher in intensity as compared to that in nontransfected cells (p < 0.0001). B, TrkB-EGFP transfected and non-transfected cells have the same density of TrkB puncta. Thus, TrkB-EGFP has the same distribution as endogenous TrkB. C-E, The distribution of TrkB-EGFP also highly correlates with the distribution of VAMP2, similar to the high colocalization for endogenous TrkB and VAMP2 (Fig. 4). C, Immunostaining of axons shows that TrkBec (red) is highly associated with VAMP2 (blue) both in non-transfected (upper panels) and TrkB-EGFP (green) transfected cells (lower panels). Scale bar, 5µm. D, Line scans of fluorescence intensities of TrkBec (red), VAMP2 (blue) and TrkB-EGFP (green) in the axons shown in C. Top graph, untransfected; bottom graph, transfected. E, The colocalization of endogenous TrkB or TrkB-EGFP with VAMP2 is similar. Cross-covariagrams of endogenous TrkB and VAMP2 in non-transfected axons (upper graph) and TrkB-EGFP and VAMP2 in transfected axons (lower graph) had peaks of similar height, centered around the same value.
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Supplemental Figure 3. VAMP2-DsRed is transported with another SV protein, synaptophysin-EGFP. Axon from a cotransfected neuron showing a punctum of VAMP2-DsRed and synaptophysin-EGFP moving together (orange arrow - left panels). The white arrow indicates the original position of the synaptic protein precursor. Scale bar, 10 µm. Right panels show the corresponding intensity profiles of a line draw through the axon shown on the left. Intensity profiles show colocalization and co-movement (arrowhead) of peaks of intensity for synaptophysin-EGFP (green line) and VAMP2-DsRed (red line) that correspond to the puncta in the left panels.
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Supplemental Movie 1. A dendrite from a pyramidal neuron expressing TrkB-EGFP. Although it is impossible to track individual TrkB-EGF puncta due to their high density in dendrites, it is clear that a high level of trafficking of TrkB-EGFP puncta occurs in both anterograde and retrograde directions. Note the high density of lightly labeled TrkB-EGFP puncta and the streaming of those smallest particles. Frames were collected every 15 s, and the total movie length is 11 min and 15 s. Scale bar, 10 µm.
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Supplemental Movie 2. An axon from a pyramidal neuron expressing TrkB-EGFP. TrkB-EGFP puncta move anterogradely, retrogradely and bidirectionally. Frames were collected every 15 s, and the total movie length is 9 min. Scale bar, 10 µm.
