JCS01511 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Biological activity of TrkB.FL-GFP fusion protein, of neurotrophins and of inhibitors of TrkB signalling. PC12 cells were transfected with TrkB.FL-GFP, and fibre outgrowth was induced by adding neurotrophins. After 3 days, the percentage of transfected cells with fibre outgrowths longer than their respective soma diameter was determined (see Materials and Methods). (A) PC12 cells expressing either the fusion protein (TrkB.FL-GFP), wild-type TrkB without GFP tag (wt TrkB.FL) or untransfected control cells were stimulated with NGF (100 ng/ml), BDNF or NT-4/5 (100 ng/ml) or were left unstimulated (w/o NT). The efficiency of fibre outgrowth induced by TrkB.FL-GFP was indistinguishable from the results obtained with wt TrkB.FL, indicating intact biological function of the TrkB.FL-GFP receptor. 1 mg/ml TrkB-Fc significantly inhibited fibre outgrowth of 100 ng/ml BDNF in this assay. (* P< 0.01). (B) Typical PC12 cell, showing TrkB.FL-GFP expression, and neurite outgrowth induced by NT-4/5. Scale bar: 10 mm.

• Supplemental Figure 2 -

  Fig. S2. Similar outline of dendritic filopodia by DsRed and TrkB.T1-GFP. Mouse hippocampal neurons were cotransfected at 8 DIV with TrkB.T1 and DsRed (A; DNA ratio 3:1) or with GFP and DsRed (B; DNA ratio 3:1) and living neurons were analysed 2 days after transfection. Lower panels in A and B show higher magnification of the boxed areas. Yellow arrows indicate filopodia that have moved between the two exposures (5-second intervals). The larger number of dendritic filopodia in A than in B is clearly visible also in the DsRed only channel, indicating that the lower number of filopodia in GFP controls is not an artefact of dendritic outline. (C) Quantification of filopodia in the red channel (three independent experiments with n>28 cells in each group, *significantly different with P<0.0001). Scale bars: 10 mm (A,B).

• Supplemental Figure 3 -

  Fig. S3. Additional axonal targeting of all TrkB constructs. Rat hippocampal neurons were transfected at 2 DIV with TrkB.T1-GFP (A), TrkB.FL-GFP (B), and TrkB.T2-GFP (C) and processed for anti-tau immunocytochemistry 1 day after transfection. Arrows indicate tau immunopositive axons. Cells in A and C, are reconstructed from overlapping pictures. Bars represent 10 µm.

• Supplemental Figure 4 -

  Fig. S4. TrkB.T1 induced growth of filopodia is independent of the GFP tag. Rat hippocampal neurons were transfected at 8 DIV with either wt TrkB.T1, TrkB.T1-GFP or with GFP, and a DsRed expression plasmid was coexpressed (DNA ratio: TrkB/GFP:DsRed, 3:1). (A) For all conditions, neurons were analyzed in the red fluorescent channel 2 days after transfection. Lower panels show higher magnification of boxed areas in upper panels. (B) Quantification of filopodia in the red channel (two independent experiments with n=40 cells in each group). Untagged and GFP-tagged TrkB.T1 both showed a significant (**significantly different from GFP with P<0.001) increase in dendritic filopodia, indicating that the induction of filopodia is not dependent on the GFP tag. Scale bars: 10 mm.