Volume 8 issue 12 December 2007
Neurotrophins redirect p75^NTR from a clathrin-independent to a clathrin-dependent endocytic pathway coupled to axonal transport
Katrin Deinhardt, Alessandra Reversi, Otto Berninghausen, Colin R. Hopkins and Giampietro Schiavo

Supplemental Figures

Supplemental Figure 1: Time course of p75^NTR internalization. MN were incubated on ice with p75^NTR(EC), washed and shifted to 37°C in presence of 100 ng/ml NGF for the indicated times. Cells were then acid washed, fixed and stained with a secondary anti-rabbit IgG antibody to visualize internalized p75^NTR(EC). No internalized p75^NTR was noticeable at time points earlier than 15 min.

Figure 1 (.jpg)

Supplemental Figure 2: Internalization of p75^NTR is independent of NGF. (A) Surface-bound, but not internalized Cy3-p75^NTR(EC) is removed by an acid treatment. MN were incubated with 0.4 µg/ ml Cy3-p75^NTR(EC) for 30 min either on ice (a-d) or at 37°C (e, f). Cells were then acid washed to remove antibody still present on the plasma membrane (c-f), fixed and imaged. All surface-bound antibody was removed with this treatment (c), while the internalized Cy3-p75^NTR(EC) was protected (e). (B) Endocytosis of p75^NTR is NGF-independent upon starvation. Cells were starved for 5 h and then incubated: (a) with control medium; (b) with p75^NTR(EC) and NGF simultaneously on ice for 30 min; (c) with p75^NTR(EC) followed by the addition of NGF and (d) with NGF followed by addition of p75^NTR(EC), washed and shifted to 37°C for 45 min. Cells were acid washed, fixed and stained with a secondary antibody to visualize internalized p75^NTR(EC). A scheme of the experiments is shown on the left. (C) NGF-independent internalization of p75^NTR is not triggered by the p75^NTR(EC) antibody. MN were incubated on ice with IgG192, washed and shifted to 37°C for 45 min with or without 100 ng/ml NGF. MN were acid washed, fixed and stained with a secondary anti-rabbit IgG antibody to visualize internalized p75^NTR(EC). p75^NTR is internalized into neurites and the soma in both conditions. Scale bars, 10 µm.

Figure 2 (.jpg)

Supplemental Figure 3: Distribution of LAMP-1 positive compartments in MN. (A) MN were fixed and stained for LAMP-1. Very few LAMP-1 positive organelles are visible in axons (arrowheads) compared to the dendrites and soma. (B-E) p75^NTR does not colocalize with lysosomal markers. MN were incubated with Cy3-labelled p75^NTR(EC) for 90 min (B, C) or 180 min (D, E), acid washed, fixed and stained for LAMP-1. No overlap between LAMP-1 and internalized p75^NTR was observed with or without NGF. Scale bars, 10 µm (A-C) and 5 µm (D, E).

Figure 3 (.jpg)

Supplemental Figure 4: p75^NTR recycling is detected with the IgG192 antibody. MN were incubated with IgG192 antibody on ice for 30 min, washed and shifted to 37°C with or without 100 ng/ml NGF for 30 min, acid washed and incubated for further 60 min at 37°C in presence of Alexa555-coupled anti-mouse IgG with or without NGF. Cells were fixed and imaged by confocal microscopy. A significant proportion of p75^NTR recycles both in absence and presence of NGF, as shown by the labeling with the IgG192 antibody. These results confirm those obtained with the p75^NTR(EC) antibody (Figure 2).

Figure 4 (.jpg)

Supplemental Figure 5: AP180-C expressing neurons display functional mitochondria. Immediately after transport analysis (Figure 6B), MN were incubated with 5 nM TMRE for 5 min, washed, and the imaged axon was scanned again at low laser power. Mitochondrial morphology and potential were unaffected in the AP180-C expressing axon. Scale bar, 10 µm.

Figure 5 (.jpg)

Supplemental Figure 6: Expression of GFP-Rab^wt or GFP-Rab5^Q79L does not alter p75^NTR transport. (A) MN were microinjected with a plasmid encoding GFP-Rab5^Q79L. After overnight expression, MN were incubated with 0.4 mg/ml Cy3-p75^NTR(EC) and 100 ng/ml NGF for 30 min at 37 °C, washed and imaged by time-lapse confocal microscopy. Just like GFP-Rab5^wt (Figure 7), GFP-Rab5^Q79L does not interfere with p75^NTR transport and localizes to oscillating p75^NTR-positive compartments (see asterisks), but not to p75^NTR-carriers (see arrowheads). (B) Kinetic analysis of p75^NTR carriers in MN expressing Rab5 constructs. GFP-Rab5^wt and GFPRab5^Q79L overexpression do not affect p75^NTR retrograde transport. Kinetic analysis of p75^NTR carriers in control MN (black squares; 365 single movements, 47 carriers), MN overexpressing GFP-Rab5^wt (green triangles; 523 single movements, 63 carriers) and the constitutively-active mutant GFP-Rab5^Q79L (blue squares; 782 single movements, 75 carriers) are shown. Speed profiles were assembled by binning the speed values using a 0.2 µm/s interval and calculating the relative frequencies as described in (17). Retrograde transport is conventionally shown as positive and pauses during movement are grouped at 0 µm/s.

Figure 6 (.jpg)

Supplemental Videos

Supplemental Video 1: p75^NTR transport in control MN. MN were incubated with 0.4 mg/ml Cy3-p75^NTR(EC) and 100 ng/ml NGF for 30 min at 37 °C, washed and imaged by time-lapse confocal microscopy. Shown is a movie consisting of 23 frames, played at 5 frames/s. The soma is located out of view to the right. The image size is 73.1 µm x 11.4 µm. See also Figure 6A.

Video 1 (.avi)

Supplemental Video 2: p75^NTR transport is blocked in MN expressing AP180-C. 24 h after microinjection, AP180-C and GFP-expressing MN were incubated with 0.4 mg/ml Cy3-p75^NTR(EC) and 100 ng/ml NGF for 30 min at 37 °C, washed and imaged by time-lapse confocal microscopy. Shown is the red channel (p75^NTR signal) of a movie consisting of 50 frames, played at 5 frames/s. The soma is located out of view to the right. The image size is 55.4 µm x 9.1 µm. See also Figure 6B.

Video 2 (.avi)

Supplemental Video 3: The role of GFP-Rab5^wt in p75^NTR transport. GFP-Rab5^wt does not interfere with p75^NTR transport, and localizes to oscillating p75^NTR-positive compartments, but not to p75^NTR-carriers. GFP-Rab5^wt-expressing MN were incubated with 0.4 mg/ml Cy3-p75^NTR(EC) and 100 ng/ml NGF for 30 min at 37 °C, washed and imaged by time-lapse confocal microscopy. Shown is a movie consisting of 150 frames, played at 5 frames/s. The soma is located out of view to the right. The image size is 71.4 µm x 14.8 µm. See also Figure 7B.

Video 3 (.avi)

Supplemental Video 4: Expression of GFP-Rab^N133I blocks axonal retrograde transport of p75^NTR. After 16 h expression, cells were incubated with 0.4 mg/ml Cy3-p75^NTR(EC) and 100 ng/ml NGF for 30 min at 37 °C, washed and imaged by time-lapse confocal microscopy. This movie consists of 119 frames played at 5 frames/s. The soma is located out of view to the right. The image size is 71.9 µm x 15.9 µm. See also Figure 7C.

Video 4 (.avi)