by Neurobasal medium (supplemented with B27, 0.5mM glutamine, and 100 U/mL

Transcription

1 Supplementary Materials and methods Neuronal cultures and transfection The hippocampus was dissected from E8 rat embryos, dissociated, and neurons plated onto glass coverslips coated with poly-ornithine (Sigma) at a density of 8, cells per coverslip. After the neurons attached ( h after plating) they were transfected using Lipofectamine (Invitrogen) as described. After an incubation for h, the transfection medium was replaced by Neurobasal medium (supplemented with B7,.5mM glutamine, and U/mL penicillin/streptomycin; Invitrogen). The cells were detached after the transfection by moderate pipetting and replated onto new coverslips at a lower density (,-6, cells per coverslip in a -well plate). Alternatively, neurons were transfected by electroporation using the Rat Neuron Nucleofector Kit (Amaxa). For electroporation,.5 x 6 cells were resuspended in µl Nucleofectamin solution, transfected according to the manufacturer's recommendations, and plated at. cells per coverslip. Neurons were fixed at d.i.v. with % paraformaldehyde and 5% sucrose in phosphatebuffered saline (PBS) for min at o C or (for staining with anti-smurf and anti-smurf antibodies) with methanol/acetone (:) for min at - o C. For immunofluorescence, hippocampal neurons were permeabilised with.% Triton X- in PBS, blocked with % FCS in PBS (blocking buffer), and incubated with primary and secondary antibodies in blocking buffer. The following antibodies were used: Tau-, anti-synapsin I (Chemicon; #MAB, dilution :; AB5, dilution :), anti-map (Chemicon; #AB56, :5), anti-gfp (BabCo; #MMS-9P, :5), rabbit anti-par (Upstate; #7-, :), monoclonal anti- Rap (BD Biosciences; #695, :), polyclonal anti-smurf (#APa, :) and anti- Smurf (Abgent; #AP5a, :), and Alexa-conjugated secondary antibodies (Molecular

2 Probes; :). The specificity of the Smurf and Smurf antibodies was confirmed in Western blots (not shown) and by immunofluorescence (Supplementary Figure S5). The anti- Rap antibody recognises both RapA and RapB (95% amino acid sequence identity) but not other GTPases. Hippocampal neurons express mainly RapB (Schwamborn and Püschel, ). FM-6 dye uptake Uptake of FM-6 was analysed as described previously (Jiang et al., 5). At d.i.v., neurons were incubated with 5 mm K + solution in the presence of µm FM-6FX (Invitrogen) for min. Subsequently, neurons were washed for 5 min with a mm K + solution and fixed or incubated with 9 mm K + for 5 min before fixation. All solutions contained mm kynurenic acid (Calbiochem). References Jiang, H., Guo, W., Liang, X. and Rao, Y. (5) Both the establishment and the maintenance of neuronal polarity require active mechanisms: critical roles of GSK-beta and its upstream regulators. Cell,, -5. Schwamborn, J.C. and Püschel, A.W. () The sequential activity of the GTPases RapB and Cdc determines neuronal polarity. Nat. Neurosci., 7, 9-99.

3 Supplementary Figure Legends Supplementary Figure S Inhibition of the proteasome disrupts neuronal polarity. (A) Quantification of RapB immunoreactivity in the growth cones marked in Fig. A. (B) Quantification of the RapB signal from the Western Blot shown in Fig. E. (C-D) Hippocampal neurons were incubated with solvent (DMSO) or.5µm Lactacystin (dissolved in DMSO) for 8 h, and analysed at d.i.v. by staining with the Tau- (C, blue) and an anti-par antibodies (red). Axons identified by Tau- immunoreactivity are marked by asterisks. The marked growth cones are shown at a higher magnification. Treatment with Lactacystin induced the formation of multiple axons that were all positive for Par (arrow). Marked growth cones (, ) are shown at a higher magnification. Scale bars are µm. (D) The length of minor neurites was not changed by treatment with solvent (D, DMSO) Lactacystin (L), ALLN (A), or MG- (M; means ± s.e.m.; p<. compared to treatment with DMSO; n= independent experiments). (E-I) Hippocampal neurons were cultured in the presence of solvent (DMSO) or µm ALLN (dissolved in DMSO) for 8 h and analysed at d.i.v. (stage ) by staining with (E) an anti-map (red) and the Tau- antibody (blue), (F) rhodamine-phalloidin (red), the Tau- (blue), and an anti-par antibody (green), or (H) rhodamine-phalloidin (red), the Tau- (blue), and an anti-rap antibody (green). Axons identified by Tau- immunoreactivity are marked by asterisks. One neuron is shown at a higher magnification. Treatment with ALLN induced the formation of multiple axons, that were positive for Par and RapB (F, H; H, arrows). (G) Quantification of RapB immunoreactivity in the growth cones labeled in F. (H) Insets show higher magnifications of the marked growth cones. (I) Quantification of RapB immunoreactivity in the growth cones labeled in (H). (J) Hippocampal neurons were incubated overnight with solvent (DMSO, -) or µm ALLN (+). RapB was precipitated from cell lysates using an anti-rap antibody (xip) and analysed by Western blot using anti-ubiquitin, anti-rap, anti-α-tubulin, and the

4 Tau- antibodies (WB). Luminescence signals from Western blots were collected for second (s) and min to detect poly-ubiquitinated RapB visible after proteasome inhibition. Staining for tubulin confirmed the loading of comparable amounts of protein. The slight increase in the amount of α-tubulin upon inhibition of the proteasome is consistent with the formation of supernumerary axons. The Tau- antibody revealed a small increase in the level of the hypo-phosporylated tau isoform detected by this antibody after inhibition of the proteasome. Supplementary Figure S In vitro ubiquitination is specific for GDP-bound RapB. (A) HEK 9T cells were transfected with expression vectors for myc-tagged wild type RapB (wt), constitutively active RapB (V), or dominant negative RapB (N7). The cells were treated with. µm 8-CPT-Me-cAMP (8-CPT) as indicated which activates RapB through Epac. The amount of active RapB was determined by a GST pull-down assay using bacterially expressed GST-RalGDS-RBD coupled to glutathion-sepharose. Bound RapB was detected in Western blots using an anti-myc antibody. The expression of comparable amounts of RapB and GST-RalGDS-RB was confirmed by Western blot. (B) HEK 9T cells were transfected with expression vectors for Flag-SmurfC76A (F/SmurfCA) or HA-Epac (H/Epac; as a control for GDP-loading) and the cell lysates incubated with bacterially expressed GST-RapB coupled to glutathion-sepharose and preloaded with.7 mm GTPγS or.7 mm GDP as indicated. Bound Smurf (left panel) or Epac (right panel) were detected in Western blots using an anti-flag or anti-ha antibody. The expression of equivalent amounts of Smurf, Epac, and GST-RapB was confirmed by Western blot. Supplementary Figure S GST-RalGDS-RBD is specific for active RapB. (A, B) COS-7 cells were fixed 5 min after treatment with DMSO (-) or. µm 8-CPT-Me-cAMP (8- CPT), incubated with bacterially expressed GST (negative control) or GST-RalGDS-RBD

5 that specifically binds RapB-GTP and bound GST was detected in situ with an anti-gst antibody (green). RapB was detected with an anti-rap antibody (red). GST immunofluorescence intensity was color-coded. Blue indicates weak staining, white strong labeling. RapB immunofluorescence and RalGDS-RBD binding showed a high degree of colocalization. RapB was recruited to the cell membrane upon activation of Epac by 8-CPT, as previously described (LaFuente et al., ). (C) The specificity of the immunostaining for bound GST-RalGDS-RBD (Fig. ) was confirmed by incubation of stage and stage hippocampal neurons with GST and staining with an anti-gst antibody. Cells were labeled with the cell volume marker CMFDA (green). Neurons were analysed by confocal microscopy. Projections of z-stacks that contain the complete cell are shown. Pictures were taken with the same exposure time that was used for Fig. C. GST immunofluorescence intensity was color-coded. Blue indicates weak staining, white strong labeling. The cell body shows only a very weak staining after incubation with GST and no signals were detectable in neurites or growth cones. Scale bars are µm. Supplementary Figure S Smurf expression increases neurite length. (A-C) Hippocampal neurons were transfected h after plating with expression vectors for EGFP or EGFP and Smurf, SmurfC699A, Smurf, or SmurfC76A. Neurons were analysed at d.i.v. by staining with Tau- and anti-map antibodies. The total number of neurites (A, axon plus minor neurites), axon length (B) and the length of minor neurites (C) were determined (means ± s.e.m.; p<. compared to EGFP; n= independent experiments). Supplementary Figure S5 Efficiency of the shrnas. (A-F) Hippocampal neurons were transfected with pshag- (Mock) or expression vectors for anti-smurf, anti-smurf, anti- SmurfMut (inactive control vector), or anti-smurfmut shrnas and EGFP (A and B, green). Transfected cells were analysed at d.i.v. by staining with anti-smurf or anti-smurf

6 antibodies (A, red) or by staining with Tau- (blue) and anti-map antibodies (B, red). (A) Expression of anti-smurf or anti-smurf shrnas but not the control shrnas resulted in an almost complete loss of Smurf or Smurf from neurites and cell bodies. Only weak residual staining was visible in the cell bodies. Neurons expressing anti-smurfmut or anti- SmurfMut shrnas and EGFP showed normal Smurf or Smurf staining (A, arrows) and developed a normal polarity with one axon (B, asterisks) and several minor neurites. The number of minor neurites (C), the total number of neurites (D), axon length (E), and the length of minor neurites (F) were determined (means ± s.e.m.;, p<. compared to Mock; n= independent experiments). Higher magnifications of the growth cones marked in (A) are shown. Scale bars are µm. (G-N) To confirm the specificity of the Smurf knock-down, a second RNAi construct based on the psm vector (Silva et al., 5) and directed against a different target sequence in Smurf was used. Hippocampal neurons were transfected with EGFP (green) and psm or the expression vector for the anti-smurf shrna- (G, L). Transfected cells were analysed at d.i.v. by staining with the Tau- (blue) and anti-map antibodies (G, red) or an anti-smurf antibody (L, red). Tau- positive axons are marked by asterisks and axons formed by untransfected neurons by arrowheads (G) The knock-down of Smurf induced the formation of multiple axons (G, H), while the number of minor neurites remained unchanged (I). The length of axons was slightly reduced (J) while the length of the minor neurites was not affected significantly (K) (means ± s.e.m.;, p<. compared to control (psm); n= independent experiments). (L) The expression of the anti-smurf shrna- but not transfection with the vector psm resulted in an almost complete loss of Smurf from neurites and growth cones. The marked growth cones (: transfected cell, : untransfected cell) are shown at a higher magnification. (M-P) Hippocampal neurons were transfected h after plating with an expression vector for EGFP and psm or the vector for the anti-smurf shrna-. Transfected cells were analysed at 6 d.i.v. by staining with the Tau- (M, blue), anti-gfp (green) and anti-map antibodies (M, red). Because the density of

7 the neurites at this stage prevented the reliable and unambiguous assignment of Tau- positive neurites to a cell body (arrow), neuronal polarity was analysed only by morphology (N). Long neurites were scored as axons (asterisks) while short neurites were counted as dendrites. The number of axons (O) and dendrites (P) per cell was determined (means ± s.e.m.; p<. compared to psm; independent experiments).(q, R) Hippocampal neurons were transfected at d.i.v. with an expression vector for EGFP (green) and pshag-magic (EGFP) or the expression vector for the anti-smurf shrna- (Smurf-RNAi) and analysed at 5 d.i.v. by staining with the Tau- (Q, blue) and an anti-map antibody (Q, red). Neurons were plated at a lower density (, cells per coverslip) than for the experiments shown in (M-P). The number of axons and dendrites per cell is shown (means ± s.e.m.; p<. compared to EGFP/pSM; independent experiments). Controls (EGFP) formed. ±. axons and 6. ±. dendrites per cell (n=). Suppression of Smurf at this stage did not affect neuronal polarity (. ±. axons and 5. ±. dendrites per cell; n=). (S, T) Solvent (DMSO) or nm Lactacystin (dissolved in DMSO) was added to cultures of hippocampal neurons at d.i.v. and neurons analysed at 5 d.i.v. by staining with the Tau- (S, blue) and an anti-map antibody (S, red). The number of axons and dendrites per cell is shown (means ± s.e.m.; p<. compared to DMSO; independent experiments). Lactacystin induced the formation of multiple axons (Lactacystin:. ±. axons and.7 ±.9 dendrites per cell; n=5). (U) Hippocampal neurons were transfected with expression vectors for an shrna directed against Smurf (S RNAi) and EGFP (green). As control, a construct for an inactive shrna (S RNAi mut) was used. Transfected cells were analysed at d.i.v. (stage ) by staining with anti-synapsin I (blue) and anti-map antibodies (red). (V, W) Hippocampal neurons were transfected with expression vectors for EGFP and an shrna directed against Smurf (Smurf RNAi) or an inactive shrna as control (Smurf RNAi mut). At d.i.v. (stage ), the uptake of the dye FM-6 by transfected cells was analysed to confirm that axons show active synaptic vesicle recycling. In control neurons (Smurf RNAi 5

8 mut), all axons (marked with an asterisk) showed uptake of FM-6 after stimulation with K + (a higher magnification of the axon marked is shown) whereas little uptake was detectable in minor neurites (). Suppression of Smurf by RNAi induced the formation of supernumerary axons (marked with an asterisk). All axons (identified by their length) were positive for FM-6 uptake (higher magnification:, ) whereas minor neurites showed little dye uptake (). After a second stimulation with K +, most of the dye was released from axons (not shown). These results confirm that the supernumerary axons induced by suppression of Smurf are functional in vesicle recycling. The scale bars are µm. (W) The number of neurites per cell positive (black bars) or negative for FM-6 (white bars) is shown (Smurf RNAi mut:. ±. neurites/cell positive for FM-6 uptake,. ±. negative, n = 9; Smurf RNAi:. ±. positive,. ±. negative, n = ; means ± s.e.m.; p<. compared to Smurf RNAi mut). Supplementary Figure S6 Distribution of active RapB after expression of Smurf. (A, B) Hippocampal neurons were transfected h after plating with expression vectors for EGFP, EGFP and Smurf, or EGFP and SmurfC76A (green). Neurons were analysed at d.i.v. by incubation with bacterially expressed GST (A, negative control) or GST-RalGDS-RBD (B) that specifically binds RapB-GTP. GST was detected in situ with an anti-gst antibody (red). As an additional control, the growth cones of untransfected neurons in the vicinity of transfected cells are shown in (B). Neurons were analysed by confocal microscopy. Projections of z-stacks that contain the complete cell are shown. Incubation with RalGDS- RBD confirmed that the supernumerary axons induced by expression of SmurfC76A contained active RapB. The marked growth cones are shown at a higher magnification (picture shows growth cones from untransfected cells in the upper and middle panel of (B)). Scale bars are µm. 6

9 Supplementary Figure S7 Expression of RapBR5 and Smurf deletion constructs. (A) Alignment of RhoA and RapB N-terminal sequences. Conserved lysines are indicated in red. (B) HEK 9T cells were transfected with expression vectors for myc-rapb (M/RapB) or myc-rapbr5 (M/RapBR5) and the cell lysates incubated with bacterially expressed GST- SmurfC76A coupled to glutathion-sepharose. Bound RapB and RapBR5 (top panel) were detected in Western blots using an anti-myc antibody. The expression of equivalent amounts of RapB, RapBR5, and GST-SmurfC76A was confirmed by Western blot. (C) HEK 9T cells were transfected with expression vectors for myc-rapb (M/RapB) or myc- RapBR5 (M/RapBR5) and the cell lysates incubated with bacterially expressed GST- RalGDS-RBD coupled to glutathion-sepharose to determine the amount of active GTPase. Bound RapB and RapBR5 (top panel) were detected in Western blots using an anti-myc antibody. The expression of equivalent amounts of RapB, RapBR5, and GST-RalGDS- RBD was confirmed by Western blot with anti-gst and anti-myc antibodies. Equivalent amounts of active RapB and RapBR5 were detected showing that the R5 mutation does not increase Rap activity. (D) Hippocampal neurons were transfected h after plating with expression vectors for RapB, RapBR5, RapB and EGFP-Smurf, or RapBR5 and EGFP- Smurf. Transfected cells were analysed at d.i.v. by staining with the Tau- and anti-map antibodies, and neuronal morphology was analysed by determining the number of minor neurites per cell (means ± s.e.m.; p<. compared to EGFP; n= independent experiments). (E) Hippocampal neurons were transfected with expression vectors for EGFP (green) or EGFP and RapBR5. Transfected cells were analysed at d.i.v. (stage ) by staining with anti-synapsin I (blue) and anti-map antibodies (red). (F, G) Hippocampal neurons were transfected with expression vectors for EGFP or EGFP-RapBR5. At d.i.v. (stage ), the uptake of the dye FM-6 by transfected cells was analysed to confirm that axons show active synaptic vesicle recycling. In control neurons (GFP), all axons (marked with an asterisk) showed uptake of FM-6 after stimulation with K + (a higher magnification 7

10 of the axon marked is shown) whereas little dye uptake was detectable in minor neurites (). Transfection of neurons with an expression vector for RapBR5 induced the formation of supernumerary axons (marked with an asterisk). All axons (identified by their length) were positive for FM-6 uptake (higher magnification:, ) whereas minor neurites showed little dye uptake (). After a second stimulation with K +, most of the dye was released from the axons (not shown). These results confirm that the supernumerary axons induced by suppression of Smurf are functional in vesicle recycling. The scale bars are µm. (G) The number of neurites per cell positive (black bars) or negative for FM-6 (white bars) is shown (EGFP:. ±. neurites/cell positive for FM-6 uptake,. ±. negative, n = ; RapBR5:.9 ±. positive,.5 ±. negative, n = ; means ± s.e.m.; p<. compared to EGFP). 8

11 Suppl. Figure S A Rap fluorescence intensity (a.u.) DMSO Lactacystin B Rap amount (a.u.) RapB ohne DMSO RapB mit ALLN C Par Tau- Overlay DMSO Lactacystin D Length minor neurites (µm) DMSO Lacta D L A M

12 E MAP Tau- Overlay DMSO ALLN F Actin Tau- Par Overlay DMSO ALLN 6 5 G Par fluorescence intensity (a.u.) DMSO ALLN Suppl. Figure S

13 Suppl. Figure S H Actin Tau- Rap Overlay DMSO 5 ALLN I Rap fluorescence intensity (a.u.) DMSO ALLN

14 J s ALLN - + min ALLN - + WB: kd (Ub) n -RapB α-ub kd Rap kd α-rap tubulin xip: α-rap 55kD α-tubulin tau Lysate 55kD Tau- Suppl. Figure S

15 A M/RapB 8CPT RapB GST/RalGDS-RBD WT WT N7 V WB: α-myc GST/RalGDS-RBD α-gst RapB GST pull-down α-myc Lysate B GTPγS GDP F/Smurf / H/Epac GST/RapB F/Smurf CA GST/RapB H/Epac WB: + + α-flag / α- HA α-gst F/Smurf / H/Epac GST pull-down Lysate α-flag / α- HA Suppl. Figure S

16 Suppl. Figure S A Rap GST GST Overlay 8CPT - 8CPT - B Rap RalGDS-RBD RalGDS-RBD Overlay

17 Suppl. Figure S C CMFDA GST Overlay Stage Stage

18 Suppl. Figure S A Total no. neurites 9 7,5 6,5,5 EGFP Smurf Smurf CA Smurf Smurf CA B Length axons (µm) EGFP Smurf Smurf CA Smurf Smurf CA C Length minor neurites (µm) 8 6 EGFP Smurf Smurf CA Smurf Smurf CA

19 Suppl. Figure S5 A EGFP Smurf Overlay EGFP Smurf S RNAi Mock Smurf RNAi Mut Mock Smurf RNAi Mut EGFP Smurf Overlay EGFP Smurf Smurf RNAi B EGFP MAP Tau- Overlay Smurf RNAi Mut Smurf RNAi Mut

20 Suppl. Figure S5 C 6 D 7 No. minor neurites 5 Total no. of neurites 6 5 pshag Smurf RNAi Smurf RNAiMut Smurf RNAi Smurf RNAiMut pshag Smurf RNAi Smurf RNAiMut Smurf RNAi Smurf RNAiMut E 6 F 5 Length axons (µm) 8 6 pshag Smurf RNAi Smurf RNAiMut Smurf RNAi Smurf RNAiMut Length minor neurites (µm) pshag Smurf RNAi Smurf RNAiMut Smurf RNAi Smurf RNAiMut

21 Suppl. Figure S5 G EGFP MAP Tau- Overlay S RNAi- MOCK L S RNAi- MOCK EGFP Smurf Overlay H No. minor neurites No. Axons J minor neurite (µm) axons (µm) ILength K psm psm psm Smurf RNAi- Smurf RNAi- Smurf RNAi- psm Smurf RNAi-

22 Suppl. Figure S5 M MAP Tau- Overlay EGFP Smurf-RNAi- EGFP

23 N EGFP Smurf-RNAi- Suppl. Figure S5 EGFP

24 Suppl. Figure S5 O No. of axons EGFP Smurf-RNAi- P 7 No. of dendrites 6 5 EGFP Smurf-RNAi-

25 Suppl. Figure S5 Q MAP Tau- Overlay EGFP EGFP Smurf-RNAi R No. of dendrites No. of axons EGFP Smurf-RNAi EGFP Smurf-RNAi

26 S MAP Tau- Overlay DMSO Lactacystin T No. of axons DMSO Lactacystin 8 7 No. of dendrites 6 5 DMSO Lactacystin Suppl. Figure S5

27 U GFP Synapsin I Map Overlay Smurf RNAi Smurf RNAi mut Suppl. Figure S5

28 V GFP FM-6 Smurf RNAimut Smurf RNAi W # neurites/cell,5,5,5 FM-6 uptake no FM-6 uptake,5 Smurf RNAi mut Smurf RNAi Supplementary Figure S5

29 A EGFP GST Overlay Suppl. Figure S6 EGFP SmurfCA Smurf EGFP B EGFP RalGDS-RBD Overlay Smurf SmurfCA

30 B A RhoA MAAIRKKLVIVGD RapB MREYKLVVLGS GST/SmurfCA M/RapB WT R5 WB: M/RapB WT R5 WB: RapB α-myc RapB α-myc C GST/RalGDS-RBD Smurf α-gst RalGDS-RBD α-gst GST pull-down GST pull-down RapB α-myc RapB α-myc D No. minor neurites Lysate 6 EGFP RapB RapB5R RapB + Smurf RapB5R + Smurf Lysate E GFP Synapsin I MAP Overlay RapB R5 EGFP Suppl. Figure S7

31 F GFP FM-6 RapBR5 GFP G # neurites/cell,5,5,5 FM-6 uptake no FM-6 uptake,5 GFP RapB R5 Supplementary Figure S7