Figure 2.
Proteasome retention and local protein degradation in the AIS require Ecm29 interaction with AnkG. (A) In vivo protein binding assays in HEK293T cells transfected with a plasmid encoding FLAG-tagged Ecm291–1,840 (FLAG-Ecm29FL) and plasmids encoding 190AnkG-GFP (AnkG-GFP) or control GFP vectors, as indicated. Cell lysates were IP by FLAG M2 antibodies and blotted with indicated antibodies. Histograms at right show relative protein levels based on immunoblotting of GFP, Arp1, Rpt1, and KLC1 coIP by FLAG antibodies (±SEM; n = 4 independent experiments; *, P < 0.05; ns, not significant, by t test). (B) cAMP increases facilitate Ecm29–AnkG interaction. (B1) Schematic showing design of AIS-mimicking ratiometric FRET sensors. AIS molecular dock: NF-186. Donor molecule: 190AnkG-GFP. Acceptor molecule: Ecm29ΔC-mCherry or Ecm29ΔN-mCherry. (B2) Time-lapse FRET images of HEK293T cells cotransfected with plasmids encoding 190AnkG-GFP, NF-186-HA, and N- or C-terminal Ecm29-mCherry (Ecm29ΔC-mCherry or Ecm29ΔN-mCherry) in the presence of 20 µM forskolin, with or without 30 min of pretreatment with the PKA inhibitor KT5720, as indicated. White dashed line passes through the center of the cell, with points a and b as intersection points. Scale bar, 10 µm. (B3) Quantitative measurement of FRET signals (±SEM, n ≥ 13 cells for each group) at different times before and after addition of forskolin (20 µM) in experiments as described in B. Arrow in right panel denotes forskolin addition. Norm., normalized. (C) Ecm29 expression is required for proteasome association with the detergent-resistant AIS structure. (C1) Representative images of 7-DIV hippocampal neurons transfected with SC-shRNA or AnkG-targeting siRNA (AnkG-siRNA) at 3 DIV, with or without nocodazole (Noco; 1 µM for 3 h) or latrunculin A (Lat A; 1 µM for 6 h), followed by detergent extraction with 0.1% Triton X-100 at 37°C for 2 min before immunostaining for the AIS marker AnkG/Trim46 (red, merge panels), proteasome subunit Rpt1/Rpt5 (green, merge panels), and the proteasome adaptor Ecm29 (green, merge panels). (C2) Quantitative measurement of immunostaining intensities (±SEM, n = 15–35 cells) along AIS segments. (D) Local protein turnover at the AIS requires Ecm29 expression and proteasome activity. (D1) Images of 7-DIV hippocampal neurons expressing an AIS-located protein degradation reporter, NavII-III–tagged GFPu, showing more durable GFPu accumulation after MG132 pretreatment (2.5 µM, 30 min) or shRNA-mediated knockdown of Rpt-1 or Ecm29 expression, as indicated. Scale bar, 15 µm. (D2) Quantitative measurement of fluorescence intensities in the AIS (top panel) and soma (bottom panel) at different time points based on all experiments performed as described in D1 (±SEM, n > 6 cells for each group, one-way ANOVA followed by Dunnett’s multiple comparison test. *, P < 0.05; ns, not significant).

Proteasome retention and local protein degradation in the AIS require Ecm29 interaction with AnkG. (A) In vivo protein binding assays in HEK293T cells transfected with a plasmid encoding FLAG-tagged Ecm291–1,840 (FLAG-Ecm29FL) and plasmids encoding 190AnkG-GFP (AnkG-GFP) or control GFP vectors, as indicated. Cell lysates were IP by FLAG M2 antibodies and blotted with indicated antibodies. Histograms at right show relative protein levels based on immunoblotting of GFP, Arp1, Rpt1, and KLC1 coIP by FLAG antibodies (±SEM; n = 4 independent experiments; *, P < 0.05; ns, not significant, by t test). (B) cAMP increases facilitate Ecm29–AnkG interaction. (B1) Schematic showing design of AIS-mimicking ratiometric FRET sensors. AIS molecular dock: NF-186. Donor molecule: 190AnkG-GFP. Acceptor molecule: Ecm29ΔC-mCherry or Ecm29ΔN-mCherry. (B2) Time-lapse FRET images of HEK293T cells cotransfected with plasmids encoding 190AnkG-GFP, NF-186-HA, and N- or C-terminal Ecm29-mCherry (Ecm29ΔC-mCherry or Ecm29ΔN-mCherry) in the presence of 20 µM forskolin, with or without 30 min of pretreatment with the PKA inhibitor KT5720, as indicated. White dashed line passes through the center of the cell, with points a and b as intersection points. Scale bar, 10 µm. (B3) Quantitative measurement of FRET signals (±SEM, n ≥ 13 cells for each group) at different times before and after addition of forskolin (20 µM) in experiments as described in B. Arrow in right panel denotes forskolin addition. Norm., normalized. (C) Ecm29 expression is required for proteasome association with the detergent-resistant AIS structure. (C1) Representative images of 7-DIV hippocampal neurons transfected with SC-shRNA or AnkG-targeting siRNA (AnkG-siRNA) at 3 DIV, with or without nocodazole (Noco; 1 µM for 3 h) or latrunculin A (Lat A; 1 µM for 6 h), followed by detergent extraction with 0.1% Triton X-100 at 37°C for 2 min before immunostaining for the AIS marker AnkG/Trim46 (red, merge panels), proteasome subunit Rpt1/Rpt5 (green, merge panels), and the proteasome adaptor Ecm29 (green, merge panels). (C2) Quantitative measurement of immunostaining intensities (±SEM, n = 15–35 cells) along AIS segments. (D) Local protein turnover at the AIS requires Ecm29 expression and proteasome activity. (D1) Images of 7-DIV hippocampal neurons expressing an AIS-located protein degradation reporter, NavII-III–tagged GFPu, showing more durable GFPu accumulation after MG132 pretreatment (2.5 µM, 30 min) or shRNA-mediated knockdown of Rpt-1 or Ecm29 expression, as indicated. Scale bar, 15 µm. (D2) Quantitative measurement of fluorescence intensities in the AIS (top panel) and soma (bottom panel) at different time points based on all experiments performed as described in D1 (±SEM, n > 6 cells for each group, one-way ANOVA followed by Dunnett’s multiple comparison test. *, P < 0.05; ns, not significant).

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal