Article Figures & Data
Figures
- Figure 1.
CIE of AMPARs. A, Neurons were incubated with Tf-555 and anti-GluA2 antibody for the indicated times, fixed, permeabilized, and stained for iGluA2. Arrows denote overlapping puncta visible at 20 min, but not 5 min, of internalization. n = 3. B, C, Neurons were transfected with CHC shRNA or an empty GFP vector and live labeled with Tf-555 or anti-GluA1 for 30 min 4 d after transfection. Control, Empty GFP vector. CHC shRNA blocks uptake of Tf-555 but not iGluA1. Arrows denote puncta of internalized label. n = 4. D, As above but with AP180Cmyc construct. Control, Untransfected cells. n = 3. E, Treatment with 20 μm Pitstop 2 blocks Tf-555 but not iGluA1. n = 4. Control, 0 μm Pitstop2 (DMSO). Scale bars, 10 μm.
- Figure 2.
Dynamin-independent internalization of AMPARs. A, Neurons were transfected with Dyn2wt–GFP or Dyn2K44A–GFP and labeled with Tf-555 or anti-GluA1 2 d after transfection. Mutant Dyn2 reduces Tf-555 but not iGluA1. Control, Dyn2wt-expressing cells. n = 4. B, Overexpression of dynamin 1 and 3 mutants has no effect on iGluA1. Control, Dyn1wt and Dyn3wt-expressing cells, respectively. n = 3 each. C, Neurons were pretreated with DMSO (veh) or 75 μm dynasore (ds) for 10 min and allowed to endocytose Tf-555, anti-GluA1, or anti-GluA2 for 30 min in the presence of dynasore. Ds blocks Tf-555 but not iGluA1 or iGluA2. Control, Vehicle-treated samples. n = 4. D, Surface biotinylation-based assay for internalization in cortical neurons. Total, Total protein levels in neuronal lysate; Eluate, protein levels eluted from streptavidin-conjugated beads; No strip, nonstripped control; 0′, 0 time (no endocytosis) control. Ds blocks internalization of TfR but not of GluA2. Control, Vehicle-treated samples. n = 3. E, Neurons expressing Dyn2wt–GFP or Dyn2K44A–GFP were live labeled with anti-GluA1, fixed, and stained for iGluA1 and either EEA1 or TfR. iGluA1 localizes to EEA1- and TfR-positive puncta (arrows) in both cells expressing wild-type and mutant dynamin. F, Neurons expressing a dominant-negative Rab5Q79L–GFP construct accumulate AMPARs in GFP-positive enlarged endosomes (arrows). Scale bars, 20 μm.
- Figure 3.
CME is required for NMDAR–LTD but not for basal synaptic transmission or synaptic downscaling. A, Treatment with dynasore does not affect basal excitatory synaptic transmission. Time course of EPSC amplitude evoked at 0.2 Hz at Schaffer collateral–CA1 synapses after infusion of control (n = 6), 100 μm dynasore (Ds; n = 8), vehicle (Veh, 0.1% DMSO; n = 6), or 50 μm QVPSRPNRAP (DIP; n = 6) by inclusion in the intracellular solution. B, The pairing protocol effectively induces LTD with control intracellular solution (Control; n = 12) but intracellular infusion of dynasore (n = 9) inhibits LTD in the test pathway. Right, Representative current traces from time points shown (1, black: 1 min before pairing; 2, gray: 25 min after pairing). C, Application of 50 μm myristoylated DIP (myrDIP) blocks AMPAR internalization in the live-labeling assay. Control, Vehicle-treated samples. n = 3. D, Overexpression of the Dyn2–K44A construct does not affect homeostatic downscaling. Hippocampal neurons (12–14 d in vitro) transfected with Dyn2wt–GFP or Dyn2K44A–GFP were treated with 15 mm KCl and 40 μm bicuculline for 48 h (Scaled) and labeled to quantify sGluA1 and sGluA2. Control, Dyn2wt-expressing cells under control conditions. n = 3. Scale bar, 20 μm.
- Figure 4.
Nonstructural F-actin regulates AMPAR trafficking. A, Pharmacological inhibition of Rac1 inhibits AMPAR trafficking. Neurons were treated overnight with vehicle (DMSO) or 100 μm NSC23766, and sGluA2 and iGluA2 levels were quantified. Control, DMSO. n = 4. B, Treatment with 0.5 μm LatA does not affect F-actin content but upregulates AMPAR trafficking. Neurons were treated overnight with vehicle (DMSO) or 5 or 0.5 μm LatA, and sGluA2, iGluA2, and F-actin levels were quantified. Phalloidin–Alexa Fluor 568 was used to label F-actin. Control, DMSO. n = 6 (sGluA2 and iGluA2), n = 3 (F-actin). C, Stabilization of F-actin by Jasplakinolide downregulates AMPAR trafficking. Neurons were treated overnight with vehicle (DMSO) or 1 μm Jasplakinolide (Jas) and iGluA2 levels were quantified. Control, DMSO. n = 4. D, Treatment with 0.5 μm LatA increases bulk membrane uptake but not CME. Neurons (14–21 d in vitro) were treated as in B and then incubated with either FM1-43fx or Tf-555 for 30 min. Control, DMSO. n = 3. E, LatA at 0.5 μm disrupts synaptic downscaling of GluA2. Hippocampal neurons were treated with 15 mm KCl and 40 μm bicuculline for 48 h (Scaled) in the presence of 0.5 μm LatA, and sGluA2 levels were quantified. Control, DMSO-treated cells under control conditions. n = 3. Scale bars, 20 μm.
