Figure 10. AIM-100-induced DAT endocytosis is independent of Ack1 and clathrin.

(A-D) PAE/YFP-HA-DAT cells were transfected twice with non-targeting (NT), clathrin heavy chain (CHC) or Ack1 siRNAs. After 3–5 days, the cells were lysed and tested for the efficiency of knock-downs (A) or used for microscopy imaging (B-D). Asterisk in (A) indicates non-specific band recognized by Ack1 antibodies. (B) Cells were incubated with vehicle (DMSO) for 2 hr at 37°C in the presence of 5 µg/ml Tfn-TxR. 3D images were acquired through 488 nm (YFP) and 561 nm (TxR) channels. Merged images of maximal intensity projections of 3D images are shown. Insets show corresponding Tfn-TxR images to better demonstrate inhibition of Tfn-TxR internalization in CHC-depleted cells. Corresponding full-size images are presented in Figure 10—figure supplement 1A. (C) Cells incubated with 20 µM AIM-100 for 2 hr at 37°C in the presence of 5 µg/ml Tfn-TxR, were imaged as in (B). Merged images of YFP-HA-DAT and Tfn-TxR are presented. Insets show high magnification of the regions marked by white rectangle to demonstrate co-localization of Tfn-TxR and YFP-HA-DAT. (D) Quantification of co-localization of YFP-HA-DAT with Tfn-TxR in experiments represented in (B) and (C). Results are presented as mean values of the fraction of YFP-HA-DAT co-localized with Tfn-TxR (±SD; n = 3-7). P values are ‘AIM-100’ compared to ‘vehicle’. (E) Cells were incubated with 20 µM AIM-100, vehicle (DMSO) or 20 µM KRCA-0008 for 2 hr at 37°C, fixed, and 3D images of YFP were acquired. Maximal intensity projection images are presented. Scale bars, 10 µm.

Figure 10—figure supplement 1. Endocytosis of Tfn-TxR in the presence of AIM-100 in cells depleted of Ack1 or CHC.

(A) Single-channel images of Tfn-TxR (561 filter channel; maximal intensity projections of 3D images) corresponding to the merged images shown in Figure 10B and C are presented. Scale bars, 10 µm. (B) Quantification of the amount of vesicular (endosomal) Tfn-TxR in 3D images from the experiment presented in (A). Bars represent mean value of arbitrary linear units of fluorescence intensity (a.l.u.f.i.) of Tfn-TxR (±SD; n = 3–4). P value is calculated for ‘vehicle’ versus ‘AIM-100’. The experiment is representative of at least five independent experiments.

Figure 10—figure supplement 2. AIM-100-induced DAT oligomerization is not induced by KRCA-0008 and not affected by CHC knockdown.

(A) PAE/YFP-HA-DAT cells were incubated with vehicle (DMSO), 20 µM AIM-100, 10 µM cocaine plus 20 µM AIM-100 or 20 µM KRCA-0008 for 2 hr at 37°C, lysed, and YFP-HA-DAT was detected by blotting with the GFP antibody. M, monomers; O, oligomers. (B) PAE/YFP-HA-DAT cells were transfected twice with non-targeting (NT) or clathrin heavy chain (CHC). After 5 days, the cells were incubated with DMSO or AIM-100 as in (A), lysed, and probed for DAT oligomerization (GFP antibody) and the efficiency of knock-downs (CHC antibody). All lanes are from the same blot. M, monomers; O, oligomers.

Figure 10—figure supplement 3. Endosomal accumulation of DAT in the presence of monensin is partically clathrin-dependent.

(A) PAE/YFP-HA-DAT cells were transfected with non-targeting (NT) or clathrin heavy chain (CHC) siRNAs. After 3 days, the cells were incubated with HA11 for 30 min at 37°C, and then further incubated with vehicle (DMSO) or 25 µM monensin for 60 min at 37°C. After fixation, cultures were stained with secondary anti-mouse antibodies conjugated with Cy5 (surface YFP-HA-DAT), permeabilized with Triton X-100 and stained with secondary anti-mouse conjugated with Cy3 (internalized YFP-HA-DAT). 3D images were acquired through 488 (YFP, not shown), 561 (Cy3, red) and 640 nm (Cy5, cyan) channels. Individual confocal sections are shown. Scale bars, 10 µm. (B) The Cy3/Cy5 ratio was calculated in HA11 uptake experiments exemplified in (A). Results are presented as mean values (±SD, n = 5-6).