Optogenetically-induced multimerization of the dopamine transporter increases uptake and trafficking to the plasma membrane

Abstract

The dopamine transporter (DAT) is essential for the reuptake of the released neurotransmitter dopamine (DA) in the brain. Psychostimulants, methamphetamine and cocaine, have been reported to induce the formation of DAT multimeric complexes in vivo and in vitro. The interpretation of DAT multimer function has been primarily in the context of compounds that induce structural and functional modifications of the DAT, complicating the understanding of the significance of DAT multimers. To examine multimerization in the absence of DAT ligands as well as in their presence, we developed a novel, optogenetic fusion chimera of cryptochrome 2 and DAT with an mCherry fluorescent reporter (Cry2-DAT). Using blue light to induce Cry2-DAT multimeric protein complex formation, we were able to simultaneously test the functional contributions of DAT multimerization in the absence or presence of substrates or inhibitors with high spatiotemporal precision. We found that blue light-stimulated Cry2-DAT multimers significantly increased IDT307 uptake and MFZ 9-18 binding in the absence of ligands as well as after methamphetamine and nomifensine treatment. Blue light-induced Cry2-DAT multimerization increased colocalization with recycling endosomal marker Rab11 and had decreased presence in Rab5-positive early endosomes and Rab7-positive late endosomes. Our data suggest that the increased uptake and binding results from induced and rapid trafficking of DAT multimers to the plasma membrane. Our data suggest that DAT multimers may function to help maintain DA homeostasis.

Keywords: cryptochrome 2 (Cry2); dopamine transporter (DAT); methamphetamine (METH); multimer; oligomer; optogenetics.

Figure 1

The Novel Cry2–DAT optogenetic construct encodes a DAT with normal cell-surface expression and uptake activity.A, YFP–DAT and Cry2–DAT construct maps display the DAT location in respect to YFP or Cry2PHR–mCh. The cartoons to the right of the construct maps demonstrate the N-terminal location of the YFP and Cry2–mCherry. For the Cry2–DAT construct, the location of mCherry is the same as YFP, whereas Cry2 is N-terminal to mCherry. B, confocal images of live HEK293 cells expressing the Cry2–mCh empty vector show a cytoplasmic localization. Live-cell confocal imaging of HEK293 cells expressing Cry2–DAT localizes primarily to the plasma membrane. C, YFP–DAT and Cry2–DAT coexpressing HEK293 cells show strong colocalization with a Pearson’s correlation coefficient of 0.87. D, the Cry2–mCh vector, FLAG–DAT, or Cry2–DAT was expressed in HEK293 cells before imaging IDT307 uptake. The Cry2–mCh empty vector has minimal uptake of IDT307. Cry2–DAT and FLAG–DAT have the same uptake capacity for IDT307 (p < 0.0001, n = 19). Significance was determined using one-way ANOVA with Tukey’s multiple comparisons test. The scale bar represents 5 μm. Cry2, cryptochrome 2; DAT, dopamine transporter; mCh, mCherry.

Figure 2

Light-induced Cry2–DAT multimers mimic DA- and METH-induced YFP–DAT multimers.A, HEK cells stably expressing YFP–DAT were treated with 10 μM DA or (B) 10 μM METH for the indicated times. After DA or METH treatment, CuP was used to crosslink the DAT to retain multimer bands. The Mem-PER Plus Membrane Protein Extraction kit was used to isolate the plasma membrane DAT. Membrane fractions were run on an 8% polyacrylamide gel. Data shown in panels A and B are representative Western blots showing time-dependent formation of DAT multimers from treatment with DA or METH. C, HEK cells were transfected with Cry2–DAT and incubated in the dark (control) or exposed to blue light for 30 s. Cry2–DAT biotinylated samples were then run on a 4–15% gradient polyacrylamide gel. D, a representative Western blot of cell surface biotinylation of Cry2–DAT with and without blue light exposure shows DAT multimer formation after exposure to blue light for 30 s. CuP, copper phenanthroline; Cry2, cryptochrome 2; Cry2–DAT, cryptochrome 2 and DAT with an mCherry fluorescent reporter; DA, dopamine; DAT, dopamine transporter; METH, methamphetamine.

Figure 3

YFP–DAT can be kinetically trapped in Cry2–DAT multimers.A, the schematic depicts the incorporation of YFP–DAT into Cry2–DAT multimers after blue light stimulation. B, Cry2–DAT or CFP–DAT was transfected into HEK cells stably expressing YFP–DAT. FRET for CFP–DAT and YFP–DAT was used as a basis of comparison to test if blue light multimerization of Cry2–DAT could kinetically trap YFP–DAT into Cry2–DAT multimers by measuring FRET efficiencies. The increases in FRET efficiencies for YFP–DAT and Cry2–DAT compared with CFP–DAT and YFP–DAT suggest that YFP–DAT is incorporated into Cry2–DAT multimers (n = 3–5, 0.001 > p < 0.05). One-way ANOVA with Tukey’s multiple comparisons test was applied to determine significance of data. C and D, the lateral mobility of YFP–DAT was tested in the absence or presence of Cry2–DAT to determine if Cry2–DAT multimers would incorporate YFP–DAT to impact its (C) diffusion coefficient (D) and (D) mobile fraction (M_f). HEK cells stably expressing YFP–DAT alone or transfected with Cry2–DAT were used for FRAP. A circular ROI with a 5-μm diameter was placed at the plasma membrane for photobleaching. (n = 8–11, 0.05 > p < 0.001). Mixed-effects analysis with Tukey’s multiple comparison test was used to determine significance of data. Cry2, cryptochrome 2; Cry2–DAT, cryptochrome 2 and DAT with an mCherry fluorescent reporter; DAT, dopamine transporter; FRAP, fluorescence recovery after photobleaching; ROI, region of interest.

Figure 4

Blue light–stimulated Cry2–DAT multimers have increased uptake activity and an outward-facing conformation at the plasma membrane. HEK cells stably expressing FLAG–DAT were used to as a comparison for Cry2–DAT uptake in HEK cells transiently transfected with Cry2–DAT. A, FLAG–DAT or (B) Cry2–DAT uptake of IDT307 was evaluated after pretreatment with vehicle, 10 μM METH, or 10 μM NOM. Where indicated, cells were also exposed to 488-nm light (n = 24–30, 0.05 > p < 0.001). C, FLAG–DAT or (D) Cry2–DAT [3H]-DA-uptake assay was evaluated after pretreatment with the vehicle, 10 μM METH, or 10 μM NOM. Where indicated, cells were also exposed to 488-nm light (n = 30–40, 0.05 > p < 0.001). E, FLAG–DAT or (F) Cry2–DAT binding of MFZ 9-18 was evaluated after pretreatment with vehicle, 10 μM METH, or 10 μM NOM. Where indicated, cells were also exposed to 488-nm light (n = 30–40, 0.05 > p < 0.001). Cry2, cryptochrome 2; Cry2–DAT, cryptochrome 2 and DAT with an mCherry fluorescent reporter; DAT, dopamine transporter; METH, methamphetamine; NOM, nomifensine.

Figure 5

Blue light-stimulated multimerization of Cry2–DAT induces rapid trafficking to the plasma membrane.A, representative images from live confocal time lapse imaging before and after 488-nm light pulse for 30 s. After the 488-nm light pulse, Cry2–DAT showed an increased membrane expression (yellow arrows) and reduced in intracellular pools of Cry2–DAT (white arrows). B, representative cell-surface biotinylation Western blot of Cry2–DAT–expressing HEK cells. C, analysis of immunoreactivity of biotinylated Cry2–DAT Western blots in panel B show a statistically significant decrease in the cell-surface DAT after treatment with 10 μM METH or 10 μM NOM. A statistically significant increase in the cell-surface DAT was detected after the 488-nm light pulse and METH treatment (n = 3, ∗0.05 > p < 0.0001∗∗). D, HEK cells transfected with Cry2–DAT were treated with cytochalasin D to inhibit actin polymerization. The cells were then with treated with the vehicle, 10 μM METH, 10 μM NOM, or blue light as indicated (n = 5–9, p < 0.0001). E, IDT307 uptake shows that 488-nm pulse before 10 μM METH or 200 nM PMA treatment does not inhibit downregulation of multimerized Cry2–DAT. IDT307 uptake is reduced when 488-nm pulse is followed with 10 μM METH or 200 nM PMA treatment (n = 15, p ≤ 0.0001). Cry2, cryptochrome 2; Cry2–DAT, cryptochrome 2 and DAT with an mCherry fluorescent reporter; DAT, dopamine transporter; METH, methamphetamine; NOM, nomifensine; PMA, phorbol 12-mristate 13-acetate.

Figure 6

Blue light–multimerized Cry2–DAT colocalizes with Rab11. HEK cells were transfected with Cry2–DAT and treated with vehicle or 10 μM METH and exposed with 488-nm light where indicated. A, Cry2–DAT (red) and Rab5 (green), (B) Rab11 (green), and (C) Rab7 (green) was used to test colocalization (yellow) with 10 μM METH treatment and a 488-nm light pulse. D, Pearson’s correlation coefficient was used to determine the extent of colocalization of Cry2–DAT with Rab5, (E) Rab11, and (F) Rab7 (n = 7–10). The scale bar represents 10 μm. Cry2–DAT, cryptochrome 2 and DAT with an mCherry fluorescent reporter; METH, methamphetamine.

Figure 7

Blue light stimulation induces exocytic trafficking.A, HEK cells were transfected with Cry2–DAT, and IDT307 uptake was measured in cells pretreated with monensin (2.5 μM), dynasore (80 μM), and nocodazole (2 μM) before 488-nM blue light pulse. (n = 12–28, 0.05 > p < 0.01). B, HEK cells were transfected with Cry2–DAT, and IDT307 uptake was measured after pretreatment with calphostin C (1 μM), bafilomycin A1 (0.06 nM), and LY333531 (0.6 nM) and exposed to a 488-nm light pulse where indicated. (n = 11–28, 0.05 > p < 0.01). C, HEK cells were transfected with Cry2–DAT, and MFZ 9-18 binding was measured after pretreatment with METH, and PMA followed by LY333531 (0.6 nM), and exposed to a 488-nm light pulse where indicated (n = 12–28, 0.05 > p < 0.01). D, HEK cells were transfected with Cry2–DAT and treated with the vehicle or 10 μM METH and exposed with 488-nm light where indicated. Pearson’s correlation coefficient was used to determine the extent of colocalization of Cry2–DAT with Rab11 with pretreatment of LY333531 (0.6 nM), exposed to 488-nm light for 30 s, or allowed to incubate for 30 min after treatment as indicated (n = 6–10). E, representative images of Cry2–DAT (red) and Rab11 (green) was used to test colocalization (yellow) with pretreatment of LY333531 (0.6 nM), exposed to 488-nm light for 30 s, or allowed to incubate for 30 min after treatment as indicated. The scale bar represents 10 μm. Cry2–DAT, cryptochrome 2 and DAT with an mCherry fluorescent reporter; METH, methamphetamine; n.s., not significant; PMA, phorbol 12-mristate 13-acetate.

Figure 8

Novel Cry2–DAT–multimerized uptake and trafficking mechanisms are confirmed in SHSY-5Y differentiated cells.A, immunofluorescence staining of differentiated SH-SY5Y cells show the endogenous DAT (green) and Cry2–DAT (red) colocalization. B, uptake of IDT307 was measured in differentiated SH-SY5Y cells treated with vehicle or 10 μM METH and exposed to a 488-nm light pulse where indicated (n = 9–10, 0.01> p < 0.001). C, uptake of IDT307 was measured in differentiated SH-SY5Y cells expressing Cry2–DAT and treated with vehicle or 10 μM METH and exposed to a 488-nm light pulse where indicated (n = 9–10, 0.01 > p < 0.001). D, uptake of IDT307 was measured in differentiated SH-SY5Y cells expressing Cry2–DAT and treated with vehicle or 10 μM METH followed by LY333531 exposed to a 488-nm light pulse where indicated (n = 9–10, 0.01 > p < 0.001). E, differentiated SH-SY5Y cells were transfected with Cry2–DAT (red) and imaged for colocalization with Rab5 (green) and (G) Rab11 (green). F, Pearson’s correlation coefficient was used to determine the extent of colocalization of Cry2–DAT with Rab5 and (H) Rab11 (n = 5, p < 0.0001). The scale bar represents 10 μm. Cry2–DAT, cryptochrome 2 and DAT with an mCherry fluorescent reporter; METH, methamphetamine.

Figure 9

Proposed mechanism for Cry2–DAT multimer–induced increase in uptake and binding. Cry2–DAT constitutively traffics to Rab5 early endosomes, Rab11 recycling endosomes, and back to the plasma membrane. Treatment with 10 μM METH or 200 nM PMA induces Cry2–DAT downregulation from the plasma membrane to traffic to Rab5- and Rab7-associated endosomes. Light stimulation (488 nm) of Cry2–DAT mobilizes Cry2–DAT to traffic from Rab11 endosomes to the plasma membrane to increase Cry2–DAT uptake and binding. This can be impeded by inhibiting the maturation of recycling endosomes (using Cal C or Baf A1) or inhibiting PKCβ. Baf A1, bafilomycin A1; Cal C, calphostin C; Cry2–DAT, cryptochrome 2 and DAT with an mCherry fluorescent reporter; METH, methamphetamine; PMA, phorbol 12-mristate 13-acetate.