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Flotillin-1 is essential for PKC-triggered endocytosis and membrane microdomain localization of DAT.

Cremona ML, Matthies HJ, Pau K, Bowton E, Speed N, Lute BJ, Anderson M, Sen N, Robertson SD, Vaughan RA, Rothman JE, Galli A, Javitch JA, Yamamoto A - Nat. Neurosci. (2011)

Bottom Line: Stimuli including protein kinase C (PKC) activation can lead to the internalization of some NTTs and a reduction in neurotransmitter clearance capacity.We found that the protein Flotillin-1 (Flot1), also known as Reggie-2, was required for PKC-regulated internalization of members of two different NTT families, the DA transporter (DAT) and the glial glutamate transporter EAAT2, and we identified a conserved serine residue in Flot1 that is essential for transporter internalization.In sum, our findings provide evidence for a critical role of Flot1-enriched membrane microdomains in PKC-triggered DAT endocytosis and the actions of amphetamine.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Columbia University, College of Physicians and Surgeons, New York, New York, USA.

ABSTRACT
Plasmalemmal neurotransmitter transporters (NTTs) regulate the level of neurotransmitters, such as dopamine (DA) and glutamate, after their release at brain synapses. Stimuli including protein kinase C (PKC) activation can lead to the internalization of some NTTs and a reduction in neurotransmitter clearance capacity. We found that the protein Flotillin-1 (Flot1), also known as Reggie-2, was required for PKC-regulated internalization of members of two different NTT families, the DA transporter (DAT) and the glial glutamate transporter EAAT2, and we identified a conserved serine residue in Flot1 that is essential for transporter internalization. Further analysis revealed that Flot1 was also required to localize DAT within plasma membrane microdomains in stable cell lines, and was essential for amphetamine-induced reverse transport of DA in neurons but not for DA uptake. In sum, our findings provide evidence for a critical role of Flot1-enriched membrane microdomains in PKC-triggered DAT endocytosis and the actions of amphetamine.

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PKC triggers endocytosis of heterologously and endogenously expressed DATa,b. EM4-YFP-DAT internalizes (white arrows) into EEA1-positive vesicles after exposure to 0.1 µM PMA for 30 min. Cells exposed to PMA were fixed and immunostained for EEA1 as described. Alexa-Fluor 633 labeled secondary antibodies were used to prevent overlap with YFP. Scale bar = 10 µm. c. Endogenous DAT internalizes in response to PMA in primary dopaminergic neurons. Midbrain cultures were treated with vehicle (Ctrl, n = 32) or 1 µM PMA (+PMA, n = 35) for 30 min, fixed and immunostained for DAT (intracellular epitope, Chemicon), and Alexa-Fluor 568 secondary. PMA led to significant internalization of DAT (‘Internalization Index,’ as described in Methods, Ctrl: 0.076±0.013; vs. PMA: 0.309±0.052). (One-way ANOVA; p < 0.001). Scale bar = 20 µm. d. Internalization of endogenous DAT in striatal slice preparations (n = 6). Slices were treated with 10 µM PMA or Ctrl for 1 hr, then cell surface biotinylated to determine DAT surface levels. ‘DAT expression levels (% Surface)’ indicates the relative amount of DAT at the cell surface, and was calculated as the ratio between the integrated densities of the surface levels of DAT after treatment (corrected for total levels of DAT) to the surface levels of DAT before treatment (corrected for total levels of DAT). Bars represent Mean + St. Dev. PMA treatment significantly decreased DAT surface levels (p = 0.0330). Complete blots can be found in Figure S9.
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Figure 1: PKC triggers endocytosis of heterologously and endogenously expressed DATa,b. EM4-YFP-DAT internalizes (white arrows) into EEA1-positive vesicles after exposure to 0.1 µM PMA for 30 min. Cells exposed to PMA were fixed and immunostained for EEA1 as described. Alexa-Fluor 633 labeled secondary antibodies were used to prevent overlap with YFP. Scale bar = 10 µm. c. Endogenous DAT internalizes in response to PMA in primary dopaminergic neurons. Midbrain cultures were treated with vehicle (Ctrl, n = 32) or 1 µM PMA (+PMA, n = 35) for 30 min, fixed and immunostained for DAT (intracellular epitope, Chemicon), and Alexa-Fluor 568 secondary. PMA led to significant internalization of DAT (‘Internalization Index,’ as described in Methods, Ctrl: 0.076±0.013; vs. PMA: 0.309±0.052). (One-way ANOVA; p < 0.001). Scale bar = 20 µm. d. Internalization of endogenous DAT in striatal slice preparations (n = 6). Slices were treated with 10 µM PMA or Ctrl for 1 hr, then cell surface biotinylated to determine DAT surface levels. ‘DAT expression levels (% Surface)’ indicates the relative amount of DAT at the cell surface, and was calculated as the ratio between the integrated densities of the surface levels of DAT after treatment (corrected for total levels of DAT) to the surface levels of DAT before treatment (corrected for total levels of DAT). Bars represent Mean + St. Dev. PMA treatment significantly decreased DAT surface levels (p = 0.0330). Complete blots can be found in Figure S9.

Mentions: Studies using heterologous systems have shown that DAT and EAAT2 rapidly internalize into endocytic structures in response to activation of PKC by the phorbol ester PMA1–6. As shown by both confocal microscopy and cell surface biotinylation, HEK293-derived EM4 cells stably expressing eYFP-tagged DAT (EM4-YFP-DAT)2 or HeLa cell lines stably expressing monomeric YFP24 (mYFP)-tagged DAT (HeLa-mYFP-DAT) or eGFP-tagged EAAT2 (HeLa-eGFP-ET2) demonstrate robust and rapid internalization of the transporter into early endosomal antigen 1 (EEA1)-positive vesicles in response to a 30 min exposure of PMA (Fig.1a–b, S1). It has previously been shown that the murine homolog of EAAT2, GLT-1, internalizes in response to PMA in neuronal-astrocyte co-cultures4, 6. To ascertain if this PKC-triggered event also is observed with endogenous DAT, we exposed primary dopaminergic neurons to 1 µM PMA (direct PKC activation) for 30 min and immunostained for DAT. Confocal imaging revealed that PKC activation led to significant redistribution of endogenous DAT to an internal compartment in primary neurons (Fig.1c). Consistent with these findings, PMA treatment of mouse striatal slices led to a significant internalization of endogenous DAT, as shown by cell-surface biotinylation (Fig.1d). Thus, PKC-triggered internalization of DAT is evident not only in a heterologous system but also in primary neurons and brain slice preparations expressing endogenous DAT.


Flotillin-1 is essential for PKC-triggered endocytosis and membrane microdomain localization of DAT.

Cremona ML, Matthies HJ, Pau K, Bowton E, Speed N, Lute BJ, Anderson M, Sen N, Robertson SD, Vaughan RA, Rothman JE, Galli A, Javitch JA, Yamamoto A - Nat. Neurosci. (2011)

PKC triggers endocytosis of heterologously and endogenously expressed DATa,b. EM4-YFP-DAT internalizes (white arrows) into EEA1-positive vesicles after exposure to 0.1 µM PMA for 30 min. Cells exposed to PMA were fixed and immunostained for EEA1 as described. Alexa-Fluor 633 labeled secondary antibodies were used to prevent overlap with YFP. Scale bar = 10 µm. c. Endogenous DAT internalizes in response to PMA in primary dopaminergic neurons. Midbrain cultures were treated with vehicle (Ctrl, n = 32) or 1 µM PMA (+PMA, n = 35) for 30 min, fixed and immunostained for DAT (intracellular epitope, Chemicon), and Alexa-Fluor 568 secondary. PMA led to significant internalization of DAT (‘Internalization Index,’ as described in Methods, Ctrl: 0.076±0.013; vs. PMA: 0.309±0.052). (One-way ANOVA; p < 0.001). Scale bar = 20 µm. d. Internalization of endogenous DAT in striatal slice preparations (n = 6). Slices were treated with 10 µM PMA or Ctrl for 1 hr, then cell surface biotinylated to determine DAT surface levels. ‘DAT expression levels (% Surface)’ indicates the relative amount of DAT at the cell surface, and was calculated as the ratio between the integrated densities of the surface levels of DAT after treatment (corrected for total levels of DAT) to the surface levels of DAT before treatment (corrected for total levels of DAT). Bars represent Mean + St. Dev. PMA treatment significantly decreased DAT surface levels (p = 0.0330). Complete blots can be found in Figure S9.
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Figure 1: PKC triggers endocytosis of heterologously and endogenously expressed DATa,b. EM4-YFP-DAT internalizes (white arrows) into EEA1-positive vesicles after exposure to 0.1 µM PMA for 30 min. Cells exposed to PMA were fixed and immunostained for EEA1 as described. Alexa-Fluor 633 labeled secondary antibodies were used to prevent overlap with YFP. Scale bar = 10 µm. c. Endogenous DAT internalizes in response to PMA in primary dopaminergic neurons. Midbrain cultures were treated with vehicle (Ctrl, n = 32) or 1 µM PMA (+PMA, n = 35) for 30 min, fixed and immunostained for DAT (intracellular epitope, Chemicon), and Alexa-Fluor 568 secondary. PMA led to significant internalization of DAT (‘Internalization Index,’ as described in Methods, Ctrl: 0.076±0.013; vs. PMA: 0.309±0.052). (One-way ANOVA; p < 0.001). Scale bar = 20 µm. d. Internalization of endogenous DAT in striatal slice preparations (n = 6). Slices were treated with 10 µM PMA or Ctrl for 1 hr, then cell surface biotinylated to determine DAT surface levels. ‘DAT expression levels (% Surface)’ indicates the relative amount of DAT at the cell surface, and was calculated as the ratio between the integrated densities of the surface levels of DAT after treatment (corrected for total levels of DAT) to the surface levels of DAT before treatment (corrected for total levels of DAT). Bars represent Mean + St. Dev. PMA treatment significantly decreased DAT surface levels (p = 0.0330). Complete blots can be found in Figure S9.
Mentions: Studies using heterologous systems have shown that DAT and EAAT2 rapidly internalize into endocytic structures in response to activation of PKC by the phorbol ester PMA1–6. As shown by both confocal microscopy and cell surface biotinylation, HEK293-derived EM4 cells stably expressing eYFP-tagged DAT (EM4-YFP-DAT)2 or HeLa cell lines stably expressing monomeric YFP24 (mYFP)-tagged DAT (HeLa-mYFP-DAT) or eGFP-tagged EAAT2 (HeLa-eGFP-ET2) demonstrate robust and rapid internalization of the transporter into early endosomal antigen 1 (EEA1)-positive vesicles in response to a 30 min exposure of PMA (Fig.1a–b, S1). It has previously been shown that the murine homolog of EAAT2, GLT-1, internalizes in response to PMA in neuronal-astrocyte co-cultures4, 6. To ascertain if this PKC-triggered event also is observed with endogenous DAT, we exposed primary dopaminergic neurons to 1 µM PMA (direct PKC activation) for 30 min and immunostained for DAT. Confocal imaging revealed that PKC activation led to significant redistribution of endogenous DAT to an internal compartment in primary neurons (Fig.1c). Consistent with these findings, PMA treatment of mouse striatal slices led to a significant internalization of endogenous DAT, as shown by cell-surface biotinylation (Fig.1d). Thus, PKC-triggered internalization of DAT is evident not only in a heterologous system but also in primary neurons and brain slice preparations expressing endogenous DAT.

Bottom Line: Stimuli including protein kinase C (PKC) activation can lead to the internalization of some NTTs and a reduction in neurotransmitter clearance capacity.We found that the protein Flotillin-1 (Flot1), also known as Reggie-2, was required for PKC-regulated internalization of members of two different NTT families, the DA transporter (DAT) and the glial glutamate transporter EAAT2, and we identified a conserved serine residue in Flot1 that is essential for transporter internalization.In sum, our findings provide evidence for a critical role of Flot1-enriched membrane microdomains in PKC-triggered DAT endocytosis and the actions of amphetamine.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Columbia University, College of Physicians and Surgeons, New York, New York, USA.

ABSTRACT
Plasmalemmal neurotransmitter transporters (NTTs) regulate the level of neurotransmitters, such as dopamine (DA) and glutamate, after their release at brain synapses. Stimuli including protein kinase C (PKC) activation can lead to the internalization of some NTTs and a reduction in neurotransmitter clearance capacity. We found that the protein Flotillin-1 (Flot1), also known as Reggie-2, was required for PKC-regulated internalization of members of two different NTT families, the DA transporter (DAT) and the glial glutamate transporter EAAT2, and we identified a conserved serine residue in Flot1 that is essential for transporter internalization. Further analysis revealed that Flot1 was also required to localize DAT within plasma membrane microdomains in stable cell lines, and was essential for amphetamine-induced reverse transport of DA in neurons but not for DA uptake. In sum, our findings provide evidence for a critical role of Flot1-enriched membrane microdomains in PKC-triggered DAT endocytosis and the actions of amphetamine.

Show MeSH
Related in: MedlinePlus