Quantum dots reveal heterogeneous membrane diffusivity and dynamic surface density polarization of dopamine transporter

Autoři: Oleg Kovtun aff001;  Ian D. Tomlinson aff001;  Riley S. Ferguson aff001;  Sandra J. Rosenthal aff001
Působiště autorů: Department of Chemistry, Vanderbilt University, Nashville, Tennessee, United States of America aff001;  Vanderbilt Institute of Nanoscale Science and Engineering, Vanderbilt University, Nashville, Tennessee, United States of America aff002;  Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, United States of America aff003;  Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, Tennessee, United States of America aff004;  Department of Physics and Astronomy, Vanderbilt University, Nashville, Tennessee, United States of America aff005;  Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, Tennessee, United States of America aff006
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0225339


The presynaptic dopamine transporter mediates rapid reuptake of synaptic dopamine. Although cell surface DAT trafficking recently emerged as an important component of DAT regulation, it has not been systematically investigated. Here, we apply our single quantum dot (Qdot) tracking approach to monitor DAT plasma membrane dynamics in several heterologous expression cell hosts with nanometer localization accuracy. We demonstrate that Qdot-tagged DAT proteins exhibited highly heterogeneous membrane diffusivity dependent on the local membrane topography. We also show that Qdot-tagged DATs were localized away from the flat membrane regions and were dynamically retained in the membrane protrusions and cell edges for the duration of imaging. Single quantum dot tracking of wildtype DAT and its conformation-defective coding variants (R60A and W63A) revealed a significantly accelerated rate of dysfunctional DAT membrane diffusion. We believe our results warrant an in-depth investigation as to whether compromised membrane dynamics is a common feature of brain disorder-derived DAT mutants.

Klíčová slova:

Cell membranes – Cytoskeleton – Dopamine – Cholesterol – Mass diffusivity – Membrane proteins – Membrane trafficking – Dopamine transporters


1. Giros B, Giros B, Caron MG. Molecular characterization of the dopamine transporter. Trends Pharmacol Sci. 1993; 14: 43–49. doi: 10.1016/0165-6147(93)90029-j 8480373

2. Girault J-A, Greengard P. The neurobiology of dopamine signaling. Arch Neurol. 2004; 61: 641–644. doi: 10.1001/archneur.61.5.641 15148138

3. Gether U, Andersen PH, Larsson OM, Schousboe A. Neurotransmitter transporters: molecular function of important drug targets. Trends in Pharmacol Sci. 2006; 27: 375–383.

4. Torres GE, Gainetdinov RR, Caron MG. Plasma membrane monoamine transporters: structure, regulation and function. Nature Rev Neurosci. 2003; 4: 13–25.

5. Greenwood TA, Alexander M, Keck PE, McElroy S, Sadovnick AD, Remick RA, et al. Evidence for linkage disequilibrium between the dopamine transporter and bipolar disorder. Am J Med Genet. 2001; 105: 145–151. doi: 10.1002/1096-8628(2001)9999:9999<::aid-ajmg1161>3.0.co;2-8 11304827

6. Swanson JM. Dopamine genes and ADHD. Neurosci Biobehav Rev. 2000; 24: 21–25. doi: 10.1016/s0149-7634(99)00062-7 10654656

7. Kirley A, Lowe N, Hawi Z, Mullins C, Daly G, Waldman I, et al. Association of the 480 bp DAT1 allele with methylphenidate response in a sample of Irish children with ADHD. Am J Med Genet Part B. 2003; 121B: 50–54. doi: 10.1002/ajmg.b.20071 12898575

8. Kurian MA, Zhen J, Cheng SY, Li Y, Mordekar SR, Jardine P, et al. Homozygous loss-of-function mutations in the gene encoding the dopamine transporter are associated with infantile parkinsonism-dystonia. J Clin Invest. 2009; 119: 1595–1603. doi: 10.1172/JCI39060 19478460

9. Gowrishankar R, Hahn MK, Blakely RD. Good riddance to dopamine: roles for the dopamine transporter in synaptic function and dopamine-associated brain disorders. Neurochem Int. 2014; 73: 42–48. doi: 10.1016/j.neuint.2013.10.016 24231471

10. Kaya C, Cheng MH, Block ER, Bartol TM, Sejnowski TJ, Sorkin A, et al. Heterogeneities in axonal structure and transporter distribution lower dopamine reuptake efficiency. eNeuro 2018; 5: e0298-17.2017, 1–21.

11. Adkins EM, Samuvel DJ, Fog JU, Eriksen J, Jayanthi LD, Vaegter CB, et al. Membrane mobility and microdomain association of the dopamine transporter studied with fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. Biochemistry. 2017; 46: 10484–10497.

12. Eriksen J, Jorgensen TN, Gether U. Regulation of dopamine transporter function by protein-protein interactions: new discoveries and methodological challenges. J Neurochem. 2010; 113: 27–41. doi: 10.1111/j.1471-4159.2010.06599.x 20085610

13. Cremona ML, Matthies HJ, Pau K, Bowton E, Speed N, Lute BJ, et al. Flotillin-1 is essential for PKC-triggered endocytosis and membrane microdomain localization of DAT. Nature Neuroscience. 2011; 14: 469–477. doi: 10.1038/nn.2781 21399631

14. Eriksen J, Rasmussen SG, Rasmussen TN, Vaegter CB, Cha JH, Zou MF, et al. Visualization of dopamine transporter trafficking in live neurons by use of fluorescent cocaine analogs. J Neurosci, 2009; 29: 6794–6808. doi: 10.1523/JNEUROSCI.4177-08.2009 19474307

15. Rahbek-Clemmensen T, Lycas MD, Erlendsson S, Eriksen J, Apuschkin M, Vilhardt F, et al. Super-resolution microscopy reveals functional organization of dopamine transporters into cholesterol and neuronal activity-dependent nanodomains. Nat Commun. 2017; 8: 740. doi: 10.1038/s41467-017-00790-3 28963530

16. Sorkina T, Richards TL, Rao A, Zahniser NR, Sorkin A. Negative regulation of dopamine transporter endocytosis by membrane-proximal N-terminal residues. J Neurosci. 2009; 29: 1361–1374. doi: 10.1523/JNEUROSCI.3250-08.2009 19193883

17. Caltagarone J, Ma S, Sorkin A. Dopamine transporter is enriched in filopodia and induces filopodia formation. Mol Cell Neurosci. 2015; 68: 120–130. doi: 10.1016/j.mcn.2015.04.005 25936602

18. Ma S, Cheng MH, Guthrie DA, Newman AH, Bahar I, Sorkin A. Targeting of dopamine transporter to filopodia requires an outward-facing conformation of the transporter. Sci Rep. 2017; 14:5339.

19. Sakrikar D, Mazei-Robison MS, Mergy MA, Richtand NW, Han Q, Hamilton PJ, et al. Attention deficit/hyperactivity disorder-derived coding variation in the dopamine transporter disrupts microdomain targeting and trafficking regulation. J Neurosci. 2012; 32: 5385–5397. doi: 10.1523/JNEUROSCI.6033-11.2012 22514303

20. Kovtun O, Tomlinson ID, Sakrikar DS, Chang JC, Blakely RD, Rosenthal SJ. Visualization of the cocaine-sensitive dopamine transporter with ligand-conjugated quantum dots. ACS Chem Neurosci. 2011; 2: 370–378. doi: 10.1021/cn200032r 22816024

21. Kovtun O, Sakrikar D, Tomlinson ID, Chang JC, Arzeta-Ferrer X, Blakely RD, et al. Single-quantum-dot tracking reveals altered membrane dynamics of an attention-deficit/hyperactivity-disorder-derived dopamine transporter coding variant. ACS Chem Neurosci. 2015; 6: 526–534. doi: 10.1021/cn500202c 25747272

22. Rosenthal SJ, Chang JC, Kovtun O, McBride JR, Tomlinson ID. Biocompatible quantum dots for biological applications. Chem Biol. 2011; 18: 10–24. doi: 10.1016/j.chembiol.2010.11.013 21276935

23. Chang JC, Tomlinson ID, Warnement MR, Ustione A, Carneiro AM, Piston DW, et al. Single molecule analysis of serotonin transporter regulation using antagonist-conjugated quantum dots reveals restricted, p38 MAPK-dependent mobilization underlying uptake activation. J Neurosci. 2012; 32: 8919–8929. doi: 10.1523/JNEUROSCI.0048-12.2012 22745492

24. Pinaud F, Clarke S, Sittner A, Dahan M. Probing cellular events, one quantum dot at a time. Nat Methods. 2010; 7: 275–285. doi: 10.1038/nmeth.1444 20354518

25. Kovtun O, Tomlinson ID, Bailey DM, Thal LB, Ross EJ, Harris L, et al. Single quantum dot tracking illuminates neuroscience at the nanoscale. Chem Phys Lett. 2018; 706: 741–752. doi: 10.1016/j.cplett.2018.06.019 30270931

26. Bailey DM, Catron MA, Kovtun O, Macdonald RL, Zhang Q, Rosenthal SJ. Single quantum dot tracking reveals serotonin transporter diffusion dynamics are correlated with cholesterol-sensitive threonine 276 phosphorylation status in primary midbrain neurons. ACS Chem Neurosci. 2018; 9:2534–2541. doi: 10.1021/acschemneuro.8b00214 29787674

27. Mason JN, Farmer H, Tomlinson ID, Schwartz JW, Savchenko V, DeFelice LJ, et al. Novel fluorescence-based approaches for the study of biogenic amine transporter localization, activity, and regulation. J Neurosci Methods. 2005; 143: 3–25. doi: 10.1016/j.jneumeth.2004.09.028 15763132

28. Orndorff RL, Rosenthal SJ. Neurotoxin quantum dot conjugates detect endogenous targets expressed in live cancer cells. Nano Lett. 2009; 9: 2589–2599. doi: 10.1021/nl900789e 19507837

29. Rosenthal SJ, Tomlinson I, Adkins EM, Schroeter S, Adams S, Swafford L, et al. Targeting cell surface receptors with ligand-conjugated nanocrystals. J Am Chem Soc. 2002; 124: 4586–4594. doi: 10.1021/ja003486s 11971705

30. Gussin HA, Tomlinson ID, Little DM, Warnement MR, Qian H, Rosenthal SJ, et al. Binding of muscimol-conjugated quantum dots to GABAC receptors. J Am Chem Soc. 2006; 128: 15701–15713. doi: 10.1021/ja064324k 17147380

31. Gussin HA, Tomlinson ID, Cao D, Qian H, Rosenthal SJ, Pepperberg DR. Quantum dot conjugates of GABA and muscimol: binding to α1β2γ2 and ρ1 GABA(A) receptors. ACS Chem Neurosci. 2013; 4: 435–443. doi: 10.1021/cn300144v 23509979

32. Tomlinson ID, Kovtun O, Rosenthal SJ. Biotinylated-spiperone ligands for quantum dot labeling of the Dopamine D2 receptor (D2 DR) in live cell cultures. Bioorg Med Chem Lett. 2019; 29:959–964. doi: 10.1016/j.bmcl.2019.02.024 30808590

33. Jaqaman K, Loerke D, Mettlen M, Kuwata H, Grinstein S, Schmid SL et al. Robust single particle tracking in live cell time-lapse sequences. Nat Methods. 2008; 5: 695–702. doi: 10.1038/nmeth.1237 18641657

34. Michalet X, Berglund AJ. Optimal diffusion coefficient estimation in single-particle tracking. Phys Rev E Stat Nonlin Soft Matter Phys. 2010; 85: 061916.

35. Kusumi A, Sako Y, Yamamoto M. Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovid microscopy). Effects of calcium-induced differentiation in cultured epithelial cells. Biophys J. 1993; 65: 2021–2040. doi: 10.1016/S0006-3495(93)81253-0 8298032

36. Crane JM, Verkman AS. Long-range nonanomalous diffusion of quantum dot-labeled aquaporin-1 water channels in the cell plasma membrane. Biophys J. 2008; 94: 702–713. doi: 10.1529/biophysj.107.115121 17890385

37. Monnier N, Barry Z, Park HY, Su KC, Katz Z, English BP, et al. Inferring transient particle transport dynamics in live cells. Nat Methods. 2015; 12: 838–840. doi: 10.1038/nmeth.3483 26192083

38. Wu S, Fagan RR, Uttamapinant C, Lifshitz LM, Fogarty KE, Ting AY, et al. The dopamine transporter recycles via a retromer-dependent postendocytic mechanism: tracking studies using a novel fluorophore-coupling approach. J Neurosci. 2017; 37: 9438–9452. doi: 10.1523/JNEUROSCI.3885-16.2017 28847807

39. Murphy-Royal C, Dupuis JP, Varela JA, Panatier A, Pinson B, Baufreton J, et al. Surface diffusion of astrocytic glutamate transporters shapes synaptic transmission. Nat Neurosci. 2015; 18: 219–226. doi: 10.1038/nn.3901 25581361

40. Vuorenpää A, Jørgensen TN, Newman AH, Madsen KL, Scheinin M, Gether U. Differential internalization rates and postendocytic sorting of the norepinephrine and dopamine transporters are controlled by structural elements in the N termini. J Biol Chem. 2016; 291: 5634–5651. doi: 10.1074/jbc.M115.702050 26786096

41. Nirenberg MJ, Chan J, Vaughan RA, Uhl GR, Kuhar MJ, Pickel VM. Immunogold localization of the dopamine transporter: an ultrastructural study of the rat ventral tegmental area. J Neurosci. 1997; 17: 5255–5262. doi: 10.1523/JNEUROSCI.17-14-05255.1997 9204909

42. Winckler B, Forscher P, Mellman I. A diffusion barrier maintains distribution of membrane proteins in polarized neurons. Nature. 1999; 397: 698–701. doi: 10.1038/17806 10067893

43. Cowan AE, Myles DG, Koppel DE. Lateral diffusion of the PH-20 protein on huinea pig sperm: Evidence that barriers to diffusion maintain plasma membrane domains in mammalian sperm. J Cell Biol. 1987; 104: 917–923. doi: 10.1083/jcb.104.4.917 3558486

44. Rasband MN. The axon initial segment and the maintenance of neuronal polarity. 2010; 11: 552–562.

45. Peter BJ, Kent HM, Mills IG, Vallis Y, Butler PJ, Evans PR, et al. BAR domains as sensors of membrane curvature: the amphiphysin BAR structure. Science. 2004; 303: 495–499. doi: 10.1126/science.1092586 14645856

46. Arizono M, Bannai H, Nakamura K, Niwa F, Enomoto M, Matsu-Ura T, et al. Receptor-selective diffusion barrier enhances sensitivity of astrocytic processes to metabotropic glutamate receptor stimulation. Sci Signal 2012; 5: ra27. doi: 10.1126/scisignal.2002498 22472649

47. Singer SJ, Nicolson GL. fluid mosaic model of the structure of cell membranes. Science. 1972; 175: 720–731. doi: 10.1126/science.175.4023.720 4333397

48. Gustafsson MG. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc. 2000; 198: 82–87. doi: 10.1046/j.1365-2818.2000.00710.x 10810003

49. Hong WC, Amara SG. Membrane cholesterol modulates the outward facing conformation of the dopamine transporter and alters cocaine binding. J Biol Chem. 2010; 285: 32616–32626. doi: 10.1074/jbc.M110.150565 20688912

50. Zeppelin T, Laderfoged LK, Sinning S, Periole X, Schiøtt B. A direct interaction of cholesterol with the dopamine transporter prevents its out-to-inward transition. PLoS Comput Biol. 2018; 14:e1005907. doi: 10.1371/journal.pcbi.1005907 29329285

51. Hamilton PJ, Belovich AN, Khelashvili G, Saunders C, Erreger K, Javitch JA, et al. PIP2 regulates psychostimulant behaviors through its interaction with a membrane protein. Nat Chem Biol. 2014; 10:582–589. doi: 10.1038/nchembio.1545 24880859

52. Gabriel LR, Wu S, Kearney P, Bellvé KD, Standley C, Fogarty KE, et al. Dopamine transporter endocytic trafficking in striatal dopaminergic neurons: differential dependence on dynamin and the actin cytoskeleton. J Neurosci. 2013; 33: 17836–17846. doi: 10.1523/JNEUROSCI.3284-13.2013 24198373

53. Wheeler DS, Underhill SM, Stolz DB, Murdoch GH, Thiels E, Romero G, et al. Amphetamine activates Rho GTPase signaling to mediate dopamine transporter internalization and acute behavioral effects of amphetamine. Proc Natl Acad Sci USA. 2015; 112: E7138–7147. doi: 10.1073/pnas.1511670112 26553986

54. Penmatsa A, Wang KH, Gouaux E. X-ray structure of dopamine transporter elucidates antidepressant mechanism. Nature. 2013; 503:85–90. doi: 10.1038/nature12533 24037379

55. Liu JJ, Hezghia A, Shaikh SR, Cenido JF, Stark RE, Mann JJ. Regulation of monoamine transporters and receptors by lipid microdomains: implications for depression. Neuropsychopharmacology. 2018; 43: 2165–2179. doi: 10.1038/s41386-018-0133-6 30022062

56. Chaudhuri A, Bhattacharya B, Gowrishankar K, Mayor S, Rao M. Spatiotemproal regulation of chemical reactions by active cytoskeletal remodeling. PNAS. 2011;108: 14825–14830. doi: 10.1073/pnas.1100007108 21873247

57. Rosholm KR, Leijinse N, Mantsiou A, Tkach V, Pedersen SL, Wirth VF, et al. Membrane curvature regulates ligand-specific membrane sorting of GPCRs in living cells. Nat Chem Biol. 2017; 13:724–729. doi: 10.1038/nchembio.2372 28481347

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2019 Číslo 11