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. Author manuscript; available in PMC: 2022 Oct 29.
Published in final edited form as: Cell Rep. 2022 Sep 27;40(13):111431. doi: 10.1016/j.celrep.2022.111431

Figure 2. Nanodomain-localized DAT is preferentially in an inward-facing conformation.

Figure 2.

(A–D) dSTORM on DA neurons and DAT-expressing CAD cells labeled with the fluorescent cocaine analog DG3–63 (10 nM).

(A) Axonal extension.

(B) Varicosity (box in A).

(C) CAD cell.

(D) ROI from (C).

(E–H) The density-based association of DG3–63 with mEOS2-DAT.

(E) Cartoon of the labeling strategy.

(F) dSTORM/PALM image of a CAD cell expressing mEOS2-DAT (red) and labeled with DG3–63 (10 nM) (blue).

(G) Close up dSTORM/PALM image of CAD cell expressing mEOS2-DAT (red) and labeled with DG3–63 (10 nM) (blue).

(H) The DG3–63 signal is blocked by 100 μM cocaine.

(I–L) Voronoi tessellation-based colocalization algorithm. Tessellation is performed on the mEOS2-DAT data, and areas are marked when the mEOS2-DAT localization has a DG3–63 localization within 25 nm.

(L) Left: total mEos2-DAT Voronoi tesselated area distribution (red) (probability density function [PDF]) and tessellated areas of mEos2-DAT associated with DG3–63 (blue). Right: tessellated areas for mEos2-DAT associated with DG3–63 (blue) compared with that associated in the presence of saturating cocaine.

(M–O) Total internal reflection fluorescence (TIRF)/dSTORM of WT DAT and the inward-facing mutant (D421N) expressed in CAD cells.

(M) Example image of DAT expressed in CAD cells.

(N) Example images of DAT clusters identified by DBSCAN in WT DAT and D421N D expressing cells; clusters with diameter >75 nm in green, clusters <75 nm in magenta.

(O) Normalized fraction of clusters with a diameter greater than 75 nm (percent). Means ± S.E. from 38 WT DAT cells and 42 D421N cells, 3 transfections, ***p < 0.001, unpaired t test.

(P–R) dSTORM of WT DAT and the inward-facing mutant (D421N) expressed in CAD cells.

(P) Number of localizations per square meter for WT DAT and D421N. Means ± S.E., unpaired t test, p = 0.4.

(Q) Example images of DAT clusters identified by DBSCAN in WT DAT and D421N D expressing cells; dense clusters (>80 localizations within a radius of 50 nm) in red.

(R) Fraction of localizations in dense clusters (in percent of WT DAT). Means ± S.E. from 38 WT DAT cells and 42 D421N cells, 3 transfections, **p < 0.01, unpaired t test.

See also Figure S3.