Disrupting primary cilia does not attenuate the D1R-mediated cAMP response.

<p><b>A</b>. Representative fluorescence micrographs showing localization of surface-labeled Flag-D1 dopamine receptors (‘FD1R’, green) relative to acetylated tubulin (‘AcTub’, red) and DAPI (blue), in IMCD3 cells transfected with non-silencing RNA duplex (‘Control’, top row) or siRNA targeting IFT88 (‘IFT88’, bottom row). In control cells FD1R was concentrated in cilia marked by AcTub and also localized in the plasma membrane outside cilia (overlay shown in Merge, with an example in inset). In IFT88 knockdown cells, FD1R was diffusely localized in the plasma membrane and cilia were not detectable by either FD1R or AcTub localization. Scale bar, 10 um. Inset is displayed at 3× increased magnification. <b>B</b>. Quantification of the percentage of cells, marked by DAPI, displaying a primary cilium, marked by AcTub, in control (left bar) and IFT88 knockdown (right bar) specimens (n = 3 experiments, 200 cells counted per experiment; p value as indicated). <b>C</b>. Flow cytometric quantification of surface FD1R immunoreactivity in control relative to IFT88 knockdown cells (n = 3 experiments, each condition determined in triplicate, 10,000 cells per determination; p value as indicated). <b>D</b>. Concentration-response relationship for FD1R-mediated accumulation of cAMP measured in cell lysates using enzyme-linked immunosorbent assay.</p

GPR88 localizes to cilia and mutationally activated GPR88 inhibits D1R-dependent cAMP accumulation.

<p><b>A</b>. Representative fluorescence micrograph of a cultured striatal neuron showing Flag-GPR88 (green, left panel), the primary cilium marker adenylyl cyclase III, (‘AC3’ red, second panel from left), nuclei are marked by DAPI staining (blue, third panel from left) and merge image showing ciliary GPR88 localization (right panel). Inset shows a higher magnification view of a primary cilium labeled with GPR88. Scale bar, 10 um. Inset is displayed at 3× increased magnification. <b>B</b>. Representative fluorescence micrograph showing Flag-GPR88 localization to cilia also in IMCD3 cells. Flag-GPR88 is displayed in green, the ciliary marker acetylated tubulin in red, DAPI in blue, and merge image is at right. Inset shows a representative example at higher magnification. Scale bar, 10 um. Inset is displayed at 4.5× increased magnification. <b>C</b>. Time course of ICUE3-Cilia nFRET change outside of cilium measured in IMCD3 cells co-expressing the sensor and Flag-D1R together with vector control (‘D1R+Vector”, black line), HA-tagged wild type GPR88 (‘D1R+GPR88’, blue line) or HA-tagged activated (G283H) GPR88 allele (‘D1R+GPR88_G283H’, red line), and with 1 uM SKF81297 added at the second frame (40 sec). Results shown are from 8 experiments (≥2 cells imaged per experiment, error bars indicate S.E.M. across experiments). <b>D</b>. Time course of ICUE3-Cilia nFRET change inside the cilium, determined from the same sets of image series as used to generate the data in panel C, and using the same color scheme for data display. Mutant GPR88 inhibited D1R-mediated cAMP accumulation estimated by nFRET change in both ciliary and extra-ciliary compartments. Results shown are from 8 experiments (≥2 cells imaged per experiment, error bars indicate S.E.M. across experiments).</p

A FRET-based biosensor for local analysis of cAMP dynamics in individual cells.

<p><b>A</b>. Schematic representation of ICUE3-Cilia design. The biosensor consists of a ligand binding-defective SSTR3 to achieve ciliary localization fused to ICUE2 except with the C-terminal citrine present in ICUE2 replaced by a circularly permuted YFP variant to increase the overall fluorescence signal. <b>B</b>. Representative fluorescence micrograph of IMCD3 cells transfected with ICUE3-Cilia, showing YFP fluorescence (left panel, yellow) relative to AcTub immunoreactivity (right panel, red). ICUE3-Cilia localized both to the primary cilium (arrowhead) and was detectable in the extra-ciliary plasma membrane (arrow). Scale bar, 10 um. <b>C</b>. YFP image of a representative ICUE3-Cilia -transfected cell (left panel), with selected regions of plasma membrane outside of cilium (‘cell’, red outline) and including the primary cilium (‘cilia’, blue outline) indicated. nFRET determinations in each region were carried out as described in Experimental Procedures. Scale bar, 10 um. <b>D</b>. Time course of nFRET change in the indicated regions of IMCD3 cells expressing ICUE3-Cilia, with 5 uM forskolin or vehicle (DMSO) applied at t = 40 sec. The black line shows vehicle control indicating minimal photobleaching over the period of nFRET determination. The red line indicates extra-ciliary (‘outside cilium’) nFRET and blue line indicates ciliary (‘cilium’) nFRET calculated from the same image series (n = 3 experiments, ≥3 cells imaged per experiment, error bars indicate S.E.M. across experiments).</p

D1R activation drives cAMP accumulation in both ciliary and extra-ciliary domains.

<p><b>A</b>. Representative fluorescence micrograph of an IMCD3 cell co-expressing FD1R (left panel, red) and ICUE3-Cilia (right panel, yellow), showing that each localize to the cilium. Blue and black outlines indicate representative examples of ciliary (‘cilium’) and extra-ciliary (‘outside cilium’) ROIs, respectively, used for nFRET determination. Scale bar, 10 um. <b>B</b>. Time course of nFRET change in the indicated regions of IMCD3 cells expressing ICUE3-Cilia and Flag-D1R with 1 uM SKF81297 added at the second frame (40 sec). Black line indicates nFRET calculated from the extra-ciliary ROI (‘outside cilum’) and blue line indicates ciliary nFRET (‘cilium’). Results shown are from 8 experiments (≥2 cells imaged per experiment, error bars indicate S.E.M. across experiments). <b>C</b>. Representative dual channel fluorescence micrographs showing that HA-B2AR is not enriched in cilia (left panel, red) and ICUE3-Cilia is distributed both inside and outside of cilia (right panel). Red and black outlines indicate representative examples of ciliary (‘cilium’) and extra-ciliary (‘outside cilium’) ROIs, respectively, used for nFRET determination. Scale bar, 10 um. <b>D</b>. Time course of nFRET change in the indicated regions of IMCD3 cells co-expressing ICUE3-Cilia and HA-B2AR with 10 uM isoproterenol added at the second frame (40 sec). Black line indicates nFRET calculated from the extra-ciliary ROI (‘outside cilum’) and red line indicates ciliary nFRET (‘cilium’). Results shown are from 8 experiments (≥2 cells imaged per experiment, error bars indicate S.E.M. across experiments). <b>E</b>. Representative fluorescence micrograph of HA-B2AR (left panel, green), acetylated tubulin (second panel from left, red), DAPI (third panel from left, blue) and merge (right panel), showing that B2AR is not concentrated in cilia. Scale bar, 10 um. Inset is displayed at 3× increased magnification.</p

Retromer Mediates a Discrete Route of Local Membrane Delivery to Dendrites

A fundamental and still largely unresolved question is how neurons achieve rapid delivery of selected signaling receptors throughout the elaborate dendritic arbor. Here we show that this requires a conserved sorting machinery called retromer. Retromer-associated endosomes are distributed within dendrites in ∼2 μm intervals and supply frequent membrane fusion events into the dendritic shaft domain immediately adjacent to (&lt;300 nm from) the donor endosome and typically without full endosome discharge. Retromer-associated endosomes contain β-adrenergic receptors as well as ionotropic glutamate receptors, and retromer knockdown reduces extrasynaptic insertion of adrenergic receptors as well as functional expression of AMPA and NMDA receptors at synapses. We propose that retromer supports a broadly distributed network of plasma membrane delivery to dendrites, organized in micron-scale axial territories to render essentially all regions of the postsynaptic surface within rapid diffusion distance of a local exocytic event