Figure 1.
Localization of DISC1-GFP near the base of primary cilia.
(A) NIH3T3 cells transfected with DISC1-GFP were fixed and immunolabeled for acetylated tubulin to mark primary cilia. An example of such a double-labeled cell is shown, with DISC1-GFP in green and acetylated tubulin in red in the merged image. Arrow indicates indicates the concentration of DISC1-GFP observed near the ciliary base. (B) The same experiment conducted using DISC1-HA. (C) Triple localization of DISC1-GFP, endogenous PCM1, and acetylated tubulin verifying localization of DISC1 in a centrosomal region near the ciliary base. (D) Merged image from the triple localization with DISC1-GFP in green, PCM1 in red, and acetylated tubulin in blue. (E) The region indicated in panel D displayed at higher magnification. Scale bar, 10 µm.
Figure 2.
Depletion of endogenous DISC1 and IFT88 by RNA interference.
NIH3T3 cells were transfected with the indicated siRNA duplexes (sequences are listed in Materials and Methods). Total cell extracts were prepared 72 hours later, and levels of DISC1 (A) or IFT88 (B) protein were assessed by immunoblot using antibodies recognizing the endogenous proteins. Representative immunoblots are shown. Densitometric scanning across multiple experiments verified >80% depletion of both proteins by the respective siRNA duplexes.
Figure 3.
DISC1 knockdown reduces cilia number.
(A) NIH3T3 cells were transfected with the indicated RNA duplexes and then stained with anti-acetylated tubulin (to mark primary cilia, left panels) and DAPI (to mark nuclei, middle panels). Merged images (right panels) show acetylated tubulin and DAPI staining in green and blue, respectively. Cilia were prominently observed in the majority of cells transfected with control (non-silencing) RNA duplexes (top row of images, an example is indicated by arrow). In cells transfected with either siRNA targeting DISC1, the vast majority of cells did not project a detectable primary cilium. Depleting IFT88 (bottom row of images) produced a similar effect. (B) Quantification of cilia loss by counting the number of cells extending a primary cilium, as marked by acetylated tubulin immunostaining, observed in blinded analysis of the indicated populations of siRNA-transfected cells. Bars represent the mean fraction of cells with a visible cilium, averaged across 5 experiments counting ≥250 cells/condition in each. Error bars represent the s.e.m. calculated across the experiments (***, p<0.0001).
Figure 4.
Rescue of the DISC1 knockdown phenotype by human-derived DISC1-GFP.
NIH3T3 cells were transfected with siRNA targeting endogenous murine DISC1 and transduced with lentiviral particles encoding human-derived DISC1-GFP lacking the target sequence. (A) Example images from the rescue experiment. Top panels show the negative control condition, in which cells were transfected with scrambled (non-silencing) siRNA and infected with a lentivirus encoding GFP. Middle panels show the knockdown condition, in which cells were transfected with DISC1 siRNA and infected with lentivirus encoding GFP. Bottom paneles show the rescue condition, in which cells were transfected with DISC1 siRNA and infected with lentivirus encoding the siRNA-resistant DISC1-GFP construct. Left panels show acetylated tubulin immunoreactivity, middle panels show GFP fluorescence, and right panels show the merge image with inset as indicated by box. (B) Quantification of the rescue effect by cilia count (n = 4 experiments).
Figure 5.
Subtype-specific localization of dopamine receptors to the primary cilium.
NIH3T3 cells were transfected with expression construct encoding the indicated Flag-tagged dopamine receptor or transferrin receptor. 48 hours after transfection, cells were fixed and receptors present in the plasma membrane were selectively labeled by incubating non-permeabilized cells in the presence of anti-Flag antibody, and cells were then permeabilized to immunolabel acetylated tubulin. Each row of images shows representative cells expressing the indicated Flag-tagged receptor construct. The merged image in each row shows surface receptor immunoreactivity in red and acetylated tubulin in green to mark the primary cilium. Insets show the indicated region at higher magnification, scale bar corresponds to 10 µm.
Figure 6.
Subtype-selective localization of dopamine receptors to cilia in striatal neurons.
Examples of ciliary localization of Flag-D1R and Flag-D2R, but not Flag-D4R, in primary striatal neurons. The left panels indicate surface Flag immunoreactivity marking receptors present in the plasma membrane. Middle panels indicate ACIII immunoreactivity marking cilia. The merged image is shown in the right panels, with the region of cell body containing the cilium displayed at higher magnification in the inset.
Figure 7.
DISC1 knockdown and rescue in striatal neurons.
(A) Example immunoblot showing depletion of endogenous DISC1 detected in PC12 cells using the C-terminal antibody (see Materials and Methods). The immunoreactive band corresponding to the major full length DISC1 species is shown. (B) Example of DISC1 depletion in striatal cultures, detected using the C-terminal (C-term) antibody and verified using the midi antibody, as indicated. (C) Quantification of knockdown and rescue phenotypes, based on count of cilia number compiled from 8 independent experiments. (D) Examples of the phenotypes observed in the indicated conditions (indicated above each column). The rescue condition (right column of images) shows the localization of DISC1-GFP rather than GFP. Insets indicate relevant regions of the cell body at higher magnification.