Single molecule imaging reveals a major role for diffusion in the exploration of ciliary space by signaling receptors
- Stanford University School of Medicine, United States
- Stanford University, United States
Figures
Figure 1 with 3 supplements
Real-time imaging of single signaling receptors in cilia of live cells.
(A) Schematic of single molecule labeling strategy. SSTR3 or Smo were fused at the extracellular N-terminus to an acceptor peptide (AP) for the biotin ligase BirA. Biotinylated AP-SSTR3 and AP-Smo …
Figure 1—figure supplement 1
Additional kymographs.
Additional kymographs representing the movement of single mSA-Alexa647 labeled AP-SSTR3-GFP (A) and AP-Smo-YFP (B). The tip (T) and the base (B) of the cilium are indicated. Scale bars, 2 μm (y), 4 …
Figure 1—figure supplement 2
Additional dual channel kymographs.
Additional kymographs of simultaneous live cell imaging of TagRFP.T-IFT88 (RFP-IFT88, IFT complex) and single molecule SSTR3 (SSTR3:mSA-A647). The processive movement of mSA labeled SSTR3 (red …
Figure 1—figure supplement 3
Half-cilium FRAP.
(A) Representative example of a half-cilium FRAP. (B) Kinetics of fluorescence recovery of photobleached region (red curve), unbleached region (green curve), and the total cilium (blue curve). (C) …
Figure 2
Functionality of the AP-SSTR3-GFP fusion protein.
(A) Saturated labeling of biotinylated AP-SSTR3-GFP (SSTR3-GFP) with 20 nM of mSA-Alexa647 (mSA) for 1 hr. Scale bar, 2 μm. (B) Time series montage represents the mobility of ciliary AP-SSTR3-GFP …
Figure 3 with 1 supplement
Single molecule imaging in ATP-depleted cells reveals the receptor population undergoing active transport in live cells.
(A) Kymographs of GFP-IFT88 foci movements after Antimycin A and 2-deoxyglucose (2DG) treatment. Scale bar, 2 μm. (B) Kymographs of simultaneous live cell imaging of tagRFP.T-IFT88 (IFT88) and …
Figure 3—figure supplement 1
Additional kymographs.
Additional kymographs representing the movement of single mSA-Alexa647 labeled AP-SSTR3-GFP in cells treated with Antimycin A and deoxyglucose. The tip (T) and the base (B) of the cilium are …
Figure 4 with 3 supplements
Single molecule imaging in digitonin-permeabilized cells reveals the receptor population undergoing active transport in live cells.
(A) Kymographs of simultaneous live cell imaging of TagRFP.T-IFT88 (RFP-IFT88, IFT trains) and single molecule SSTR3 (SSTR3:mSA-A647) movements in digitonin semi-permeabilized cells. The …
Figure 4—figure supplement 1
Measurement of ATP levels.
ATP levels quantified by luciferin-luciferase bioluminescence assay normalized to the levels in control-treated cells (ATP level [%]). To deplete intracellular ATP, IMCD3 cells were permeabilized …
Figure 4—figure supplement 2
Additional kymographs.
Additional kymographs representing the movement of single mSA-Alexa647 labeled AP-SSTR3-GFP (A) and AP-Smo-YFP (B) in cells permeabilized with digitonin. The tip (T) and the base (B) of the cilium …
Figure 4—figure supplement 3
Digitonin permeabilization does not affect the mobility of SSTR3 in cilia.
(A) Averaged fitted curves of SSTR3-GFP half-cilium FRAP in different conditions. (B) The diffusion coefficients quantified from the FRAP recovery curves in (A). More than 10 cilia were analyzed for …
Figure 5 with 2 supplements
Ciliobrevin treatment abolishes IFT train movement but not active transport of SSTR3.
(A) Kymographs of simultaneous live cell imaging of TagRFP.T-IFT88 (RFP-IFT88, IFT complex) and single molecule SSTR3 (SSTR3:mSA-A647) movements in ciliobrevin treated cells (>30 min). The mobility …
Figure 5—figure supplement 1
Additional kymographs.
Additional kymographs representing the movement of single mSA-Alexa647 labeled AP-SSTR3-GFP in cells treated with ciliobrevin. The tip (T) and the base (B) of the cilium are indicated. Scale bars, 2 …
Figure 5—figure supplement 2
Ciliobrevin treatment does not affect the mobility of SSTR3 in cilia.
(A) Averaged fitted curves of SSTR3-GFP half-cilium FRAP in different conditions. (B) The diffusion coefficients quantified from the FRAP recovery curves in (A). More than 10 cilia were analyzed for …
Figure 6 with 2 supplements
Effect of immobilizing membrane proteins on IFT train movements.
(A) Kymographs of simultaneous live cell imaging of mCherry-IFT88 (IFT complex) and single molecule SSTR3 (SSTR3:mSA-A647) movements in WGA treated cells (>7 min). The immobilization of ciliary …
Figure 6—figure supplement 1
Additional kymographs.
Additional kymographs representing the movement of single mSA-Alexa647 labeled AP-SSTR3-GFP in cells treated with WGA. The tip (T) and the base (B) of the cilium are indicated. Scale bars, 2 μm (y), …
Figure 6—figure supplement 2
WGA treatment stops the mobility of SSTR3 in cilia.
Averaged fitted curves of SSTR3-GFP half-cilium FRAP in different conditions.
Videos
Video 1
Live cell imaging of a single ciliary SSTR3 molecule.
IMCD3 cells stably expressing AP-SSTR3-GFP (pseudo-colored red) were transfected with BirA-ER to biotinylate AP-SSTR3-GFP. Biotinylated SSTR3 was detected with mSA-Alexa647 (mSA, pseudo-colored …
Video 2
Live cell imaging of a single ciliary Smo molecule.
IMCD3 cells stably expressing AP-Smo-YFP (Smo, pseudo-colored red) were transfected with BirA-ER to biotinylate AP-Smo-YFP. Biotinylated Smo was detected with mSA-Alexa647 (mSA, pseudo-colored …
Video 3
Representative examples of half-cilium FRAP in different conditions.
The distal half of the cilia were photobleached and the recovery rate of the SSTR3-GFP fluorescent signal in the bleached region was recorded at 1 s interval. Scale bar, 2 μm.
Video 4
Live cell imaging of GFP-IFT88 fluorescent foci movement before (−WGA) and after 10 min of WGA treatment (+WGA) in IMCD3 cells.
Scale bar, 2 μm.
