Figure 3. Specificity of ciliary ectocytosis.

(A–B) Comparison of on- and off-pathway ectocytosis. (A) β-arrestin2^−/− IMCD3-[^APGPR161^NG3] and β-arrestin2^−/− IMCD3-[^APSSTR3^NG] cells were tracked in the NeonGreen channel following Hh pathway activation. GPR161 but not SSTR3 is highly enriched in ectosomes compared to cilia. Ectosomes are circled. Scale bar: 4 µm. (B) The NeonGreen intensity of ectosomes observed in experiments presented in Fig. 3A and 1E was quantified and normalized to the total NeonGreen intensity of the parent cilium. (n=24 ectosomes).

(C) Diagram illustrating how activated GPCRs (red) are enriched in ectosomes while bystander proteins (green) undergo bulk flow ectocytosis.

(D) Arl6^−/− cells undergo both signal-dependent and constitutive ectocytosis. SA647-labeled WT or Arl6^−/− IMCD3-[^APSSTR3^NG] cells were imaged following addition of sst or antagonist (ACQ090). The number of cilia with a nearby SSTR3 focus was scored. Data was fit to a Hill equation of no theoretical significance. (n=76–89 cilia).

(E) Packaging efficiencies of constitutive and signal-dependent ectocytosis. Ectosomes observed in experiments presented in Fig. 3D were analyzed following the procedure used in Fig. 3B. (n=38–43 ectosomes).

(F) Signal-dependent ectocytosis from β-arrestin2^−/− cells. Ectosomes from SA647-labeled IMCD3-[^APSSTR3^NG]; β-arrestin2^−/− cells were counted following addition of sst, SAG, ACQ090, or vehicle. Analysis and fitting was as in panel (D). (n=51–141 cilia).

(G) Constitutive ectocytosis removes measurable levels of ciliary proteins. ^APSSTR3^NG was pulse-labeled by SA647 and analyzed by fixed imaging after 6 or 9 h treatment with sst, vehicle, or SAG (see Fig. S3F). Data were fit to an exponential decay and the rate constants for SSTR3 removal are shown in the table. (n=222–327 cilia measured per rate constant).