A series of GBP fusions reveals the size limit of the ciliary diffusion barrier. (A) Digitonin-permeabilized cells were incubated at room temperature for 10 min with the indicated dye-labeled GBP fusion proteins. Insets show cilia in GBP channel only. Trx, thioredoxin; MBP, maltose-binding protein; NusA, N-using substance A; Luc, firefly luciferase; LacZ, β-galactosidase. Bars: (main images) 5 µm; (insets) 1 µm. (B) Properties of GBPs including molecular mass (MM), Stokes radius, and degree of ciliary entry in digitonin-permeabilized IMCD3 cells expressing Sstr3-GFP. The relative degree of entry seen in A is denoted by the number of + symbols or by a − symbol in cases where no entry was detected. ZZ, tandem Z domain from S. aureus protein A; IgG, immunoglobulin G. (C) Kinetic analysis of GBP entry into cilia of permeabilized IMCD3 cells. GBP alone, Trx-GBP, and MBP-GBP were added at 110 nM, and capture by ciliary Sstr3-GFP was monitored by confocal microscopy. Bar, 1 µm. (D) The GBP signal at the most proximal region of the cilium is plotted versus time for GBP alone, Trx-GBP, and MBP-GBP. Traces for individual cilia are shown in thin lines (n ≥ 10). Fitted exponential curves corresponding to the mean entry rate are shown in thick lines. Inset graph at right shows fitted rate constants (plotted on a logarithmic scale) versus measured Stokes radius. Error bars indicate standard deviations.