Figure 1.
Figure 1. Ciliary proteins are not accessible to antibodies in digitonin-permeabilized cells. (A) IMCD3 cells were fixed, permeabilized with 30 µg/ml digitonin, and incubated for 10 min with antibodies to ciliary proteins and to nucleoporins (Nups). After removal of unbound antibodies, cells were permeabilized with Triton X-100 and stained with additional antibodies. Left insets show primary cilia with channels shifted to aid visualization. Arrowheads point to the cilium base in the acetylated tubulin channel (left) or the Arl13b channel (right). Right insets show staining of Nups and DNA. Ac-tub, acylated tubulin. (B) Overview of assay for soluble protein entry into primary cilia. IMCD3 cells are first treated with digitonin or PFO, which permeabilizes the plasma membrane while leaving the ciliary membrane intact. An exogenously added small GBP can cross from the cytoplasmic compartment into the cilium (left), where it is captured by GFP fused to the intracellular tail of the ciliary GPCR Sstr3. (right) A large GBP (e.g., an antibody) cannot enter the cilium and only accesses the pool of Sstr3-GFP present in the plasma membrane. (C) IMCD3 cells expressing Sstr3-GFP were stained with an anti-GFP antibody following a conventional immunofluorescence protocol using Triton X-100 for permeabilization (left) or in live cells permeabilized with digitonin (middle) or PFO (right). Insets show primary cilia with GFP and anti-GFP channels shifted to aid visualization. Arrowheads point to the base of the cilium in the GBP channel. Ab, antibody; dia., diameter. (D) Cells expressing Sstr3-GFP were stained with GBP labeled with Alexa Fluor 647 as in C. Insets show primary cilia with channels shifted. PFO- and digitonin-treated cells exhibit a gradient of GBP signal from cilium base (marked by γ-tubulin in white) to tip. (E) Line graphs of ciliary signals of GFP binders reveal that the base-to-tip gradient of GBP distribution is specific to semipermeabilized cells and that the anti-GFP antibody is absent from semipermeabilized cells. Lines were drawn through cilia from images in C and D, and intensity relative to background was measured with ImageJ. The data shown are from a representative cell, with >10 cells analyzed in each of four experiments. Bars: (main images) 5 µm; (insets) 1 µm.

Ciliary proteins are not accessible to antibodies in digitonin-permeabilized cells. (A) IMCD3 cells were fixed, permeabilized with 30 µg/ml digitonin, and incubated for 10 min with antibodies to ciliary proteins and to nucleoporins (Nups). After removal of unbound antibodies, cells were permeabilized with Triton X-100 and stained with additional antibodies. Left insets show primary cilia with channels shifted to aid visualization. Arrowheads point to the cilium base in the acetylated tubulin channel (left) or the Arl13b channel (right). Right insets show staining of Nups and DNA. Ac-tub, acylated tubulin. (B) Overview of assay for soluble protein entry into primary cilia. IMCD3 cells are first treated with digitonin or PFO, which permeabilizes the plasma membrane while leaving the ciliary membrane intact. An exogenously added small GBP can cross from the cytoplasmic compartment into the cilium (left), where it is captured by GFP fused to the intracellular tail of the ciliary GPCR Sstr3. (right) A large GBP (e.g., an antibody) cannot enter the cilium and only accesses the pool of Sstr3-GFP present in the plasma membrane. (C) IMCD3 cells expressing Sstr3-GFP were stained with an anti-GFP antibody following a conventional immunofluorescence protocol using Triton X-100 for permeabilization (left) or in live cells permeabilized with digitonin (middle) or PFO (right). Insets show primary cilia with GFP and anti-GFP channels shifted to aid visualization. Arrowheads point to the base of the cilium in the GBP channel. Ab, antibody; dia., diameter. (D) Cells expressing Sstr3-GFP were stained with GBP labeled with Alexa Fluor 647 as in C. Insets show primary cilia with channels shifted. PFO- and digitonin-treated cells exhibit a gradient of GBP signal from cilium base (marked by γ-tubulin in white) to tip. (E) Line graphs of ciliary signals of GFP binders reveal that the base-to-tip gradient of GBP distribution is specific to semipermeabilized cells and that the anti-GFP antibody is absent from semipermeabilized cells. Lines were drawn through cilia from images in C and D, and intensity relative to background was measured with ImageJ. The data shown are from a representative cell, with >10 cells analyzed in each of four experiments. Bars: (main images) 5 µm; (insets) 1 µm.

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