PMC

Decreased polarity of endogenous basolateral markers in μ1B-KD MDCK. The steady-state surface distribution of some basolateral and apical membrane proteins in fully polarized (4.5-d) parental, control, and μ1B-KD MDCK cells was determined by using Western blot analysis after domain-selective biotinylation.

μ1B-KD MDCK cells display apical TfR and LDLR. Control and μ1B-KD cells were infected with Ad-hTfR (A) or Ad-hLDLR (B) 3d after plating, fixed 36 h later, and processed for surface immunofluorescence with monoclonal antibodies against lumenal domains of TfR and LDLR, followed by brief permeabilization with Saponin 0.075% and immunostaining of β-catenin and ZO-1. (Top) XY sections at the apical membrane (μ1B-KD, Left) or at midcell (Control, Right). (Middle and Bottom) XZ sections.

Sedimentation analysis of AP1B distribution. MDCK cells, confluent for 0 (C-0 d) or 4 d (C-4 d) were homogenized and analyzed by sucrose density centrifugation, as described in Materials and Methods. Gradient fractions were analyzed by SDS/PAGE and Western blot analysis (original gels in SI Fig. 11). The values represent the percent of the various markers in a given fraction. Note that μ1B sediments preferentially in higher-density fractions that contain TfR.

Biosynthetic delivery of VSVG protein is disrupted in fully polarized (4.5 d) confluent μ1B-KD MDCK cells. (A) Parental and μ1B-KD MDCK cells were infected for 24 h with Ad-VSVG, pulsed with [³⁵S]Met/Cys, chased for the times indicated, and biotinylated from the apical or basolateral sides. VSVG from each sample was immunoprecipitated and processed for a biotin-targeting assay (). Value represents the percent at apical or basolateral surfaces of total VSVG. Note the polarized basolateral delivery of VSVG in parental MDCK cells and the depolarized delivery of VSVG in μ1B-KD MDCK cells. (B) Shows the average ± standard deviation values of the apical/basolateral delivery ratio after a 2-h chase at 32°C for μ1B-KD and parental MDCK cells.

Construction of permanently μ1B knockdown MDCK clones. (A) Rabbit antibodies were raised against μ1A and μ1B and tested by Western blot analysis against GST-tagged antigen and various tissue lysates (50 μg of protein). The anti-μ1B antibody showed no reactivity against rat (kidney, liver), human, or dog μ1A protein. (B) MDCK cells were transfected with μ1B-M16 or GL2-siRNA by electroporation and processed 72 h later for RT-PCR and Western blot. These cells are efficiently depleted of μ1B mRNA and protein. (C) MDCK cells were electroporated with m1B-M16 or GL2-siRNA three consecutive times at 3-d intervals and analyzed by Western blot. m1B-M16 siRNA results in undetectable levels of μ1B and increased levels of μ1A without altering γ-adaptin levels. (D) Puromycin-resistant clones were generated upon MDCK transfection with pSuper-μ1B-M16 vector. The extent of μ1B silencing was determined by RT-PCR and Western blot analysis. Two thirds of the screened clones displayed an effective depletion of μ1B. Each clone is infected with AdTfR for 30 h, and the fraction of cell displaying apical TfR is scored.

Biosynthetic delivery and recycling of TfR in control and μ1B-KD MDCK. (A) Assays to quantify biosynthetic delivery or recycling of TfR. MDCK cells infected with AdTfR are pulsed with [³⁵S]Met/Cys for 13 min. For biosynthetic delivery (Left), cells are immediately chased with excess unlabeled met/cys in the presence of Biotin-Tf (B-Tf) added to apical or basal side. For recycling (Right) a 3.5-h chase follows the pulse before incubation with B-Tf. Cells are lysed at pH 5.5, and the B-Tf/TfR complex was retrieved with Avidin-Sepharose. (B) Biosynthetic sorting of TfR in 4.5-d confluent MDCK cells. Control and parental MDCK cells targeted 12–15% of newly synthesized TfR to the apical surface. μ1B-KD MDCK cells targeted ≈23% of newly synthesized TfR to the apical surface (see text and SI Table 1). (C) Recycling of TfR. Whereas control and parental MDCK cells recycled >80% of TfR to the basolateral membrane, μ1B-KD cells recycled more than ≈50% of TfR to the apical membrane (see SI Table 1). (D) Modified version of recycling assay. Biotin-Tf was added to both apical and basal media during the last 90 min of the 3.5-h chase. Subsequently, cells were exposed to an excess of Tf added to either apical or basolateral side. In parental and control cells, B-Tf is displaced by Tf efficiently only when added basolaterally, whereas in μ1B-KD MDCK cells, >45% of B-Tf was displaced by Apical Tf. (E) Biosynthetic delivery of TfR in 1- to 1.5-d confluent MDCK cells. Confluent parental, control, and μ1B-KD MDCK cells were infected with Ad-TfR, trypsinized 6 h later, plated at confluency on polycarbonate filters, and used to measure biosynthetic delivery of TfR 24 h later, as in A Left. (F) Alternatively, cells after 8 h of plating were infected with Ad-hTfR for 24 h and then assayed for biosynthetic delivery of TfR. Note the nonpolarized delivery of TfR by μ1B-KD MDCK in E and F. (G) Monolayers formed by μ1B-KD, control and parental MDCK cells under conditions similar to those used in E and F display similar low permeability to [³H]inulin.