EMK1 promotes “hepatic-type” luminal targeting in nonpolarized cells. Contact-naïve control and EMK1-MDCK monolayers were generated as in Fig. 4. (A) Untransduced cells were incubated with antibodies against the ectodomains of gp135 (green) for 1 h at 37°C. Cell-associated antibodies were detected after fixation and permeabilization with secondary antibodies (ab-uptake). In parallel, samples were fixed, permeabilized, and labeled for total gp135 (immunofluorescence). Shown are 40× wide-field images. (Right) Quantification: Total cell-associated gp135 fluorescence in 30 individual control cells was determined in two separate experiments where gp135 antibodies were added to live cells (striped bars) or after fixation and permeabilization (filled bars). The difference between “total” (filled bars) and “live”(striped bars) fluorescence is statistically significant, P < 0.005. (Schemes in Upper and Lower Left) gp135 immunofluorescence (green, Upper) and gp135 antibody binding and uptake (green and red arrows, Lower) in nonpolarized cells. (B) Cells transduced with an adenovirus encoding LDLR were incubated live with antibodies against the ectodomain of gp135 (green) and LDLR (red) as described in A. Shown are confocal x-y sections. (Bars, 10 μm.) [Image gp135 in A is reprinted from ref. 42 with permission from Elsevier (Copyright 2003, Elsevier).]

EMK1 promotes basolateral targeting of luminal secretory proteins. EMK1-MDCK cells constitutively expressing solDPPIV (B) or untransfected EMK1-MDCK cells (C) were cultured as described for Fig. 1. Cells were labeled for 2 h with ³⁵S-sulfate at 20°C and subsequently chased for 2 h in the presence of 10 mM (Na)₂SO₄. Note that EMK1-overexpressing cells had ≈50% reduced ³⁵S-sulfate incorporation compared with control conditions. (A) EMK1-MDCK monolayers before and after low Ca-incubation. (Left) Confocal x-y image of EMK1-expressing monolayers similar to those analyzed in B and C labeled with gp135, phalloidin and ZO-1. Note that >90% of cells had lateral lumina organization. (Right) After PBS treatment (2 × 20 min at 37°C), polarity is lost, cells round up, and luminal markers become accessible to the medium; gp135 (green), phalloidin (red), ZO-1 (blue). Presented are confocal x-y and x-z images. (Bars, 10 μm.) (B) SolDPPIV secretion. SolDPPIV was immunoprecipitated from the basal (bl) and luminal (lum) medium and from the cell lysate (c). Shown are triplicate samples (#1–#3) of a representative experiment and quantification by PhosphorImager analysis from three independent experiments. (C) Endogenous apical secretory proteins. Cells were treated as in B, and total protein in apical and basolateral medium was analyzed. Shown are triplicate samples (#1–#3) of a representative experiment and quantification of luminal gp80 (indicated by * in nonreduced gels) from three experiments. Bar graphs in B and C show standard errors. *, Significant difference between control and EMK1 with P > 2 × 10^–5.

Model. EMK1 promotes a hepatic-type lumen, MT, and trafficking phenotype in epithelial cells. (Upper) Polarized MDCK and other columnar epithelial cells position their luminal domain at the apex, whereas liver cells position their luminal domains at the lateral surface (bile canaliculi). Columnar cells organize their MTs (red lines) as vertical arrays with the negative ends facing the apical domain, whereas liver cells position their MTs horizontally, with the negative ends facing the lateral lumina. Many luminal proteins in MDCK cells use a direct route, whereas the same proteins use an indirect (transcytotic) route in polarized hepatocytes or liver cell lines (e.g., WIF-B). EMK1 is expressed constitutively by all epithelial cells. Overexpression of EMK1 in MDCK cells at the time they are establishing their polarized phenotype promotes the establishment of all three landmarks of hepatic polarity: lateral lumina, horizontal MTs, and indirect luminal targeting. EMK1 regulates post-Golgi exocytosis by either promoting basolateral targeting of luminal post-Golgi carriers (option A) or by preventing protein sorting at the TGN (option B). (Lower) In contrast to the epithelial-type-specific MT arrays of polarized epithelial cells, nonpolarized MDCK and hepatic epithelial cells maintain a radial MT array. In addition, under certain conditions, they generate an intracellular compartment enriched in luminal proteins (VAC and hepatocyte apical compartment). Although superficially similar, these intracellular compartments have striking morphological and trafficking differences in columnar and hepatic epithelial cells (see text). Whereas in hepatic cells luminal proteins actively recycle between this compartment and the cell surface, in kidney cells the luminal proteins do not exchange with the plasma membrane. EMK1 promotes a hepatic-type mode of luminal protein trafficking in nonpolarized MDCK cells. Ap, apical; BL, basolateral.

EMK1 promotes indirect targeting of gp135 to lateral lumina. Confluent, contact-naïve EMK1-overexpressing or control MDCK cells were polarized in a Ca-switch assay as described in Methods.(A) gp135 antibody-binding, control MDCK. gp135 antibodies were incubated with the basal (Left) or apical (Right) medium of living cells for 1 h on ice; gp135 (green), phalloidin (red), and ZO-1 (blue). Shown are confocal x-z views. (B) gp135 immunofluorescence, EMK1-MDCK. gp135 (green) in Tx-100 permeabilized and unpermeabilized cells; phalloidin (red) and ZO-1 (blue) labeling was after permeabilization in both cases. Shown are confocal x-y sections. Quantification of gp135 fluorescence indicates that only 11 ± 3% of gp135 labeled in permeabilized cells was accessible to the gp135 antibody in unpermeabilized cells. (C) gp135 antibody binding and uptake, EMK1-MDCK. Shown is the distribution of gp135 antibodies bound to living cells (Left) during a 1-h incubation on ice after a 0-h (Upper) and 2-h (Lower) chase at 37°C. Apical lumina are visualized by phalloidin staining (Right); shown are confocal x-y and x-z sections. (Bars, 10 μm.)

EMK1 promotes indirect targeting of NTRp75-GFP to lateral lumina. (A) NTR-p75-GFP transport kinetics. cDNA encoding NTRp75-GFP was microinjected into the nucleus of cells facing an apical lumen and expressed for 1 h at 37°C. At this point (time, 0 h), the addition of cyclohexamide prevented further protein synthesis. After 0, 1, 2, and 4 h of chase, cells were analyzed for GFP fluorescence (Center) and by p75 surface immunofluorescence (Left). *, Microinjected cells facing apical lumina identified by phase (Right). Note that the face and fluorescence images at 0-h and 1-h chase are taken at different focal planes. (B) p75 antibody uptake. After a 1-h chase, cells were incubated with antibodies against the ectodomain of p75 for 1 h on ice, washed, and chased for 2 h at 37°C. GFP fluorescence (Center) was compared with p75 antibody distribution (Left). (Upper and Lower) Representation of different examples of the same experimental treatment. (Bars, 10 μm.)

The intracellular apical compartment of nonpolarized EMK1-MDCK cells resembles that of hepatocytic cells. EMK1-MDCK cells maintained either in the presence (control) or absence (EMK1) of doxycycline were cultured in Ca²⁺-free medium for 12 h to generate contact-naïve monolayers. (A) gp135 colocalization with phalloidin. Confocal x-y and x-z views of permeabilized cells colabeled with anti-gp135 (green) and phalloidin (red). (B and C) gp135 colocalization with dIgA and Tf. PIgR expression in EMK1-MDCK cells was induced with 10 mM sodium butyrate overnight. Human Tf receptor was expressed by adenovirus-mediated gene transfer. Cells were incubated for 2 h with either dIgA (B) or cy3-conjugated Tf (C) before fixation; colocalization between gp135 (green) and dIgA (red; detected with rhodamine-coupled anti-IgA) or gp135 (green) and cy3-Tf (red) is shown in individual confocal x-y sections. (Bars, 10 μm.)