The Apical Submembrane Cytoskeleton Participates in the Organization of the Apical Pole in Epithelial Cells

Abstract

In a previous publication (Rodriguez, M.L., M. Brignoni, and P.J.I. Salas. 1994. J. Cell Sci. 107: 3145–3151), we described the existence of a terminal web-like structure in nonbrush border cells, which comprises a specifically apical cytokeratin, presumably cytokeratin 19. In the present study we confirmed the apical distribution of cytokeratin 19 and expanded that observation to other epithelial cells in tissue culture and in vivo. In tissue culture, subconfluent cell stocks under continuous treatment with two different 21-mer phosphorothioate oligodeoxy nucleotides that targeted cytokeratin 19 mRNA enabled us to obtain confluent monolayers with a partial (40–70%) and transitory reduction in this protein. The expression of other cytoskeletal proteins was undisturbed. This downregulation of cytokeratin 19 resulted in (a) decrease in the number of microvilli; (b) disorganization of the apical (but not lateral or basal) filamentous actin and abnormal apical microtubules; and (c) depletion or redistribution of apical membrane proteins as determined by differential apical–basolateral biotinylation. In fact, a subset of detergent-insoluble proteins was not expressed on the cell surface in cells with lower levels of cytokeratin 19. Apical proteins purified in the detergent phase of Triton X-114 (typically integral membrane proteins) and those differentially extracted in Triton X-100 at 37°C or in n-octyl-β-d-glycoside at 4°C (representative of GPIanchored proteins), appeared partially redistributed to the basolateral domain. A transmembrane apical protein, sucrase isomaltase, was found mispolarized in a subpopulation of the cells treated with antisense oligonucleotides, while the basolateral polarity of Na+– K+ATPase was not affected. Both sucrase isomaltase and alkaline phosphatase (a GPI-anchored protein) appeared partially depolarized in A19 treated CACO-2 monolayers as determined by differential biotinylation, affinity purification, and immunoblot. These results suggest that an apical submembrane cytoskeleton of intermediate filaments is expressed in a number of epithelia, including those without a brush border, although it may not be universal. In addition, these data indicate that this structure is involved in the organization of the apical region of the cytoplasm and the apical membrane.

Intermediate Filaments Interact with Dormant Ezrin in Intestinal Epithelial Cells

“…Our laboratory is interested in the hypothesis that IFs may serve as an early scaffold organizing the apical domain in polarized epithelial cells. It was strongly supported by the phenotype of CACO-2 intestinal epithelial cells in tissue culture transiently depleted in IFs (Salas et al,1997) as well as by the phenotype of villus enterocytes in K8-null mice (Ameen et al, 2001). In both cases, a specifically apical phenotype comprising diminished or absent apical membrane proteins and disrupted orientation of microtubules (normally apico-basal) was observed.…”

“…No known domains are present in ezrin or keratins that would suggest the possibility of an interaction. Therefore, the brush-border phenotype of keratin knockdowns (Salas et al, 1997) and K8 knockout mice (Ameen et al, 2001) was a conundrum for the past few years. The data from stable transfectants here confirmed that intermediate filaments are necessary to organize the brush-border-like F-actin cytoskeleton in CACO-2 cells (Figure 1).…”

Identification of SNPs in the cystic fibrosis interactome influencing pulmonary progression in cystic fibrosis

“…48 KRT19 was shown to be a modulator of cellular differentiation processes at the apical membrane of epithelial cells. 35 Sun 36 (presented at the 23rd Annual North American Cystic Fibrosis Conference) illustrated that KRT19 affects CFTR function by stabilizing it at the plasma membrane and is able to increase its plasma membrane density by inhibiting CFTR endocytosis. It seems that homozygosity for haplotype TGC has a negative effect on protein levels and therefore worse progression of sR eff (Figure 1c).…”

“…[30][31][32] The last group consists of three genes that are involved in CFTR trafficking, AHSAI (activator of heat shock 90-kDa protein ATPase homolog 1), CALR (calreticulin) and KRT19 (cytokeratin 19). [33][34][35][36] Most candidate gene and whole-genome association studies so far focused on time point measurements of only one or two lung function parameters. To get a better insight on the influence of our single-nucleotide polymorphisms (SNPs) on CF lung disease, we used a new approach and correlated our genotype data with a wider range of lung function parameters measured over a time period to evaluate the influence of a SNP on the progression of the lung disease.…”

Teachings from the egg: New and unexpected functions of RNAs

“…Recent genome-wide screen of RNAs localized in leading edge of migrating mammalian fibroblasts showed that in response to migratory stimuli, 50 different mRNAs (besides previously identified b-actin mRNA) accumulated, via microtubule mediated transport, in the fibroblast protrusions rich in actin cytoskeletal elements (Mili et al, 2008). In epithelial cells, the apical cytoskeleton containing cytokeratin, actin, and microtubules (Salas et al, 1997;Sugimoto et al, 2008) is enriched in localized wingless mRNA (Simmonds et al, 2001;Najand and Simmonds, 2007). In neurons, b-actin mRNA and actin filaments are localized in the growth cones and synapses (Zhang et al, 1999;Specht and Triller, 2008) and a subset of mRNAs localized at the synapse is thought to be responsible for the generation of synaptic plasticity (Kiebler and DesGroseillers, 2000;Czaplinski and Singer, 2006;Bramham and Wells, 2007).…”