Polarization of Specific Tropomyosin Isoforms in Gastrointestinal Epithelial Cells and Their Impact on CFTR at the Apical Surface

Abstract

Microfilaments have been reported to be polarized in a number of cell types based both on function and isoform composition. There is evidence that microfilaments are involved in the movement of vesicles and the polarized delivery of proteins to specialized membrane domains. We have investigated the composition of actin microfilaments in gastrointestinal epithelial cells and their role in the delivery of the cystic fibrosis transmembrane conductance regulator (CFTR) into the apical membrane using cultured T84 cells as a model. We identified a specific population of microfilaments containing the tropomyosin (Tm) isoforms Tm5a and/or Tm5b, which are polarized in T84 cell monolayers. Polarization of this microfilament population occurs very rapidly in response to cell-cell and cell-substratum contact and is not inhibited by jasplakinolide, suggesting this involves the movement of intact filaments. Colocalization of Tm5a and/or Tm5b and CFTR was observed in long-term cultures. A reduction in Tm5a and Tm5b expression, induced using antisense oligonucleotides, resulted in an increase in both CFTR surface expression and chloride efflux in response to cAMP stimulation. We conclude that Tm isoforms Tm5a and/or Tm5b mark an apical population of microfilaments that can regulate the insertion and/or retention of CFTR into the plasma membrane.

Tissue-specific Tropomyosin Isoform Composition

“…We therefore conclude that Tm5a and Tm5b are the isoforms present in the cell periphery and ruffling membranes. This correlates well with the apical enrichment of these isoforms in epithelial cells (Dalby-Payne et al 2003). The WD4/9d antibody, which detects Tm4 and Tm1, was also predominantly found in stress fibers Equal loading (20 g) of total cellular protein isolated from mouse tissues was electrophoresed on 12.5% low-bis SDS gels, and individual blots were immunoblotted with (A) ␤-actin, (B) ␥-actin, and (C) ␣-smooth-muscle actin.…”

Tropomyosin – master regulator of actin filament function in the cytoskeleton

“…For instance, Ostap and co-workers have shown that Myo1b, which is involved in organelle transport, does not recognize actin filaments that contain either Tpm1.6 (also known as Tm2) or Tpm3.1 (Tang and Ostap, 2001;Kee et al, 2015;McIntosh et al, 2015). In addition, Tpm1.8 (also known as Tm5a) and Tpm1.9 regulates the recycling of the cystic fibrosis transmembrane receptor (CFTR) (Dalby-Payne et al, 2003). Tpm1.8 and Tpm1.9 are enriched at apical sites, which are also enriched for CFTR, and depletion of Tpm1.8 and Tpm1.9 leads to elevated levels of CFTR in the apical membrane.…”

Tropomyosin variants describe distinct functional subcellular domains in differentiated vascular smooth muscle cells

“…Colocalization is shown in c. Scale bar for all, 10 m. intracellular localization. Cytochalasin D treatment, for example, disintegrates the isoform-specific Tm localization pattern in several cell types (3,45). Therefore it is more likely that Tm proteins display a variant-specific intracellular localization via either their differential affinities for actin-binding proteins or actin isoforms (34).…”

“…Quick frozen tissue was homogenized in immunoprecipitation (IP) buffer (50 mM Tris·HCl pH 7.4, 1 mM EGTA, 50 mM NaCl, 3 mM MgCl 2, 1% Nonident P-40, 1% sodium deoxycholate, 3 mM ATP, 3 mM DTT, 67 M ZnCl2, 29.6 mM ␤-glycerophosphate, protease inhibitors) and then precleared with protein G-coupled dynabeads (Invitrogen). To reduce background, the antibodies used in this assay were cross-linked to protein G-coupled dynabeads (Invitrogen) using the cross-linking reagent Bis(sulfosuccinimidyl)suberate (BS 3) following the manufacturer's instructions. The precleared cell lysates were incubated for 16 h at room temperature with either the sheep polyclonal anti-Tm6 antibody (Sh X Tropomyosin) or the mouse monoclonal anti-Tm1 antibody [CG1; (7,15,16,28)], cross-linked to protein G-coupled dynabeads.…”