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First published online October 10, 2007
doi: 10.1242/10.1242/jcs.011445

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Tmod3 regulates polarized epithelial cell morphology

Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA

View larger version (126K): [in this window] [in a new window]   Fig. 1. Tmod3 localizes to lateral membranes in several epithelial cell types, including Caco-2 cells where it is the sole Tmod isoform. (A-I) XY confocal sections of LLC-PK1 CL4 (A-C), HBE (D-F), and Caco-2 (G-I) cells stained for F-actin (red, B,E,H) and Tmod3 (green, C,F,I). Tmod3 localizes to lateral membranes (arrowheads) in each of these epithelial cell lines as well as in a punctate pattern in the cytoplasm. Note the localization of Tmod3 to foci in LLC-PK1 CL4 cells (C); we have no information regarding the identification of these foci. (J) Immunoblot of Caco-2 cell lysates, human erythrocyte membranes (Mg2+ ghosts), and purified recombinant human Tmod1 or Tmod3 labeled with either anti-Tmod3 or anti-Tmod1 antibodies, demonstrating that Tmod3 is the sole Tmod isoform expressed in Caco-2 cells. (K-M) XZ confocal section of Caco-2 cells stained for F-actin (red, L) and Tmod3 (green, M). Tmod3 localizes to the subapical domain underlying the F-actin rich microvilli (arrows), to lateral membranes (arrowheads) and to cytoplasmic puncta.

View larger version (101K): [in this window] [in a new window]   Fig. 2. Tmod3 knockdown by shRNA results in altered cell morphology and decreased fluorescence intensity of F-actin at lateral membranes. (A) Transfection of pEGFP-H1RNAi encoding a hairpin sequence directed against Tmod3 results in decreased Tmod3 protein levels compared to transfection of pEGFP-H1RNAi or pEGFP-H1RNAi encoding mismatched target sequence. (B-E) XZ and (F-I) XY confocal sections stained with rhodamine-phalloidin for F-actin (C,G) shown in red in merges (B,F), and with antibodies against Tmod3 (D,H) shown in green in merged images (B,F), with transfected cells expressing GFP (E,I) shown in blue in merged images (B,F). (J-L) Quantification of cell morphology and total F-actin staining intensity shows that Tmod3 knockdown results in (J) decreased cell height, (K) increased cross-sectional area and, (L) decreased intensity of F-actin fluorescence when compared to untransfected cells in the same field of view, or to cells transfected with mismatched target sequence. (M-O) Line scans demonstrate that Tmod3 knockdown results in decreased F-actin fluorescence intensity at lateral membranes compared with untransfected cells or cells transfected with mismatched target sequence. (M,N) Representative line scans of lateral membrane F-actin fluorescence intensity and peak spacing in shRNA or mismatch transfected or in untransfected cells in the same field of view; 3.5 pixels per µm for both M and N. (O) Quantification of F-actin fluorescence intensity at lateral membranes from the area under peaks in line scans. Results are mean ± s.e.m.; *P<0.05; **P<0.005.

View larger version (64K): [in this window] [in a new window]   Fig. 3. Tmod3 knockdown by shRNA results in decreased fluorescence intensity of tropomyosin. (A-D) XZ and (E-H) XY confocal sections stained for F-actin (red, B,F), tropomyosin (green, C,G), with transfected cells expressing GFP shown in blue (D,H). (I) Transfection of pEGFP-H1RNAi encoding a hairpin sequence directed against Tmod3 results in decreased total F-actin and tropomyosin fluorescence as compared with untransfected cells. Total cellular F-actin and tropomyosin fluorescence intensity were quantified for untransfected cells and cells transfected with either pEGFP-H1RNAi encoding a hairpin sequence directed against Tmod3 or with pEGFP-H1RNAi encoding a mismatched target sequence, from images such as in F-I. Results are the mean ± s.e.m.; *P<0.05. (J) Representative line scans illustrating typical decreases in intensity of lateral membrane peaks of F-actin and tropomyosin fluorescence, corresponding with increased GFP fluorescence in cells transfected with pEGFP-H1RNAi encoding hairpin sequence directed against Tmod3; 7.8 pixels per µm.

View larger version (90K): [in this window] [in a new window]   Fig. 4. Tmod3 knockdown by shRNA does not disrupt adherens or tight junctions, as assessed by localization of E-cadherin or ZO-1, but leads to increased breadth of II-spectrin staining on lateral membranes. (A) Schematic of typical localization of E-cadherin, ZO-1 and II-spectrin in polarized epithelial cells. (B-M) XZ confocal sections of Caco-2 monolayers stained for F-actin (red, C,G,K), E-cadherin (D), ZO-1 (H), II-spectrin (L) (all shown in green), with transfected cells expressing GFP shown in blue (E,I,M). (O,P) Transfection of pEGFP-H1RNAi encoding a hairpin sequence directed against Tmod3 results in increased breadth of II-spectrin staining at lateral membranes when compared to (O) untransfected cells but has no effect on breadth of E-cadherin staining at (P) lateral membranes. No significant changes are observed in (O) II-spectrin or (P) E-cadherin on lateral membranes for cells transfected with pEGFP-H1RNAi encoding mismatched target sequence as compared to untransfected cells. Results are the mean ± s.e.m.; **P<0.005.

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