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Proteomic analysis of proteins surrounding occludin and claudin-4 reveals their proximity to signaling and trafficking networks.

Fredriksson K, Van Itallie CM, Aponte A, Gucek M, Tietgens AJ, Anderson JM - PLoS ONE (2015)

Bottom Line: When proteomic results were analyzed, the relative distribution among functional categories was similar between occludin and claudin-4 proximal proteins.However there were significant differences in the specific proteins comprising the functional categories near each of the tagging proteins, revealing spatial compartmentalization within the junction complex.Taken together, these results expand the inventory of known and unknown proteins at the tight junction to inform future studies of the organization and physiology of this complex structure.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Tight Junction Structure and Function, NHLBI, National Institutes of Health, Bethesda, MD, United States of America.

ABSTRACT
Tight junctions are complex membrane structures that regulate paracellular movement of material across epithelia and play a role in cell polarity, signaling and cytoskeletal organization. In order to expand knowledge of the tight junction proteome, we used biotin ligase (BioID) fused to occludin and claudin-4 to biotinylate their proximal proteins in cultured MDCK II epithelial cells. We then purified the biotinylated proteins on streptavidin resin and identified them by mass spectrometry. Proteins were ranked by relative abundance of recovery by mass spectrometry, placed in functional categories, and compared not only among the N- and C- termini of occludin and the N-terminus of claudin-4, but also with our published inventory of proteins proximal to the adherens junction protein E-cadherin and the tight junction protein ZO-1. When proteomic results were analyzed, the relative distribution among functional categories was similar between occludin and claudin-4 proximal proteins. Apart from already known tight junction- proteins, occludin and claudin-4 proximal proteins were enriched in signaling and trafficking proteins, especially endocytic trafficking proteins. However there were significant differences in the specific proteins comprising the functional categories near each of the tagging proteins, revealing spatial compartmentalization within the junction complex. Taken together, these results expand the inventory of known and unknown proteins at the tight junction to inform future studies of the organization and physiology of this complex structure.

No MeSH data available.


Ocln and Cldn4 biotin ligase fusion-proteins localize to the TJ and the lateral cell membrane.A. Both biotin ligase fused to the N terminus (BL-Ocln, myc) and C terminus (Ocln-BL, myc) of Ocln co-localized (Merge, top right, middle right panel) with ZO-1 (top left and middle left panel), although there is also some non-junctional immunofluorescence associated with the transgene. Biotin ligase fused to the N terminus of Cldn4 (BL-Cldn4, myc) partly co-localizes with ZO-1 (Merge, bottom right panel), and partly in the cytoplasm (Bottom middle panel). B. BL-Ocln, Ocln-BL and BL-Cldn4 (Myc signal middle panel) are distributed along the basolateral membrane. The two Ocln constructs also concentrate at the apical junction with ZO-1 (right top and middle panel). Bar, 20 microns.
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pone.0117074.g001: Ocln and Cldn4 biotin ligase fusion-proteins localize to the TJ and the lateral cell membrane.A. Both biotin ligase fused to the N terminus (BL-Ocln, myc) and C terminus (Ocln-BL, myc) of Ocln co-localized (Merge, top right, middle right panel) with ZO-1 (top left and middle left panel), although there is also some non-junctional immunofluorescence associated with the transgene. Biotin ligase fused to the N terminus of Cldn4 (BL-Cldn4, myc) partly co-localizes with ZO-1 (Merge, bottom right panel), and partly in the cytoplasm (Bottom middle panel). B. BL-Ocln, Ocln-BL and BL-Cldn4 (Myc signal middle panel) are distributed along the basolateral membrane. The two Ocln constructs also concentrate at the apical junction with ZO-1 (right top and middle panel). Bar, 20 microns.

Mentions: In order to determine the spatial specificity of the labeling method we determined both the cellular localization of the fusion proteins and the subcellular patterns of biotinylated proteins. Unlike ZO-1 which is focused at the TJ, both claudins and Ocln also show variable localization to the lateral membrane [6,7,12,19,20,33,38,40]. As integral membrane proteins they are also expected to be near proteins in biosynthetic vesicular trafficking pathways [2,25,26,41–47]. As expected, en face immunofluorescent images of the TJ protein ZO-1 (Fig. 1A, left panels) and BL-Ocln, Ocln-BL and BL-Cldn4 fusion proteins (middle panels) reveals colocalization at TJs (right panels), as reported by myc epitope staining. The biotin ligase fusion proteins are also found to a variable extent in intracellular compartments. In contrast, we have previously shown that myc-tagged biotin ligase alone is diffusely distributed throughout the cells including the nucleus [10].


Proteomic analysis of proteins surrounding occludin and claudin-4 reveals their proximity to signaling and trafficking networks.

Fredriksson K, Van Itallie CM, Aponte A, Gucek M, Tietgens AJ, Anderson JM - PLoS ONE (2015)

Ocln and Cldn4 biotin ligase fusion-proteins localize to the TJ and the lateral cell membrane.A. Both biotin ligase fused to the N terminus (BL-Ocln, myc) and C terminus (Ocln-BL, myc) of Ocln co-localized (Merge, top right, middle right panel) with ZO-1 (top left and middle left panel), although there is also some non-junctional immunofluorescence associated with the transgene. Biotin ligase fused to the N terminus of Cldn4 (BL-Cldn4, myc) partly co-localizes with ZO-1 (Merge, bottom right panel), and partly in the cytoplasm (Bottom middle panel). B. BL-Ocln, Ocln-BL and BL-Cldn4 (Myc signal middle panel) are distributed along the basolateral membrane. The two Ocln constructs also concentrate at the apical junction with ZO-1 (right top and middle panel). Bar, 20 microns.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4366163&req=5

pone.0117074.g001: Ocln and Cldn4 biotin ligase fusion-proteins localize to the TJ and the lateral cell membrane.A. Both biotin ligase fused to the N terminus (BL-Ocln, myc) and C terminus (Ocln-BL, myc) of Ocln co-localized (Merge, top right, middle right panel) with ZO-1 (top left and middle left panel), although there is also some non-junctional immunofluorescence associated with the transgene. Biotin ligase fused to the N terminus of Cldn4 (BL-Cldn4, myc) partly co-localizes with ZO-1 (Merge, bottom right panel), and partly in the cytoplasm (Bottom middle panel). B. BL-Ocln, Ocln-BL and BL-Cldn4 (Myc signal middle panel) are distributed along the basolateral membrane. The two Ocln constructs also concentrate at the apical junction with ZO-1 (right top and middle panel). Bar, 20 microns.
Mentions: In order to determine the spatial specificity of the labeling method we determined both the cellular localization of the fusion proteins and the subcellular patterns of biotinylated proteins. Unlike ZO-1 which is focused at the TJ, both claudins and Ocln also show variable localization to the lateral membrane [6,7,12,19,20,33,38,40]. As integral membrane proteins they are also expected to be near proteins in biosynthetic vesicular trafficking pathways [2,25,26,41–47]. As expected, en face immunofluorescent images of the TJ protein ZO-1 (Fig. 1A, left panels) and BL-Ocln, Ocln-BL and BL-Cldn4 fusion proteins (middle panels) reveals colocalization at TJs (right panels), as reported by myc epitope staining. The biotin ligase fusion proteins are also found to a variable extent in intracellular compartments. In contrast, we have previously shown that myc-tagged biotin ligase alone is diffusely distributed throughout the cells including the nucleus [10].

Bottom Line: When proteomic results were analyzed, the relative distribution among functional categories was similar between occludin and claudin-4 proximal proteins.However there were significant differences in the specific proteins comprising the functional categories near each of the tagging proteins, revealing spatial compartmentalization within the junction complex.Taken together, these results expand the inventory of known and unknown proteins at the tight junction to inform future studies of the organization and physiology of this complex structure.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Tight Junction Structure and Function, NHLBI, National Institutes of Health, Bethesda, MD, United States of America.

ABSTRACT
Tight junctions are complex membrane structures that regulate paracellular movement of material across epithelia and play a role in cell polarity, signaling and cytoskeletal organization. In order to expand knowledge of the tight junction proteome, we used biotin ligase (BioID) fused to occludin and claudin-4 to biotinylate their proximal proteins in cultured MDCK II epithelial cells. We then purified the biotinylated proteins on streptavidin resin and identified them by mass spectrometry. Proteins were ranked by relative abundance of recovery by mass spectrometry, placed in functional categories, and compared not only among the N- and C- termini of occludin and the N-terminus of claudin-4, but also with our published inventory of proteins proximal to the adherens junction protein E-cadherin and the tight junction protein ZO-1. When proteomic results were analyzed, the relative distribution among functional categories was similar between occludin and claudin-4 proximal proteins. Apart from already known tight junction- proteins, occludin and claudin-4 proximal proteins were enriched in signaling and trafficking proteins, especially endocytic trafficking proteins. However there were significant differences in the specific proteins comprising the functional categories near each of the tagging proteins, revealing spatial compartmentalization within the junction complex. Taken together, these results expand the inventory of known and unknown proteins at the tight junction to inform future studies of the organization and physiology of this complex structure.

No MeSH data available.