Tight junctions are membrane microdomains.
Tight junctions (TJ) of polarized epithelial cells regulate barrier function at mucosal surfaces. Structural proteins of TJs include hyperphosphorylated occludin (HO) and the peripheral membrane protein, ZO-1. Since TJs are dynamically regulated, and lipid-modified signal transduction proteins localize to TJs, we considered the possibility that the TJ itself is composed of microdomains with unique structure. Differential detergent extraction and isopycnic sucrose density gradients were utilized to isolate TJ-enriched membranes from a polarized intestinal epithelial cell line, T84. Here we report that major pools of hyperphosphorylated occludin (HO) and ZO-1 are found in raft-like membrane microdomains with characteristics of the previously described detergent-insoluble glycolipid rafts (DIGs). Properties of such gradient fractions included Triton X-100 (TX-100) insolubility, light scattering at 600 nm, buoyant density of approximately 1.08 g/cm(3) and increased cholesterol content compared to high density fractions. Similar results were obtained using natural epithelium. Unlike the TJ proteins HO and ZO-1, other basolateral transmembrane proteins including E-cadherin, c-met and &bgr; 1 integrin were not increased in DIG-like fractions. Immunoprecipitation studies revealed coprecipitation of a pool of occludin with caveolin-1, a scaffolding protein abundant in DIGs. Coprecipitation results were supported by immunofluorescence and immunogold labeling studies demonstrating caveolin-1 localization in the apical membrane and focal colocalization with occludin in TJs. TJ disassembly by calcium chelation resulted in displacement of TJ proteins from the 'raft-like' compartment. Our findings suggest that raft-like compartments play an important role in the spatial organization of TJs and probably in regulation of paracellular permeability in epithelial cells.
Nusrat
A
A
Epithelial Pathobiology Research Unit, Department of Pathology, Emory University School of Medicine, Atlanta GA, USA. anusrat@emory.edu
Parkos
C A
CA
Verkade
P
P
Foley
C S
CS
Liang
T W
TW
Innis-Whitehouse
W
W
Eastburn
K K
KK
Madara
J L
JL
D051242:Caveolin 1; D022461:Caveolins; D002462:Cell Membrane / Q000737:chemistry*; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004847:Epithelial Cells / Q000737:chemistry* / Q000648:ultrastructure; D006017:Glycolipids / Q000737:chemistry; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification; D062793:Occludin; D017830:Octoxynol / Q000494:pharmacology; D010750:Phosphoproteins / Q000737:chemistry* / Q000302:isolation & purification; D017434:Protein Structure, Tertiary / Q000187:drug effects / Q000502:physiology*; D012995:Solubility / Q000187:drug effects; D019108:Tight Junctions / Q000737:chemistry*; D062826:Zonula Occludens-1 Protein
C495540:CAV1 protein, human; D051242:Caveolin 1; D022461:Caveolins; D002614:Chelating Agents; D003902:Detergents; D006017:Glycolipids; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; D010750:Phosphoproteins; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; D004533:Egtazic Acid; D017830:Octoxynol