Tight junctions are membrane microdomains

Tight junctions (TJ) of polarized epithelial cells regulate barrier functio n at mucosal surfaces. Structural proteins of TJs include hyperphosphorylat ed occludin (HO) and the peripheral membrane protein, ZO-1, Since TJs are d ynamically regulated, and lipid-modified signal transduction proteins local ize 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 me mbranes from a polarized intestinal epithelial cell line, T84. Here we repo rt 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 g radient fractions included Triton X-100 (TX-100) insolubility, light scatte ring at 600 nm, buoyant density of approximately 1.08 g/cm(3) and increased cholesterol content compared to high density fractions. Similar results we re obtained using natural epithelium. Unlike the TJ proteins HO and ZO-1, o ther basolateral transmembrane proteins including E-cadherin, c-met and bet a 1 integrin were not increased in DIG-like fractions. Immunoprecipitation studies revealed coprecipitation of a pool of occludin with caveolin-l, a s caffolding protein abundant in DIGs, Coprecipitation results were supported by immunofluorescence and immunogold labeling studies demonstrating caveol in-l localization in the apical membrane and focal colocalization with occl udin in TJs, TJ disassembly by calcium chelation resulted in displacement o f TJ proteins from the 'raft-like' compartment, Our findings suggest that r aft-like compartments play an important role in the spatial organization of TJs and probably in regulation of paracellular permeability in epithelial cells.