T1 /Podoplanin Shows Raft-Associated Distribu- tion in Mouse Lung Alveolar Epithelial E10 Cells

Aims: T1 /(podoplanin) is abundantly expressed in the alveolar epithelial type I cells (ATI) of rodent and human lungs. Caveolin-1 is a classical primary structural protein of plasmalemal invaginations, so-called caveolae, which represent specialized lipid rafts, and which are particularly abundant in ATI cells. The biological functions of T1 in the alveolar epithelium are unknown. Here we report on the characteristics of raft domains in the microplicae/microvillar protrusions of ATI cells, which contain T1 . Methods: Detergent resistant membranes (DRMs) from cell lysates of the mouse epithelial ATI-like cell line E10 were prepared using different detergents followed by flotation in a sucrose gradient and tested by Western and dot blots with raft markers (caveolin-1, GM1) and nonraft markers (transferrin receptor, PDI and -Cop). Immunocytochemistry was employed for the localization of T1 in E10 cells and in situ in rat lungs. Results: Our biochemical results showed that the solubility or insolubility of T1 and caveolin-1 differs in Triton X100 and Lubrol WX, two distinct non-ionic detergents. Caveolin-1 was unsoluble in both detergents, whereas T1 was Triton X-100 soluble but Lubrol WX insoluble. Immunofluorescence double stainings revealed that both proteins were colocalized with GM1, while caveolin-1 and T1 were not colocalized in the plasma membrane. Cholesterol depletion modified the segregation of T1 in Lubrol WX DRMs. Cellular processes in ultrathin sections of cultured mouse E10 cells were immunogold positive. Immunoelectron microscopy (postembedding) of rat lung tissue revealed the preferential localization of T1 on apical microvillar protrusions of ATI cells. Conclusion: We conclude that T1 and caveolin-1 are located in distinct plasma membrane microdomains, which differ in their protein-lipid interactions. The raft-associated distribution of T1 may have an impact on a specific, not yet clarified function of this protein in the alveolar epithelium.