T1alpha/podoplanin shows raft-associated distribution in mouse lung alveolar epithelial E10 cells.
OBJECTIVE
T1alpha/(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 T1alpha 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 T1alpha.


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 beta-Cop). Immunocytochemistry was employed for the localization of T1alpha in E10 cells and in situ in rat lungs.


RESULTS
Our biochemical results showed that the solubility or insolubility of T1alpha and caveolin-1 differs in Triton X-100 and Lubrol WX, two distinct non-ionic detergents. Caveolin-1 was unsoluble in both detergents, whereas T1alpha was Triton X-100 soluble but Lubrol WX insoluble. Immunofluorescence double stainings revealed that both proteins were colocalized with GM1, while caveolin-1 and T1alpha were not colocalized in the plasma membrane. Cholesterol depletion modified the segregation of T1alpha 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 T1alpha on apical microvillar protrusions of ATI cells.


CONCLUSIONS
We conclude that T1alpha and caveolin-1 are located in distinct plasma membrane microdomains, which differ in their protein-lipid interactions. The raft-associated distribution of T1alpha may have an impact on a specific, not yet clarified function of this protein in the alveolar epithelium.
Barth
Kathrin
K
Institute of Anatomy, Medical Faculty Carl Gustav Carus, TU Dresden, Germany.
Bläsche
Robert
R
Kasper
Michael
M
D056809:Alveolar Epithelial Cells / Q000166:cytology*; D000818:Animals; D051242:Caveolin 1 / Q000032:analysis; D002460:Cell Line; D006801:Humans; D008168:Lung / Q000737:chemistry; D008562:Membrane Glycoproteins / Q000032:analysis*; D021962:Membrane Microdomains / Q000737:chemistry*; D051379:Mice; D017830:Octoxynol; D011092:Polyethylene Glycols; D051381:Rats; D012995:Solubility; D000098853:Podoplanin
D051242:Caveolin 1; D008562:Membrane Glycoproteins; D017830:Octoxynol; D011092:Polyethylene Glycols; C490441:Pdpn protein, mouse; D000098853:Podoplanin; C017474:Lubrol WX