T1alpha interacts with Myh9, a nonmuscle myosin, in type 1 lung epithelial cells

Abstract

T1α (also known as Podoplanin), is a highly expressed gene found in lung alveolar type I (ATI) epithelial cells and in several other organ systems including lymphatic vessels and kidney. T1α is necessary for proper alveolar development and cell proliferation in the lung. It encodes an apical surface membrane protein involved in cell migration and morphogenesis. T1α also is upregulated at the leading edge of invasive tumors, and is used as a negative prognostic for remission-free recovery in cancer patients. Although T1α is known to interact with other cell membrane proteins, its molecular function and mechanism of action is not fully understood.

To elucidate the molecular mechanisms of T1α regulation in normal lung alveolar development, we decided to identify T1α interacting proteins in lung cells. We performed mass spectrometry analysis on protein bands isolated from T1α co-immunoprecipitation, western blot, and immunofluorescence analyses.

The mass spectrometry analysis of co-immunoprecipitated proteins with T1α resulted in the identification of several T1α interacting protein candidates, most notably Myh9. Myh9 is a member of a family of non-muscle myosins which is expressed in the lung epithelium, as well as in the kidneys’ podocytes, where its absence leads to a phenotype similar to reduced expression of T1α. Moreover, we confirmed the interaction of Myh9 with T1α by co-immunoprecipitation analysis in cell lysates derived from E10, a murine ATI-like cell line. Similarly, immunofluorescence staining showed that in the murine lung Myh9 and T1α display similar patterns of expression at the edge of ATI epithelial cells. Our results suggest that T1α interaction with Myh9 may play an important role in T1α-mediated cell migration and morphogenesis.