Podoplanin function is switched by partner proteins on fibroblastic reticular cells

Lymph node expansion is pivotal during immune activation. It is controlled by interactions between CLEC-2hi migratory dendritic cells and podoplanin+ fibroblastic reticular cells (FRCs), resulting in rapid loss of actomyosin contractility permitting FRC spreading. Podoplanin is an inflammatory marker in many pathologies, reported to facilitate both contractility and cell protrusions. However, how podoplanin expression is regulated, and how this one membrane protein can elicit these opposing phenotypes is unknown. We report that CLEC-2 binding to FRCs induces transcriptional upregulation of podoplanin, increasing surface protein expression. Further, we find that tetraspanin CD82 is required for trafficking of podoplanin to the plasma membrane. At the cell surface, podoplanin elicits multiple functions, balanced by its membrane binding partners hyaluronan receptor CD44 and tetraspanin CD9. FRCs lacking expression of both CD44 and CD9 are hypercontractile, and upon binding to CLEC-2hi dendritic cells, both CD44 KO and CD9 KO FRCs exhibit defective protrusion formation and fail to spread. Podoplanin co-localises with CD44 or CD9 in distinct membrane domains, and both CD44 and CD9 are required for FRCs to spread in response to dendritic cells, however they control formation of cell protrusions via different and complimentary mechanisms. In vivo, surface expression levels of podoplanin, CD44 and CD9 are upregulated on T-cell zone FRCs in the early phase of lymph node expansion. Our data support a model whereby podoplanin resides in distinct plasma membrane do-mains, and that CLEC-2 binding serves as a molecular switch to change podoplanin function.