PB1280 - Investigation of Podoplanin Signalling Complexes in Primary Lymphatic Endothelial Cells

Background: The lymphatic system is an extensive network of vessels which plays an important role in numerous physiological and pathological conditions such as the removal of excess fluid from tissues, immune surveillance and cancer metastasis. Despite this importance little is known about its formation. However, blood platelets have been found to play a vital role in the separation of the lymphatic vasculature from blood vessels throughout embryonic development and into adulthood. This role is independent of the classical function of platelets in thrombosis and haemostasis and is mediated by the interaction of the platelet C-type lectin-like receptor, CLEC-2, with Podoplanin on lymphatic endothelial cells (LECs). The mechanism by which Podoplanin relays signals from platelets to the LECs is unclear.

Aims: This study aims to identify membrane associated Podoplanin containing signalling complexes in primary LECs and to understand the spatial regulation of these complexes.

Methods: LEC Podoplanin complexes were preserved by cell lysis in a Brij 97 buffer to preserve tetraspanin domains. Podoplanin containing complexes were immunoprecipitated, proteins separated by SDS-PAGE and the presence of complex members determined by Western blotting. Super resolution microscopy (dSTORM) was used to study the spatial distribution of these complexes on the LEC surface.

Results: Podoplanin forms membrane associated complexes with multiple proteins, including the tetraspanin CD9. The composition of Podoplanin complexes changes following stimulation of the LEC signalling receptor VEGFR3, a receptor essential for lymphangiogenesis.

Conclusion(s): Podoplanin resides in tetraspanin domains, the composition of which changes upon the initiation of VEGFR3 signalling. The functional consequence of these dynamic changes is yet to be elucidated.