supplentary and video of manuscript entitled: RalGPS2 interacts with Akt and PDK1 promoting Tunneling nanotubes formation in bladder cancer and kidney cells microenvironment

RalGPS2 is a Ras-independent Guanine Nucleotide Exchange Factor (GEF) for RalA GTPase involved in several cellular processes such as cytoskeletal organization and tunneling nanotubes (TNTs) formation, as here demonstrated in 5637 bladder cancer cells. In particular, TNTs have emerged as a novel mechanism of cell-cell communication and in tumor microenvironment they play a central role in cancer progression and metastasis. However, up to now little is known about the molecular machinery underlying nanotubes formation in tumors. In this paper, we demonstrated that mid- and high-stage bladder cancer cell lines display functional TNTs able to exchange mitochondria. Moreover, using confocal fluorescence time-lapse microscopy we observed mCherry tagged proteins RalA and the transmembrane MHC class III protein leukocyte specific transcript 1 (LST1) trafficking between 5637 cells via TNTs. Furthermore, we found that RalGPS2 is essential for nanotubes generation and stress conditions boost its expression both in 5637 and HEK293 cells. Finally, we determined that RalGPS2 interacts with Akt and PDK1 in addition to LST1 and RalA leading to a formation of a complex which promotes nanotubes formation. In conclusion, these findings suggest a molecular machinery in which RalGPS2 orchestrates the assembly of multimolecular complexes for nanotubes generation in the tumor microenvironment.