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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.

Ubiquitin-specific peptidase 8 (USP8) is a deubiquitinating enzyme that works as a regulator of endosomal sorting and vesicle morphology in cultured cells. Its function in vivo is, however, unknown as USP8 gene deletion leads to embryonic lethality. Previously, we have shown that USP8 is highly expressed in male germ cells. These cells develop a peculiar acidic vesicle that is indispensable for fertilization, the acrosome; USP8 might be involved in vivo in acrosomogenesis. The objective of this study was to test this hypothesis by determining if selective components of the early endosomal machinery interact functionally with USP8 during acrosomogenesis using protein-protein interaction assays and double/triple immunolabeling. Moreover, by exploiting the characteristic of USP8 that exhibits a microtubule interacting and trafficking/transport (MIT) domain, we verified whether USP8 effectively associates with spermatid microtubules by microtubule cosedimentation and binding assays. USP8 was able to interact with spermatid ESCRT-0 (endosomal-sorting complex required for transport-0) and microtubule structures; USP8/ESCRT-0-labeled vesicles, monitored by fluorescence microscopy, were found to contribute to acrosome formation while USP8 can directly link, via its MIT domain, the labeled vesicles/developing acrosome to microtubules, which could favor both acrosome assembly and shaping. VPS54, the vacuolar-sorting protein responsible for early endocytic retrograde transport, was here detected for the first time in male germ cells; VPS54 followed the intracellular route of USP8/ESCRT-0-labeled vesicles during acrosomogenesis. We concluded that in vivo USP8 has a role strongly associated with acrosome biogenesis and that the early endosome pathway is significantly involved in the process, which suggests that the acrosome could be a novel lysosome-related organelle.