RalGPS2 is involved in tunneling nanotubes formation in 5637 bladder cancer cells.

RalGPS2 is a Ras-independent Guanine Nucleotide Exchange Factor (GEF) for RalA containing a PH domain and an SH3-binding region and it is involved in several cellular processes, such as cytokinesis, control of cell cycle progression, differentiation, cytoskeleton organization and rearrangement. Up to now, few data have been published regarding RalGPS2 role in cancer cells, and its involvement in bladder cancer is yet to be established. In this paper we demonstrated that RalGPS2 is expressed in urothelial carcinoma-derived 5637 cancer cells and is essential for cellular growth. These cells produces thin membrane protrusions that displayed the characteristics of actin rich tunneling nanotubes (TNTs) and here we show that RalGPS2 is involved in the formation of these cellular protrusions. In fact the overexpression of RalGPS2 or of its PH-domain increased markedly the number and the length of nanotubes, while the knock-down of RalGPS2 caused a strong reduction of these structures. Moreover, using a series of RalA mutants impaired in the interaction with different downstream components (Sec5, Exo84, RalBP1) we demonstrated that the interaction of RalA with Sec5 is required for TNTs formation. Furthermore, we found that RalGPS2 interacts with the transmembrane MHC class III protein leukocyte specific transcript 1 (LST1) and RalA, leading to the formation of a complex which promotes TNTs generation. These findings allow us to add novel elements to molecular models that have been previously proposed regarding TNTs formation.

Hydrogen-peroxide and silicone-hydrogel contact lenses: Worsening of external eye condition and tear film instability.

PURPOSE: The aim is discussing the origins of worsening of external eye condition (EEC) and of tear film (TF) instability after wear of silicone-hydrogel contact lenses (CLs) with hydrogen-peroxide (H2O2) care system. METHODS: EEC and TF stability were evaluated before and after 15days of wear combined with different care systems: (1) H2O2, (2) detergent solution and H2O2, (3) multipurpose solution (MPS), (4) H2O2 and artificial tears. In-vitro cell mortality tests were performed after 24h cell incubation with CLs treated with H2O2. Photon correlation spectroscopy (PCS) was carried out on tears of non-wearers and CL wearers who used MPS or H2O2 solution. RESULTS: Worsening of EEC was observed only for the group using H2O2 (group 1). In-vitro, cell mortality was found higher for worn CL than for unworn CLs. Worsening of TF stability was observed regardless of care system and also PCS results on tears of CL wearers were found different compared to non-wearers regardless of care system. The only observed remedy for tear instability of CL wearers was found to be the administration of artificial tears. CONCLUSIONS: Worsening of EEC of CL wearers using H2O2 is attributed to H2O2 scarce cleaning efficacy, which can be solved by adding a CL detergent solution. The origin of TF instability is found to be different. A remedy was found to be the administration of artificial tears, whose effect could be attributed either to the role of specific components or to rinsing and replacement of TF during wear.

Photon correlation spectroscopy applied to tear analysis.

This study aims to deepen the knowledge on tear film properties by the development of a protocol for analyses of Photon Correlation Spectroscopy (PCS) on human tears and by the comparison between PCS results obtained on tears of contact lens wearers and non-wearers. Tears (5µL) were collected by a glass capillary. The analyses provide the hydrodynamic diameter of tear components by analyzing intensity fluctuations in time of scattered light. PCS appears a promising technique for studying tear features and for shedding light on specific eye conditions, such as on the clinical effects of CL wear. In fact, statistical difference (p<0.001) was found between the measured mean hydrodynamic diameter of tear components of wearers and non-wearers, the resulting value significantly higher for CL wearers. The scenario does not substantially change after (25±5)min from the CL removal. The difference is attributed to changes in the interactions between tear constituents due to CL wear. In order to get deeper insights on the influence of CL wear on aggregation and structure of tear components, a preliminary Electron Spin Resonance (ESR) investigation was performed, monitoring Fe3+ species. ESR spectra on tears of both CL wearers and non-wearers showed the presence of intense signals, probably associated to iron (III) centers in proteins such as lactoferrin, and a weaker resonance attributable to Fe3+ species interacting with S-S bridges of lysozyme. Differences in ESR spectra between CL wearers and non-wearers were detected and tentatively ascribed to changes in coordination or in local environment of Fe3+ centers connected to aggregation phenomena induced by CL wear, which promote their interaction with other neighboring iron species.

CK2 and GSK3 phosphorylation on S29 controls wild-type ATXN3 nuclear uptake.

In the present work we show that murine ATXN3 (ATXN3Q6) nuclear uptake is promoted by phosphorylation on serine 29, a highly conserved residue inside the Josephin domain. Both casein kinase 2 (CK2) and glycogen synthase kinase 3 (GSK3) are able to carry out phosphorylation on this residue. S29 phosphorylation was initially assessed in vitro on purified ATXN3Q6, and subsequently confirmed in transfected COS-7 cells, by MS analysis. Site-directed mutagenesis of S29 to an alanine was shown to strongly reduce nuclear uptake, in COS-7 transiently transfected cells overexpressing ATXN3Q6, while substitution with phospho-mimic aspartic acid restored the wild-type phenotype. Finally, treatment with CK2 and GSK3 inhibitors prevented S29 phosphorylation and strongly inhibited nuclear uptake, showing that both kinases are involved in ATXN3Q6 subcellular sorting. Although other authors have previously addressed this issue, we show for the first time that ATXN3 is phosphorylated inside the Josephin domain and that S29 phosphorylation is involved in nuclear uptake of ATXN3.