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Ral proteins are members of the Ras small GTPase superfamily and are involved in multiple cellular events including cytoskeletal organization and tumorigenesis “in vitro” and in animal models. RalA overexpression is associated with bladder and prostate cancer. RalGPS2 is a guanine exchange factor (GEF) for RalA belonging to RalGPS family that contains a GEF domain, a PxxP motif and a PH domain (Ceriani et al., 2007). Previous experiments have demonstrated that RalGPS2 activates RalA in vivo, while the overexpression of PH-PxxP region acts as a dominant negative for RalA activation in NIH3T3 and PC12 cells (Ceriani et al., 2010). These data suggest that the PH-PxxP region of RalGPS2 could inhibit RalA activation in tumor cell lines. The aim of this work is to analyze the role of RalGPS2 and of its PH-PxxP region in human bladder cancer cells (5637). The overexpression of PH-PxxP region of RalGPS2 reduced the level of RalA-GTP also in 5637 cells and confocal microscopy analyses revealed that there is a partial co-localization between RalA and truncated proteins containing only the PH domain or the PH-PxxP region of RalGPS2 at plasma membrane and in thin membrane protrusions. Besides the overexpression of PH domain and PH-PxxP region induced a marked cytoskeleton re-organization: the PH domain caused formation of thin vesiculating protrusion while the PH-PxxP domains caused formation of long inter-cellular structure. Morphological characterization of protrusions induced by PH domain overexpression reveled that they were tunnelling nanotubes (TNTs). TNTs are a new type of cell-cell communication between remote cells and through TNTs cells can exchange various cellular components and signals (Hase K. et al., 2009). Finally, immunoprecipitation assays reveal the presence of a complex between RalA, RalGPS2 and LST1, a transmembrane protein that induces TNTs formation.

Ral GTPases have been implicated in tumorigenesis and metastasis in vitro and animal models; furthermore Ral pathway appears to play a prominent role in transformation of human cells (Smith et al. 2007 Clin. Cancer Res. 13, 3803-3813). RalGPS2 is a GEF for RalA belonging to RalGPS family that contains a well conserved Ras-GEF domain, a PxxP motif and a PH domain. Previous experiments demonstrated that RalGPS2 activates RalA in vivo, while its PH-PxxP domain acts as a dominant negative for RalA activation in NIH3T3 and PC12 cells. These data suggest that RalGPS2 PH-PxxP domain could inhibit RalA activation influencing cytoskeleton rearrangements. The aim of this project is to analyse the role of RalGPS2 and of its PH-PxxP domain in human bladder cancer cells (5637) and rodent transformed cells (NIH3T3-k-Ras). RalA and RalGPS2 were highly expressed in 5637 cells and the overexpression of PH-PxxP domain reduced the level of RalA-GTP. Besides the overexpression of PH and PH-PxxP domains in both cell lines induced a marked cytoskeleton re-organization: in particular the PH domain caused formation of thin vesiculating protrusion while the PH-PxxP domain caused formation of long inter-cellular structure probably involved in the exchange of signals and cellular components between cells.

The vesicular trafficking inside eukaryotic cells is a highly regulated process that requires specificity at each transport step. The selective interaction between donor and acceptor membranes is mediated by tethering factors, among which GARP (Golgi associated retrograde protein) complex. The vacuolar protein sorting Vps54 is a subunit of GARP and in yeast is involved in the retrograde transport of early endosomes to the trans Golgi network. In the mouse, a single point mutation near the C-terminus of Vps54 (L967Q) gives rise to the Wobbler phenotype, characterized by defects in motor neuron function and male sterility. We took advantage of this mutant variant to investigate more in detail the role of Vps54 in vesicular trafficking of mammalian cells. In cultured HEK293 cells, Vps54-GFP colocalizes with Golgi and partially with endosomal compartment. The localization of the wobbler variant is comparable with that of wt protein, even if Vps54L967Q-GFP is slightly more diffuse into the cytoplasm. This result supports the literature data suggesting that wobbler Vps54 retains activity in trafficking. Since wobbler disease specifically affects spermatogenesis, we focused our attention on Vps54 in developing mouse germ cells. In the physiological contest, we demonstrated that Vps54 is involved in acrosome biogenesis. The acrosome is an acidic vesicle of not better-identified origin, indispensable for fertilization. Wobbler spermatozoa are resulted to lack a true acrosome: during sperm cell differentiation Vps54(L967Q) marks numerous punctuated vesicles that, on the contrary of the wt Vps54-labeled ones, are not able to coalesce and develop into the acrosome. These results were confirmed by double immunostaining with USP8, a deubiquitinating enzyme marker of the endocytic pathway. We investigated by exploiting recombinant proteins if the absence of an acrosome could depend on the failure of Vps54L967Q to interact with sperm factors involved in trafficking machinery

Ral proteins are members of the Ras GTPase superfamily and localize at the plasma membrane, in endocytic and exocytic vesicles and in synaptic vesicles. These GTPases are involved in multiple cellular events including proliferation, migration, differentiation, cytoskeletal organization, vesicular transport, exocytosis and receptor endocytosis. They are also implicated in tumorigenesis, invasion and metastasis. RalGPS2 is a guanine nucleotide exchange factor for RalA belonging to RalGPS family. It is a 590 aminoacids polypeptide that contains a well conserved Ras-GEF domain, a PxxP motif and a PH (Pleckstrin Homology) domain. Previous experiments have demonstrated that RalGPS2 can activate RalA in vivo, while the PH-PxxP domain acts as a dominant negative for RalA activation in both NIH3T3 and PC12 cells. These data suggest that the PH-PxxP domain of RalGPS2 could inhibit RalA activation, thus influencing cytoskeleton rearrangement. The principal aim of this work is to analyze the role of RalGPS2 and of its PH-PxxP domain in human and rodent cells transformation. In particular our attention was been focused on transformed murine fibroblasts (NIH3T3 k-Ras) and on human bladder cancer cells (5637 cell line). We found that RalA and RalGPS2 were highly expressed in 5637 human bladder cancer cells and that the overexpression of PHPxxP domain reduced the level of active RalA bound at GTP. Furthermore the expression of PH and PH-PxxP domains in both NIH3T3 k-ras and 5637 cell line, induced a marked cytoskeleton re-organization. In particular PH domain caused formation of thin protrusions with the presence of vesicles. PH-PxxP domain caused formation of long inter-cellular structure that are probably involved in the exchange of signals and cellular components between different cells. It will be interesting in the future to understand the molecular nature of these ‘‘hairy’’ structures that could be nanotubes.

L’osteoartrite (OA) è una delle malattie reumatiche con più alta incidenza nel mondo moderno e rappresenta la pricipale cause di disabilità. L’OA è data da un disquilibrio tra degradazione e riparazione della cartilagine, a favore della degradazione, con un incremento dell’attivita degli enzimi catabolici come le matrici metalloproteinasiche. I condrociti sono responsabili della riparazione e della biosintesi degli elementi che compongono la ECM. Diversi studi supportano l’ipotesi che il diabete è uno dei fattori che causano l’osteoartrite. Sebbene non si conoscono ancor ai meccanismi molecolari che permettano l’associazione tra diabete e OA. Abbiano analizzato la crescita cellulare, i livelli dei ROS e l’apoptosi di condrociti posti in terreno con differenti concentrazioni di glucosio. I risultati mostrano che i condrociti preferiscono la concentrazione 2.5 mM di glucosio che è stata utilizzata come concentrazione normoglicemica; mentre 25 mM di glucosio è stata utilizzata come concentrazione iperglicemica. I livelli di ROS e la morte cellulare aumentano in condrociti cresciuti in alto glucosio, anche il citoscheletro si presenta disorganizzato in cellule C28/I2 cresciute in queste condizioni. Questo correla con lo stato di attivazione della GTPasi RalA, la cui attivazione è ridotta in condizioni iperglicemiche. Infatti questa GTPasi è coinvolta nella regolazione della riorganizzazione citoscheletrica. Inoltre sono stati effettuati esperimenti utilizzando medium contenenti ITS (Insulina, transferrina e selenio) che pruomuove il differenziamento a condrocita; mentre l’interleuchina-1β per simulare l’ambiente osteoartritico. Anche in uesto caso i livelli di RalA GTP diminuiscono in cellule cresciute in 25 mM glucosio e stimolate con IL-1β. Inoltre in queste condizioni diminuiscono ache i livelli di p-ERK1/2. Sono stati valutati anche i livelli di NF-κB, iNOS e LC3II. I risultati dimostrano che l’alto glucosio blocca l’autofagia nei condrociti. Inoltre sono stati valutati anche i pathways attivati dall’alto glucosio in condrociti primari umani dopo 24 h di trattamento. I risultati mostrano che la fosforilazione di ERK1/2, p38, Akt e p65 è alterata in condrociti cresciuti in condizioni iperglicemiche tale evento è correlato con un aumento di MMP-13. Per analizzare meglio il ruolo dei ROS nei condrociti ho lavorato per 6 mesi nel laboratorio del Dr. Loeser alla scuola di medicina presso l’università della north Carolina a Chapel Hill. Ho lavorato su PRX6 che è coinvolto nella detossificazione dai ROS. Lo stato di ossidazione di PRX6 è stato valutato nei condrociti trattati con diversi stimoli come H2O2, Fn-f, menadione (men) and DMNQ. Dopo questi esperimenti abbiamo voluto osservare se PRX6 potesse influenzare i pathway delle MAPK in cellule trattate con IGF-1, menadione, combinazione tra IGF-1 e menadione e con Fn-f. è stata valutata la localizzazione di PRX6 che si è visto essere sia nucleare che citoplasmatico. Inoltre ho lavorato in collaborazione con Prof. Laura Cipolla e Prof. Maddalena Collini per sviluppare e caratterizzare nuovi idrogel fatti di gelatina usando come agente cross-linkante lo squarato. Osteoarthritis (OA) is the most common rheumatic disease in the world and represents the first cause of disability in the world. OA results from the loss of balance between degradation and repair inside cartilage, in favor of degradation, with increased activity of catabolic enzymes such as matrix metalloproteinases and decreased production of ECM proteins. Chondrocytes are responsible for the repair and biosynthesis of elements of the extracellular matrix. Experimental findings support the hypothesis that diabetes is an independent risk factors for OA. However, correct molecular mechanisms underlying the diabetes-associated OA phenotype is still largely unknown. Firstly chondrocytes cell growth, ROS levels and apoptosis were analyzed using different glucose concentration. Results shown that chondrocytes prefer 2.5 mM of glucose which was used as normal glucose concentration and 25 mM of glucose was used as high glucose concentration. ROS levels and cell death increase in chondrocytes growth in high glucose environment. Also cytoskeletal network is more disorganized in C28/I2 cells growth at high glucose concentration, this correlates with different RalA-GTP levels which is involved in the regulation of cytoskeletal organization. Experiments were performed even using medium supplemented by ITS (Insulin-transferrin-selenium) to promote chondrocyte differentiation and IL-1β was used to simulate osteoarthritic cartilage environment. Ral A-GTP levels are lower in cells grown in 25 mM of glucose and stimulated with IL1β. Levels of p-ERK1/2 decrease in cells grown at high glucose concentration and in cells stimulated with IL1β. Furthermore, NF-κB, iNOS and LC3II levels were evaluated. Results demonstrate that high glucose media block autophagic process in chondrocytes. Effect of glucose concentration on human primary chondrocytes cells was evaluated after only 24 h to understand which signaling pathways is activated by high glucose environment. Phosphorylation of ERK1/2, p38, Akt and p65 is altered in chondrocytes growth at high glucose concentration and this correlates with an increase secretion of MMP-13. To better analyze the role of ROS levels in the chondrocytes I worked for 6 months in the Dr. Loeser Lab at the School of Medicine in the University of North Carolina at Chapel Hill; one of the best lab in the cartilage biology field. I worked on PRX6 which is involved in the recovery from H2O2. Oxidation state of PRX6 was evaluated in chondrocytes treated with different stimuli, like H2O2, Fn-f, menadione (men) and DMNQ. After this experiment, we wanted to see if PRX6 could impact the MAPK signaling pathways in cells treated with IGF-1, menadione, combination of menadione and IGF-1 and with Fn-f. Localization of PRX6 was analyzed using nuclear and cytoplasm extraction. Then I worked in collaboration with Prof. Laura Cipolla and Prof. Maddalena Collini to develop and characterized a new gelatin-based hydrogel using Diethylsquarate as crosslinker.

RalGPS2 è uno scambiatore appartenente alla famiglia RalGPS, composto da un dominio catalitico Cdc25-like nella regione N-terminale, un motivo PxxP nella regione centrale, e un dominio di omologia alla Pleckstrina (PH) nella regione C-terminale. E’ stato precedentemente dimostrato che RalGPS2 attiva in “vivo” la GTPasi RalA, mentre la regione PH-PxxP si comporta da dominante negativo per l’attività di RalA in cellule NIH3T3 e PC12. Inoltre, se è overespresso RalGPS2 causa cambiamenti morfologici consistenti nelle cellule HEK293, suggerendo che esso possa avere effetti sul citoscheletro. Tutto ciò suggerisce un possibile ruolo di RalGPS2 nella riorganizzazione del citoscheletro anche in linee cellulari tumorali. A tal fine è stata scelta come modello la linea cellulare umana 5637 di cancro alla vescica, in cui la GTPasi RalA è iperattiva. Nel presente lavoro abbiamo dimostrato che RalGPS2 da solo è in grado attivare RalA in “vivo”, mentre la sua deplezione ne abbassa notevolmente i livelli. In più si è dimostrato che la regione PH-PxxP e il dominio PH di RalGPS2 si comportano da dominanti negativi per l’attività di RalA. Inoltre, analisi al confocale hanno rivelato una parziale ma marcata co-localizzazione tra RalA, RalGPS2, il dominio PH e la regione PH-PxxP a livello della membrana plasmatica e in sottili protrusioni di membrana. La presenza di queste protrusioni in cui si localizzava RalA ha suggerito che esse potessero essere nanotubi traforati (TNT). I TNT sono condotti intracellulari per il trasporto di vari componenti cellulari o segnali importanti per la comunicazione cellulare. Siccome i TNT sono stati precedentemente descritti come strutture costituite da actina ma non da tubulina, si è utilizzato questo criterio per caratterizzare tali protrusioni. L’analisi al microscopio confocale ha evidenziato la presenza di protrusioni ricche in actina ma povere in tubulina. Per valutare se effettivamente RalGPS2 e i suoi domini influenzino la formazione dei TNT, si è condotta un’analisi al microscopio confocale in cui si andava a caratterizzare le protrusioni formate dalle cellule. Un’analisi statistica dettagliata ha evidenziato che RalGPS2 supporta la formazione di TNT in cellule 5637. Successivamente si è cercato di analizzare il ruolo degli effettori di RalA nella formazione dei TNT. Un’analisi statistica accurata ha dimostrato che il blocco di Sec5 (subunià del complesso delle esocisti ed effettore di RalA) riduce fortemente la formazione dei TNT. Dunque sia Sec5 che RalGPS2 sembrano giocare un ruolo chiave nella genesi di queste strutture. Per confermare il ruolo di RalGPS2 nella formazione dei TNT e per valutare se esso cooperi assieme a Sec5 in tale processo abbiamo effettuato un saggio di co-immunoprecipiatazione. Tale analisi rivela la presenza di un complesso tra RalA, RalGPS2,LST1 (proteina che induce la formazione dei TNT) e Sec5. Inoltre è stato dimostrato che RalGPS2 supporta la formazione dei TNT maggiormente in condizioni di carenza di nutrienti. I risultati ottenuti ci suggeriscono l’esistenza di due pathway compresenti, ma maggiormente attivati in condizioni diverse. In questa proposta RalGPS2 interagendo con LST1 e RalA determina la formazione di un complesso che in condizioni di stress si attiva e permette l’interazione tra RalA e Sec5. L’interazione RalA-Sec5 determina l’assemblaggio di un complesso multi-proteico che controlla la formazione dei nanotubi. Al contrario in condizioni di stimolo proliferativo, sebbene il complesso RalGPS2-LST1-RalA sia comunque presente e in parte attivo è eclissato dall’attivazione di un altro pathway che ha come protagonisti i GEF della famiglia RalGDS, la GTPasi RalA e Sec5. In queste condizioni infatti i GEF RalGDS sono attivi e interagiscono con RalA attivandola. In questo stato attivo RalA interagisce a sua volta con Sec5 promuovendo l’assemblaggio del complesso delle esocisti e regolando così l’esocitosi. RalGPS2 is a murine guanine nucleotide exchange factor belonging to RalGPS family; that contains a well conserved CDC25-like domain in the N-terminal region, a PxxP motif in central region and a PH (Pleckstrin Homology) domain in the C-terminus. It has been demonstrated that RalGPS2 can activate RalA in vivo, while the PH-PxxP domain behaves as a dominant negative for RalA activation in NIH3T3 and PC12 cells. Furthermore, when overexpressed, RalGPS2 causes considerable morphological changes in HEK293 cells, suggesting its possible role on cytoskeleton re-organization. These data suggest us a possible role of RalGPS2 and its domains in cytoskeleton re-modelling also in tumour cell lines. For this purpose it has been chosen the human bladder cancer cell line 5637, as a model. In the present work it has been shown that RalGPS2 alone is able to activate RalA in “vivo”, while its depletion significantly lowers RalA levels. Furthermore, it has been demonstrated that PH-PxxP region and PH domain of RalGPS2 behave as dominant negatives for RalA activation. Moreover, confocal analysis reveals a partial, but marked co-localization between RalA, RalGPS2, the PH domain and the PH-PxxP region at the level of plasma membrane end in thin membrane protrusions. The presence of these protrusions in which localize the GTPase RalA suggested us that these structures could be Tunneling Nanotubes (TNTs). TNTs are intercellular conduits and have been shown to enable the transport of various cellular components and signals, they are important for cellular communication between cells. Since nanotubes were initially described to contain actin but not tubulin we used this criterion to characterize the protrusions that we have observed in 5637 cells. Confocal analysis reveals presence of protrusions rich in actin but poor in tubulin. To determinate whether RalGPS2 and its domain induce formation of TNTs, it has been made a confocal analysis in which it has been characterized protrusions formed by cells. Statistical analysis reveals that RalGPS2 supports TNTs formation in 5637 cells. Later, it has been analyzed the role of RalA effectors in TNTs formation. Statistical analysis shown that lack of interaction between RalA and Sec5 (subunit of exocyst complex and RalA effector) strongly reduces nanotubes formation. Therefore, both Sec5 and RalGPS2 seem to play a key role in generation of these structures. To confirm the role of RalGPS2 in TNTs formation and to evaluate whether it cooperates with Sec5 in this process, it has been performed an co-immunoprecipitation assay. This investigation reveals the presence of a complex between RalA,RalGPS2, LST1 (protein which induces TNTs formation) and Sec5. Moreover, it has been demonstrated that RalGPS2 supports TNT formation more in conditions of nutrient deficiency. Results obtained suggest the existence of two coexisting pathways, but more activates under different conditions. In this proposal, interaction between RalGPS2, LST1 and RalA establishes formation of a complex that under stress condition is active and allows the interaction between the RalA and Sec5. RalA-Sec5 interaction determines the assembly of multi-protein complex which controls TNTs formation. On the contrary, in proliferative stimulus conditions, while RalGPS2-LST1-RalA complex is still present and partially activated, it is outclassed by the activation of a distinct pathway in which GEFs of the RalGDS family, the RalA GTPase and Sec5 play a pivotal role. In such conditions, RalGDS GEFs are activated and interact with the RalA GTPase while promoting the GDP-GTP exchange. RalA in its active state also interacts with Sec5, allowing the assembly of the exocyst complex and so regulating the exocytosis.