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Despite advances in cancer therapies, nanomedicine approaches including the treatment of glioblastoma (GBM), the most common, aggressive brain tumor, remains inefficient. These failures are likely attributable to the complex and not yet completely known biology of this tumor, which is responsible for its strong invasiveness, high degree of metastasis, high proliferation potential, and resistance to radiation and chemotherapy. The intimate connection through which the cells communicate between them plays an important role in these biological processes. In this scenario, tunneling nanotubes (TnTs) are recently gaining importance as a key feature in tumor progression and in particular in the re-growth of GBM after surgery. In this context, we firstly identified structural differences of TnTs formed by U87-MG cells, as model of GBM cells, in comparison with those formed by normal human astrocytes (NHA), used as a model of healthy cells. Successively, we have studied the possibility to exploit U87-MG TnTs as drug-delivery channels in cancer therapy, using liposomes composed of cholesterol/sphingomyelin and surface functionalized with mApoE and chlorotoxin peptides (Mf-LIP) as nanovehicle model. The results showed that U87-MG cells formed almost exclusively thick and long protrusions, whereas NHA formed more thin and short TnTs. Considering that thick TnTs are more efficient in transport of vesicles and organelles, we showed that fluorescent-labeled Mf-LIP can be transported via TnTs between U87-MG cells and with less extent through the protrusions formed by NHA cells. Our results demonstrate that nanotubes are potentially useful as drug-delivery channels for cancer therapy, facilitating the intercellular redistribution of this drug in close and far away cells, thus reaching isolated tumor niches that are hardly targeted by simple drug diffusion in the brain parenchyma. Moreover, the differences identified in TnTs formed by GBM and NHA cells can be exploited to increase t

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 de, 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

The tropomyosin-related kinase (Trk) family of receptor tyrosine kinases controls synaptic function, plasticity and sustains differentiation, morphology, and neuronal cell survival. Understanding Trk receptors down-regulation and recycling is a crucial step to point out sympathetic and sensory neuron function and survival. PC12 cells derived from pheochromocytoma of the rat adrenal medulla have been widely used as a model system for studies of neuronal differentiation as they respond to nerve growth factor (NGF) with a dramatic change in phenotype and acquire a number of properties characteristic of sympathetic neurons. In this study we demonstrated that in PC12 cells the TrkA receptor interacts with the deubiquitinating enzyme USP8/UBPy in a NGF-dependent manner and that it is deubiquitinated in vivo and in vitro by USP8. USP8 overexpression blocked NGF-induced neurites outgrowth while the overexpression of the catalytically inactive mutant USP8/UBPyC748A caused a marked increase of cell differentiation. Localization and biochemical experiments have point out that USP8 and TrkA partially co-localize in endosomes after NGF stimulation. Finally we have studied the role played by USP8 on TrkA turnover; using specific siRNA for USP8 we found that USP8 knockdown increases TrkA half-life, suggesting that the deubiquitinating activity of USP8 promotes TrkA degradation.

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

RalGPS2 is a guanine nucleotide exchange factor for RalA GTPase characterized by a C-terminal Pleckstrin Homology (PH) domain; this GEF is endogenously expressed in PC12 cells and in rat brain but its role in PC12 cells and in cell differentiation is actually unknown. Here we have shown that transient expression of RalGPS2-PH-PxxP domain in PC12 and PC12-TrkA cells induces high level of neurite outgrowth; this differentiation is comparable with that of PC12 cells treated with RalGPS2 siRNA. Stable PC12 cell lines expressing the PH-PxxP domain of RalGPS2 have been generated; in these cell lines the PH-PxxP domain acts as a dominant negative for RalA activation, promotes cells differentiation and re-directs NGF signals towards MAPKs. Furthermore it has been also demonstrated that the PH-PxxP domain of RalGPS2 induces microspikes formation a typical feature of cells in which the Cdc42 GTPase is constitutively activated.

RalGPS2 is a murine guanine nucleotide exchange factor of the RalGPS family; it contains a Cdc25-like GEF domain and does not exhibit a Ras-binding domain. The main characteristic of RalGPS2 is its pleckstrin homology (PH) domain, present at the C terminus, that preferentially binds phosphatidylinositol-4,5-biphosphate and in HEK 293 cells localized in membranes, causing ruffling and vesiculation. Moreover, RalGPS2 contains a PxxP motif in the central part of the molecule. This motif binds in vitro and in vivo SH3 domains of Grb2 and PLCgamma. RalGPS2 and its GEF domain activate RalA in vivo while the PH-PxxP domains inhibited it behaving as a dominant negative for the RalA pathway; this activation was not inhibited by co-expression of a dominant negative Ras. RalGPS2 is physiologically expressed in testis and brain; when overexpressed, the whole RalGPS2 causes considerable morphological changes in HEK 293 cells, suggesting its possible role on cytoskeleton reorganization. This is further strengthened by data obtained in NIH3T3 cells where expression of PH-PxxP domain promotes actin depolymerization. Finally, RalGPS2 and its GEF domain induce Ras-independent transcriptional activation of the c-fos promoter in NIH3T3 cells.

The guanosine trisphosphatase Rap1 serves as a critical player in signal transduction, somatic cell proliferation and differentiation, and cell-cell adhesion by acting through distinct mechanisms. During mouse spermiogenesis, Rap1 is activated and forms a signaling complex with its effector, the serine-threonine kinase B-Raf. To investigate the functional role of Rap1 in male germ cell differentiation, we generated transgenic mice expressing an inactive Rap1 mutant selectively in differentiating spermatids. This expression resulted in a derailment of spermiogenesis due to an anomalous release of immature round spermatids from the seminiferous epithelium within the tubule lumen and in low sperm counts. These spermiogenetic disorders correlated with impaired fertility, with the transgenic males being severely subfertile. Because mutant testis exhibited perturbations in ectoplasmic specializations (ESs), a Sertoli-germ. cell-specific adherens junction, we searched for expression of vascular endothelial cadherin (VE-cadherin), an adhesion molecule regulated by Rap1, in spermatogenic cells of wild-type and mutant mice. We found that germ cells express VE-cadherin with a timing strictly related to apical ES formation and function; immature, VE-cadherin-positive spermatids were, however, prematurely released in the transgenic testis. In conclusion, interfering with Rap1 function during spermiogenesis leads to reduced fertility by impairment of germ-Sertoli cell contacts; our transgenic mouse provides an in vivo model to study the regulation of ES dynamics

Recombinant human nerve growth factor (rhNGF) is regarded as the most promising therapy for neurodegeneration of the central and peripheral nervous systems as well as for several other pathological conditions involving the immune system. However, rhNGF is not commercially available as a drug. In this work, we provide data about the production on a laboratory scale of large amounts of a rhNGF that was shown to possess in vivo biochemical, morphological, and pharmacological effects that are comparable with the murine NGF (mNGF), with no apparent side effects, such as allodynia. Our rhNGF was produced by using conventional recombinant DNA technologies combined with a biotechnological approach for high-density culture of mammalian cells, which yielded a production of approximately 21.5 +/- 2.9 mg/liter recombinant protein. The rhNGF-producing cells were thoroughly characterized, and the purified rhNGF was shown to possess a specific activity comparable with that of the 2.5S mNGF by means of biochemical, immunological, and morphological in vitro studies. This work describes the production on a laboratory scale of high levels of a rhNGF with in vitro and, more important, in vivo biological activity equivalent to the native murine protein

Msj-1 (mouse sperm cell-specific DnaJ first homologue) is a gene specifically expressed in germ cells at haploid stages. The protein first appears in round spermatids, accumulates in the periacrosomal region of elongating spermatids, and is maintained in spermatozoa. The msj-1 expression pattern is consistent with a role for this DnaJ protein in the spermiogenesis process. In this study, we used two experimental models, the anuran amphibian Rana esculenta and the wobbler mutant mouse, to explore the role of MSJ-1 during spermatogenesis, with a focus on spermiogenesis. Mice homozygous for the recessive mutation wobbler (wr/wr), a mutation of unknown identity, produce sperm cells characterized by a missing acrosome. In Rana esculenta testis, detection of high levels of MSJ-1 protein coincided with the appearance of postmeiotic germ cells during the annual sexual cycle. Conversely, elimination of the meiotic and postmeiotic stages, through gonadotropin administration at low temperature, abolished the MSJ-1 immunoreactive signal. In 20-day-old mice, when postmeiotic germ cells appeared for the first time, MSJ-1 mRNA and protein were observed in +/+ testis but were barely detectable in wr/wr testis. In adult testis, reduced MSJ-1 protein levels were observed in both +/wr and wr/wr testis, as compared with +/+ controls. Similarly, numbers of spermatids that stained by immunofluorescence for MSJ-1 appeared to be progressively reduced in adult +/+, +/wr, and wr/wr mouse testes, respectively. Characterization of the endocrine status of wobbler testis revealed reduced transcript levels of estrogen receptor alpha and reduced intratesticular androgen levels. However, androgen treatment did not affect MSJ-1 protein levels in either frogs or mice. In conclusion, our data in Rana esculenta and the wobbler mouse demonstrate a tight correlation between MSJ-1 protein expression and postmeiotic stages. In particular, the findings in the wobbler testis suggest a role for this protein i

We used yeast "two-hybrid" screening to isolate cDNA-encoding proteins interacting with the N-terminal domain of the Ras nucleotide exchange factor CDC25(Mm). Three independent overlapping clones were isolated from a mouse embryo cDNA library. The full-length cDNA was cloned by RACE-polymerase chain reaction. It encodes a large protein (1080 amino acids) highly homologous to the human deubiquitinating enzyme hUBPy and contains a well conserved domain typical of ubiquitin isopeptidases. Therefore we called this new protein mouse UBPy (mUBPy). Northern blot analysis revealed a 4-kilobase mRNA present in several mouse tissues and highly expressed in testis; a good level of expression was also found in brain, where CDC25(Mm) is exclusively expressed. Using a glutathione S-transferase fusion protein, we demonstrated an "in vitro" interaction between mUBPy and the N-terminal half (amino acids 1-625) of CDC25(Mm). In addition "in vivo" interaction was demonstrated after cotransfection in mammalian cells. We also showed that CDC25Mm, expressed in HEK293 cells, is ubiquitinated and that the coexpression of mUBPy decreases its ubiquitination. In addition the half-life of CDC25Mm protein was considerably increased in the presence of mUBPy. The specific function of the human homolog hUBPy is not defined, although its expression was correlated with cell proliferation. Our results suggest that mUBPy may play a role in controlling degradation of CDC25(Mm), thus regulating the level of this Ras-guanine nucleotide exchange factor

The CDC25 gene product is an exchange factor for Ras proteins and it activates the Ras/cAMP pathway in the yeast Saccharomyces cerevisiae. The overexpression of the CDC25 gene in S. cerevisiae cells causes a partial glucose-derepressed phenotype which is particularly evident for expression of invertase. To define domains of Cdc25 protein relevant for this derepression and to test another glucose repressed system, different to invertase, we have overexpressed different regions of the CDC25 gene under the control of a GAL-promoter. We found that a derepression of both GAL-regulated promoters and invertase was related to the overexpression of CDC25 regions that contain a functional guanine nucleotide exchange (GEF) domain. The effect on GAL-promoters was particular evident when the CDC25 gene was under the control of a UAS(gal) element and operates at transcriptional level, although a moderate derepression was found also for UAS(gal)/lacZ reporter gene. Finally, the overexpression of the GEF domain of CDC25 also caused an increase in the expression of the GAL4 regulatory gene, while a constitutive activation of the Ras/cAMP pathway did not produce any increase in GAL4 expression. These findings indicate that the overexpression of the catalytic domain of CDC25 gene is necessary and sufficient to give a glucose-derepression of GAL promoters and of invertase. They also suggest that the derepression of GAL promoters occurs through an increase of GAL4 expression in a Ras/cAMP independent way