Novel role and regulation of the WASP protein

No abstract available

Piezo acts as a molecular brake on wound closure to ensure effective inflammation and maintenance of epithelial integrity

Wound healing entails a fine balance between re-epithelialization and inflammation(1)(,)(2) so that the risk of infection is minimized, tissue architecture is restored without scarring, and the epithelium regains its ability to withstand mechanical forces. How the two events are orchestrated in vivo remains poorly understood, largely due to the experimental challenges of simultaneously addressing mechanical and molecular aspects of the damage response. Here, exploiting Drosophila’s genetic tractability and live imaging potential, we uncover a dual role for Piezo—a mechanosensitive channel involved in calcium influx(3)—during re-epithelialization and inflammation following injury in vivo. We show that loss of Piezo leads to faster wound closure due to increased wound edge intercalation and exacerbated myosin cable heterogeneity. Moreover, we show that loss of Piezo leads to impaired inflammation due to lower epidermal calcium levels and, subsequently, insufficient damage-induced ROS production. Despite initially appearing beneficial, loss of Piezo is severely detrimental to the long-term effectiveness of repair. In fact, wounds inflicted on Piezo knockout embryos become a permanent point of weakness within the epithelium, leading to impaired barrier function and reduced ability of wounded embryos to survive. In summary, our study uncovers a role for Piezo in regulating epithelial cell dynamics and immune cell responsiveness during damage repair in vivo. We propose a model whereby Piezo acts as molecular brake during wound healing, slowing down closure to ensure activation of sustained inflammation and re-establishment of a fully functional epithelial barrier

WASP family proteins and formins compete in pseudopod- and bleb-based migration

Actin pseudopods induced by SCAR/WAVE drive normal migration and chemotaxis in eukaryotic cells. Cells can also migrate using blebs, in which the edge is driven forward by hydrostatic pressure instead of actin. In Dictyostelium discoideum, loss of SCAR is compensated by WASP moving to the leading edge to generate morphologically normal pseudopods. Here we use an inducible double knockout to show that cells lacking both SCAR and WASP are unable to grow, make pseudopods or, unexpectedly, migrate using blebs. Remarkably, amounts and dynamics of actin polymerization are normal. Pseudopods are replaced in double SCAR/WASP mutants by aberrant filopods, induced by the formin dDia2. Further disruption of the gene for dDia2 restores cells’ ability to initiate blebs and thus migrate, though pseudopods are still lost. Triple knockout cells still contain near-normal F-actin levels. This work shows that SCAR, WASP, and dDia2 compete for actin. Loss of SCAR and WASP causes excessive dDia2 activity, maintaining F-actin levels but blocking pseudopod and bleb formation and migration

A conserved myotubularin-related phosphatase regulates autophagy by maintaining autophagic flux

Macroautophagy (autophagy) targets cytoplasmic cargoes to the lysosome for degradation. Like all vesicle trafficking, autophagy relies on phosphoinositide identity, concentration, and localization to execute multiple steps in this catabolic process. Here, we screen for phosphoinositide phosphatases that influence autophagy in Drosophila and identify CG3530. CG3530 is homologous to the human MTMR6 subfamily of myotubularin-related 3-phosphatases, and therefore, we named it dMtmr6. dMtmr6, which is required for development and viability in Drosophila, functions as a regulator of autophagic flux in multiple Drosophila cell types. The MTMR6 family member MTMR8 has a similar function in autophagy of higher animal cells. Decreased dMtmr6 and MTMR8 function results in autophagic vesicle accumulation and influences endolysosomal homeostasis

"Studio dei meccanismi responsabili della de-regolazione dell'espressione genica in pazienti affetti da Sindrome di Cornelia de Lange mediante un approccio 'genome wide'"

La sindrome di Cornelia de Lange (CdLS) è una condizione patologica che colpisce un neonato ogni diecimila. I soggetti affetti presentano tratti somatici tipicamente dismorfici e anomalie agli arti, in particolare quelli superiori; manifestano inoltre ritardo nello sviluppo e un deficit mentale più o meno marcato. Ad oggi sono stati identificati tre geni responsabili della CdLS: NIPBL, SMC1A ed SMC3. Mutazioni in NIPBL sono state identificate in circa il 60% dei casi, in SMC1A in circa il 5% mentre un solo probando porta la mutazione in SMC3. I geni citati codificano per proteine che fanno parte del complesso proteico della coesina o per fattori regolatori della coesina stessa. Infatti, il core della coesina è costituito da due proteine Structural Mainteinance of Chromosome (SMC, SMC1A ed SMC3) e due proteine non SMC, RAD21 e SA1/SA2. La proteina NIPBL invece è richiesta per il caricamento della coesina sulla cromatina. Il ruolo canonico della coesina è quello di presiedere alla corretta coesione tra i cromatidi fratelli, e pertanto si è ritenuto inizialmente che alla base della CdLS ci fossero dei difetti di coesione dei cromatidi fratelli. Tuttavia l'analisi di metafasi di soggetti affetti non mostra alcuna anomalia. Negli ultimi anni, numerose evidenze sperimentali suggeriscono che la coesina è in grado di svolgere anche un ruolo importante nella regolazione dell'espressione genica. Sembra infatti che la coesina possa svolgere la funzione di “isolatore” ostacolando l’interazione fisica tra il promotore e l’enhancer, impedendo così la formazione dell'apparato trascrizionale e l’avvio della trascrizione. Questo modello prevede che, per una corretta attività trascrizionale, la coesina venga rimossa o dislocata dalla cromatina, in modo che il loop originatosi consenta la giustapposizione di promotore ed enhancer. A supporto di tale modello vi sono alcuni dati sperimentali. Mutazioni nei geni della coesina conferiscono al complesso una maggiore affinità di legame al DNA rispetto alle proteine wild type, e pazienti CdLS mostrano un profilo di espressione genica alterato. Scopo di questa tesi è di investigare se mutazioni nei geni della coesina influenzano l’accessibilità della RNA polimerasi II (RNA pol II) alle regioni regolatorie. Esperimenti di sequenziamento della cromatina immunoprecipitata (ChIP-seq) eseguiti, attraverso una collaborazione scientifica, presso il laboratorio di Richard Young (Massachusetts Institute of Technology, Boston) hanno permesso di identificare sia le regioni del genoma in cui la coesina e i suoi regolatori vengono caricate co-localizzandosi con la RNA pol II, sia quelle in cui la coesina non si lega. Correlando questi dati con quelli pubblicati derivanti dall'utilizzo di microarray di espressione in cellule CdLS, sono state selezionate cinque categorie di regioni: 1) contenenti geni housekeeping, 2) contenenti geni cellula-specifici, 3) contenenti geni in cui l'arricchimento della coesina è maggiore, 4) regioni contenenti geni non legati dalla coesina, 5) regioni di deserto genico. Linee linfoblastoidi di pazienti CdLS e di soggetti sani, usate come controllo, sono state sottoposte ad immunoprecipitazione della cromatina (ChIP) al fine di ottenere un arricchimento in sequenze legate dalla RNA pol II, e sono state successivamente analizzate per le categorie di regioni precedentemente descritte tramite Real Time PCR. I risultati ottenuti mostrano una riduzione del reclutamento della RNA pol II alle regioni occupate dalla coesina nelle linee CdLS rispetto a quelle di controllo, portando nuove evidenze sperimentali a supporto dell'ipotesi che alla base della CdLS vi sia una de-regolazione della trascrizione genica

Nose-to-brain delivery of insulin enhanced by radiation-engineered nanogels

Recent evidences suggest thet insulin delivery to the brain can be an important pharmacological therapy for some neurodegenerative pathologies, including Alzheimer disease (AD

Xyloglucan-based hydrogel film for wound dressing: Structure and biological properties relationships

Crosslinked xyloglucan-poly(vinyl alcohol) based hydrogel films have been synthetized and characterized for wound healing applications. Polyvinyl alcohol (PVA) and glycerol (Gro) were added to have an optimal combination of softness, conformability and resilience. Physical hydrogel have been transformed into permanent covalent hydrogels by reaction with glutaraldehyde (GA). Chemical and physical properties of the films have been analyzed by different technique. Moreover, biocompatibility of a selected formulation was assessed in vitro by investigating different aspects, such as cell viability, oxidative stress, mitochondrial dysfunction and specific stress biomarkers. Partial adhesiveness was demonstrated by performing different attaching assays and phalloidin staining. After interaction with blood the hemocompatibility of the XG-PVA film was evaluated by using a multiparametric approach. Antimicrobial activity of the film against Escherichia coli (E.coli) was tested, and we found that XG-PVA film promotes bacterial retentivity and provides mechanical protection against bacterial infiltration. After loading the film with ampicillin, an inhibitory E. coli growth zone was observed. All together these results indicate that the manufactured film is a promising material to be tested in vivo for wound healing application

Insulin Nanogels: a New Strategy for the Treatment of Alzheimer’s Disease

A growing body of evidence shows that Insulin, Insulin Receptor (IR) and IR signaling are involved in brain cognitive functions and their dysfunction is implicated in Alzheimer’s disease (AD) degeneration. Thus, administration of insulin could be a strategy for AD treatment. For this aim we have designed, synthesized and characterized a nanogel system (NG) to deliver insulin to the brain, as a tool for the development of a new therapy for AD. A carboxyl-functionalized poly(N-vinyl pyrrolidone) nanogel system produced by ionizing radiation was chosen as substrate for the covalent attachment of insulin or fluorescent molecules relevant for its characterization. Biocompatibility of the naked carrier was demonstrated by absence of cytotoxicity, oxidative stress and mitochondrial dysfunction. Hemocompatibility was demonstrated by hemolysis, coagulation time, leukocyte proliferation and inflammatory response tests. By immonufluorescence measurements we demonstrated that insulin conjugated to the NG (NG-In) is preotected by protease degradation and is able to bind and activate insulin receptor bringing to trigger the insulin signalling via AKT activation. Moreover, to provide consistent evidence on the functionality of the conjugated insulin on the glucose levels, the effect of NG-In was tested in mice demonstrating that plasma glucose levels was reduced. Neuroprotection of NG-In against dysfunction induced by amyloid β, a peptide mainly involved in AD, was verified. Finally, the potential of NG-In to be efficiently transported across the Blood Brain Barrier was demonstrated by using an in vitro system. All together these results indicated that the synthesized NG-In was a suitable vehicle system for insulin delivery in biomedicine and a very promising tool to develop new therapies for neurodegenerative diseases. The research eas supported by MIUR, FLAGSHIP PROJECT NanoMAX