Descrizione del progetto
Le GTPasi della famiglia Rab e il loro ruolo nella compartimentazione cellulare
La compartimentazione in molti diversi organelli legati alla membrana è la caratteristica principale delle cellule eucariote. Le piccole GTPasi della famiglia Rab svolgono un ruolo essenziale in questa organizzazione, ma i meccanismi di formazione spazio-temporale del complesso ambiente cellulare non sono noti. Il progetto GTPaseNet, finanziato dall’UE, mira a introdurre un nuovo approccio sperimentale, utilizzando la biologia sintetica per ricostruire le reti di GTPasi a partire da componenti purificati e dimostrare l’auto-organizzazione in modelli spazio-temporali in vitro. Gli esperimenti di ricostituzione saranno combinati con la microscopia crioelettronica per delucidare le strutture reclutate dalla membrana. La microfabbricazione e la litografia laser consentiranno di ricostruire la cellula compartimentata, mediante modellizzazione del sistema Rab GTPase grazie al rilevamento di spunti geometrici e biochimici nella cellula vivente.
Obiettivo
Eukaryotic cells are characterized by their compartmentalization into hundreds of different membrane-bound organelles with unique biochemical identities. Small GTPases of the Rab family play a central role in this organization, but how they are able to generate spatiotemporal order in the complex cellular environment is currently not known. Most previous studies on Rab GTPases have either relied on describing their behavior in living cells or in highly reductionist biochemical assays. However, neither of these two approaches can explain the dynamic activity patterns of Rab GTPases associated with their cellular functions. It has become clear that Rab GTPases are controlled in sophisticated regulatory networks with emergent, self-organizing properties. To obtain a mechanistic understanding of these Rab GTPase systems, new experimental assays are now required. In this proposal, we will use a “bottom-up” synthetic biology approach to rebuild the biochemical networks of Rab GTPases from purified components and demonstrate their self-organization into spatiotemporal activity patterns in vitro. We will combine these reconstitution experiments with cryo-electron microscopy to elucidate the structures of membrane-recruited Rab GTPase regulators. Finally, we will use microfabrication and laser lithography to prepare a mimic for the compartmentalized cell and find out how Rab GTPase signaling systems sense and process preexisting geometric and biochemical cues as in the living cell. This project will provide novel, quantitative information from different scales, from the emergent ensemble behavior down to the molecular structure of protein complexes. Together, this data will reveal how signaling systems of Rab GTPases control membrane identities in space and time, thereby improving our understanding of the intracellular organization of the eukaryotic cell.
Campo scientifico
Programma(i)
- HORIZON.1.1 - European Research Council (ERC) Main Programme
Argomento(i)
Meccanismo di finanziamento
HORIZON-AG - HORIZON Action Grant Budget-BasedIstituzione ospitante
3400 Klosterneuburg
Austria