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Tosetto, Jessica (2008) Molecular and functional analysis of the Drosophila Dynamin-like GTPase atlastin. [Ph.D. thesis]

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Abstract (english)

Mutations in the gene encoding the large oligomeric GTPase Atlastin-1 are responsible for SPG3a, a common autosomal dominant Hereditary Spastic Paraplegia.
Recent studies carried out in our laboratory have shown that the Drosophila ortholog of human Atlastin-1 (Datlastin) is required for homotypic fusion of ER membranes. Homotypic fusion activity is critical for the both the biogenesis and maintenance of the ER and a proper ER architecture is essential for ER functionality. Datlastin specifically localizes to ER membranes and alteration of its expression levels in vivo in Drosophila has revealed that Datlastin depletion causes ER fragmentation, while its overexpression results in the formation of expanded ER elements consistent with excessive fusion of ER membranes.
Using different experimental approaches, we demonstrate that Datlastin has the ability to homo-oligomerize and that Datlastin molecules localized on distinct ER membranes can form trans-oligomeric complexes. In contrast, GTPase-deficient Datlastin is inactive and unable to form trans-oligomeric complexes due to its failure to self-associate. These results suggest that Datlastin undergoes GTPase-dependent homo-oligomerization thereby mediating the tethering of adjacent ER membranes that precedes homotypic fusion. We further show that Datlastin contains a coiled-coil region responsible for mediating oligomerization. Mutations disrupting the structure of the coiled-coil inactivate Datlastin by preventing tethering and the subsequent fusion of ER membranes. The close membrane localization of the coiled-coil Datlastin domain provides a mechanism for the very close apposition between ER membranes that must occur during Datlastin mediated fusion.
These results provide an explanation of the mechanisms underlying Datlastin mediated membrane fusion and lay the foundations for understanding the mechanics of ER biogenesis and maintenance that are presently unknown.

Abstract (italian)

Mutazioni a carico del gene spg3a codificante per Atlastina-1 sono responsabili di una forma autosomica dominante di paraplegia spastica ereditaria.
Ricerche condotte nel laboratorio in cui ho svolto il dottorato dimostrano che Datlastina, l’ortologo in Drosophila di Atlastina-1, promuove la fusione omotipica delle membrane del reticolo endoplasmico (ER).
La fusione omotipica è un processo essenziale per il corretto funzionamento del ER poiché regola la biogenesi e il mantenimento della sua complessa struttura.
Datlastina è una proteina integrale delle membrane del ER. In Drosophila, in seguito alla deplezione di Datlastina il ER risulta frammentato, mentre la sua sovraespressione induce la comparsa di cisterne di ER dilatate riconducibili ad una attività di iperfusione.
Questa tesi ha come oggetto lo studio del meccanismo con cui atlastina media la fusione omotipica delle membrane del ER. La fusione tra membrane si divide in tre fasi: l’avvicinamento, l’”ancoraggio” e la fusione finale dei due strati fosfolipidici.
Attraverso un approccio biochimico, questo lavoro dimostra che Datlastina è capace di oligomerizzare e che molecole di Datlastina presenti su distinte membrane del reticolo endoplasmico sono in grado di associarsi a formare dei complessi in trans. Al contrario, forme mutate di Datlastina prive di attività GTPasica sono inattive ed incapaci sia di associarsi, sia di formare complessi in trans. Questi risultati suggeriscono che Datlastina, attraverso una oligomerizzazione dipendente dall’attività GTPasica, è in grado di avvicinare membrane adiacenti di reticolo endoplasmico per la loro successiva fusione.
Analisi di modelling molecolare ed esperimenti di dicroismo circolare hanno permesso di identificare un dominio coiled-coil responsabile dell’oligomerizzazione di Datlastina. Mutazioni che distruggono la struttura del dominio coiled-coil inattivano Datlastina che risulta incapace sia di avvicinare sia di fondere le membrane del reticolo endoplasmico.
La stretta vicinanza richiesta durante la fusione tra due membrane è soddisfatta, nel processo mediato da Datlastina, dalla prossimità dei domini coiled-coil ai domini transmembrana.
Questi risultati forniscono una spiegazione del meccanismo con cui Datlastina opera nella fusione delle membrane del reticolo endoplasmico e consentono una maggiore comprensione dei processi biofisici coinvolti nella fusione delle membrane.

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EPrint type:Ph.D. thesis
Tutor:Cargnelli, Gabriella
Supervisor:Daga, Andrea
Ph.D. course:Ciclo 21 > Corsi per il 21simo ciclo > FARMACOLOGIA MOLECOLARE E CELLULARE
Data di deposito della tesi:27 January 2009
Anno di Pubblicazione:31 December 2008
Key Words:drosophila; endoplasmic reticulum, homotypic fusion; Hereditary Spastic paraplegia
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/13 Biologia applicata
Struttura di riferimento:Dipartimenti > pre 2012 - Dipartimento di Farmacologia ed Anestesiologia "E. Meneghetti"
Codice ID:1450
Depositato il:27 Jan 2009
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