Background: Vaults are eukaryotic ribonucleoprotein particles composed of up 78 copies of the 97 kDa major vault protein that assembles into a barrel-like, “nanocapsule” enclosing poly(ADP-ribose) polymerase, telomerase-associated protein-1 and small untranslated RNAs. Overall, the molecular mass of vault particles amounts to about 13 MDa. Although it has been implicated in several cellular functions, its physiological roles remain poorly understood. Also, the possibility to exploit it as a nanovector for drug delivery is currently being explored in several laboratories. Methods: Using the baculovirus expression system, vaults were expressed and purified by a dialysis step using a 1 MDa molecular weight cutoff membrane and a subsequent size exclusion chromatography. Purity was assessed by SDS-PAGE, transmission electron microscopy and dynamic light scattering. Particle's endocytic uptake was monitored by flow cytometry and confocal microscopy. Results: The purification protocol here reported is far simpler and faster than those currently available and lead to the production of authentic vault. We then demonstrated its clathrin-mediated endocytic uptake by normal fibroblast and glioblastoma, but not carcinoma cell lines. In contrast, no significant caveolin-mediated endocytosis was detected. Conclusions: These results provide the first evidence for an intrinsic propensity of the vault complex to undergo endocytic uptake cultured eukaryotic cells. General significance: The newly developed purification procedure will greatly facilitate any investigation based on the use of the vault particle as a natural nanocarrier. Its clathrin-mediated endocytic uptake observed in normal and in some tumor cell lines sheds light on its physiological role.

Galbiati, E., Avvakumova, S., La Rocca, A., Pozzi, M., Messali, S., Magnaghi, P., et al. (2018). A fast and straightforward procedure for vault nanoparticle purification and the characterization of its endocytic uptake. BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS, 1862(10), 2254-2260 [10.1016/j.bbagen.2018.07.018].

A fast and straightforward procedure for vault nanoparticle purification and the characterization of its endocytic uptake

Galbiati, E
Primo
;
Avvakumova, S;Colombo, M;Prosperi, D
Penultimo
;
Tortora, P
Ultimo
2018

Abstract

Background: Vaults are eukaryotic ribonucleoprotein particles composed of up 78 copies of the 97 kDa major vault protein that assembles into a barrel-like, “nanocapsule” enclosing poly(ADP-ribose) polymerase, telomerase-associated protein-1 and small untranslated RNAs. Overall, the molecular mass of vault particles amounts to about 13 MDa. Although it has been implicated in several cellular functions, its physiological roles remain poorly understood. Also, the possibility to exploit it as a nanovector for drug delivery is currently being explored in several laboratories. Methods: Using the baculovirus expression system, vaults were expressed and purified by a dialysis step using a 1 MDa molecular weight cutoff membrane and a subsequent size exclusion chromatography. Purity was assessed by SDS-PAGE, transmission electron microscopy and dynamic light scattering. Particle's endocytic uptake was monitored by flow cytometry and confocal microscopy. Results: The purification protocol here reported is far simpler and faster than those currently available and lead to the production of authentic vault. We then demonstrated its clathrin-mediated endocytic uptake by normal fibroblast and glioblastoma, but not carcinoma cell lines. In contrast, no significant caveolin-mediated endocytosis was detected. Conclusions: These results provide the first evidence for an intrinsic propensity of the vault complex to undergo endocytic uptake cultured eukaryotic cells. General significance: The newly developed purification procedure will greatly facilitate any investigation based on the use of the vault particle as a natural nanocarrier. Its clathrin-mediated endocytic uptake observed in normal and in some tumor cell lines sheds light on its physiological role.
Articolo in rivista - Articolo scientifico
Scientifica
Baculovirus; Endocytosis; Major vault protein; Transmission electron microscopy;
Baculovirus; Endocytosis; Major vault protein; Transmission electron microscopy; Biophysics; Biochemistry; Molecular Biology
English
Galbiati, E., Avvakumova, S., La Rocca, A., Pozzi, M., Messali, S., Magnaghi, P., et al. (2018). A fast and straightforward procedure for vault nanoparticle purification and the characterization of its endocytic uptake. BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS, 1862(10), 2254-2260 [10.1016/j.bbagen.2018.07.018].
Galbiati, E; Avvakumova, S; La Rocca, A; Pozzi, M; Messali, S; Magnaghi, P; Colombo, M; Prosperi, D; Tortora, P
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/10281/207984
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