Femtoliter Injection of ESCRT-III Proteins into Adhered Giant Unilamellar Vesicles.
Adhesion
Budding
ESCRT-III
Extracellular vesicles
Giant unilamellar vesicle (GUV)
Microinjection
Journal
Bio-protocol
ISSN: 2331-8325
Titre abrégé: Bio Protoc
Pays: United States
ID NLM: 101635102
Informations de publication
Date de publication:
20 Feb 2022
20 Feb 2022
Historique:
received:
04
01
2022
revised:
12
11
2021
accepted:
06
01
2022
entrez:
28
3
2022
pubmed:
29
3
2022
medline:
29
3
2022
Statut:
epublish
Résumé
The endosomal sorting complex required for transport (ESCRT) machinery mediates membrane fission reactions that exhibit a different topology from that observed in clathrin-coated vesicles. In all of the ESCRT-mediated events, the nascent vesicle buds away from the cytosol. However, ESCRT proteins are able to act upon membranes with different geometries. For instance, the formation of multivesicular bodies (MVBs) and the biogenesis of extracellular vesicles both require the participation of the ESCRT-III sub-complex, and they differ in their initial membrane geometry before budding starts: the protein complex acts either from outside the membrane organelle (causing inward budding) or from within (causing outward budding). Several studies have reconstituted the action of the ESCRT-III subunits in supported bilayers and cell-sized vesicles mimicking the geometry occurring during MVBs formation (in-bud), but extracellular vesicle budding (out-bud) mechanisms remain less explored, because of the outstanding difficulties encountered in encapsulation of functional ESCRT-III in vesicles. Here, we provide a different approach that allows the recreation of the out-bud formation, by combining giant unilamellar vesicles as a membrane model and a microinjection system. The vesicles are immobilized prior to injection via weak adhesion to the chamber coverslip, which also ensures preserving the membrane excess area required for budding. After protein injection, vesicles exhibit outward budding. The approach presented in this work can be used in the future to disentangle the mechanisms underlying ESCRT-III-mediated fission, recreating the geometry of extracellular bud production, which remains a challenge. Moreover, the microinjection methodology can be also adapted to interrogate the action of other cytosolic components on the encapsulating membranous organelle. Graphic abstract: Out-bud formation after ESCRT-III protein injection into GUVs.
Identifiants
pubmed: 35340293
doi: 10.21769/BioProtoc.4328
pii: e4328
pmc: PMC8899557
doi:
Types de publication
Journal Article
Langues
eng
Pagination
e4328Informations de copyright
Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC.
Déclaration de conflit d'intérêts
Competing interestsThe authors declare no competing interests.
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