English
 
Help Privacy Policy Disclaimer
  Advanced SearchBrowse

Item

ITEM ACTIONSEXPORT
  Femtoliter injection of ESCRT-III proteins into adhered giant unilamellar vesicles

Georgiev, V., Avalos Padilla, Y., Fernàndez-Busquets, X., & Dimova, R. (2022). Femtoliter injection of ESCRT-III proteins into adhered giant unilamellar vesicles. Bio-protocol, 12(4): e4328. doi:10.21769/BioProtoc.4328.

Item is

Basic

show hide
Genre: Journal Article

Files

show Files
hide Files
:
Article.pdf (Publisher version), 4MB
 
File Permalink:
-
Name:
Article.pdf
Description:
-
Visibility:
Restricted (Max Planck Institute of Colloids and Interfaces, MTKG; )
MIME-Type / Checksum:
application/pdf
Technical Metadata:
Copyright Date:
-
Copyright Info:
-
License:
-

Locators

show

Creators

show
hide
 Creators:
Georgiev, Vasil1, Author              
Avalos Padilla, Yunuen1, Author              
Fernàndez-Busquets, Xavier, Author
Dimova, Rumiana1, Author              
Affiliations:
1Rumiana Dimova, Theorie & Bio-Systeme, Max Planck Institute of Colloids and Interfaces, Max Planck Society, ou_1863328              

Content

show
hide
Free keywords: Giant unilamellar vesicle (GUV), Microinjection, ESCRT-III, Extracellular vesicles, Adhesion, Budding
 Abstract: 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.

Details

show
hide
Language(s): eng - English
 Dates: 2022-02-202022
 Publication Status: Published in print
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Type: -
 Identifiers: DOI: 10.21769/BioProtoc.4328
 Degree: -

Event

show

Legal Case

show

Project information

show

Source 1

show
hide
Title: Bio-protocol
Source Genre: Journal
 Creator(s):
Affiliations:
Publ. Info: Berkley : Bio Online
Pages: - Volume / Issue: 12 (4) Sequence Number: e4328 Start / End Page: - Identifier: ISSN: 2331-8325