In Vitro Polymerization of F-actin on Early Endosomes.

Details

Serval ID
serval:BIB_ED3885549030
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
In Vitro Polymerization of F-actin on Early Endosomes.
Journal
Journal of visualized experiments
Author(s)
Muriel O., Scott C.C., Larios J., Mercier V., Gruenberg J.
ISSN
1940-087X (Electronic)
ISSN-L
1940-087X
Publication state
Published
Issued date
28/08/2017
Peer-reviewed
Oui
Number
126
Pages
126
Language
english
Notes
Publication types: Journal Article ; Video-Audio Media
Publication Status: epublish
Abstract
Many early endosome functions, particularly cargo protein sorting and membrane deformation, depend on patches of short F-actin filaments nucleated onto the endosomal membrane. We have established a microscopy-based in vitro assay that reconstitutes the nucleation and polymerization of F-actin on early endosomal membranes in test tubes, thus rendering this complex series of reactions amenable to genetic and biochemical manipulations. Endosomal fractions are prepared by floatation in sucrose gradients from cells expressing the early endosomal protein GFP-RAB5. Cytosolic fractions are prepared from separate batches of cells. Both endosomal and cytosolic fractions can be stored frozen in liquid nitrogen, if needed. In the assay, the endosomal and cytosolic fractions are mixed, and the mixture is incubated at 37 °C under appropriate conditions (e.g., ionic strength, reducing environment). At the desired time, the reaction mixture is fixed, and the F-actin is revealed with phalloidin. Actin nucleation and polymerization are then analyzed by fluorescence microscopy. Here, we report that this assay can be used to investigate the role of factors that are involved either in actin nucleation on the membrane, or in the subsequent elongation, branching, or crosslinking of F-actin filaments.
Keywords
Actins/metabolism, Animals, Endosomes/metabolism, Polymerization
Pubmed
Web of science
Create date
11/12/2017 17:47
Last modification date
20/08/2019 16:15
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