Live-cell Imaging of Clathrin Coats

Overview

Journal Methods Enzymol

Specialty Biochemistry

Date 2012 Feb 1

PMID 22289448

Citations 8

Authors

Comert Kural

Tom Kirchhausen

Affiliations

Soon will be listed here.

Abstract

We compare the use of two-dimensional total internal reflection fluorescence microscopy with a rapid, simple-to-implement method for three-dimensional (3D) imaging using spinning-disk confocal microscopy suitable for reliable 3D tracking of clathrin-coated endocytic and endosomal carriers. These carriers contain about 20 EGFP (enhanced green fluorescent protein) equivalents of a chimeric fluorescent protein (either clathrin light chain or one of the clathrin adaptor subunits). Under tissue culture conditions, the clathrin-containing carriers correspond to a variable number of relatively sparse, diffraction-limited, fluorescent objects that can be identified with a spatial precision of ~30 nm or better and a temporal resolution of <1 s. The applicability of these approaches to mammalian cells in culture allows investigators detailed monitoring of the composition dynamics of the clathrin-containing carriers which can then be used to study in living cells the molecular mechanisms required for the formation and traffic of clathrin-coated pits and vesicles.

Citing Articles

Vesiculation pathways in clathrin-mediated endocytosis.

Wang X, Berro J, Ma R bioRxiv. 2024; .

PMID: 39185216 PMC: 11343097. DOI: 10.1101/2024.08.13.607731.

Endocytosis at extremes: Formation and internalization of giant clathrin-coated pits under elevated membrane tension.

Akatay A, Wu T, Djakbarova U, Thompson C, Cocucci E, Zandi R Front Mol Biosci. 2022; 9:959737.

PMID: 36213118 PMC: 9532848. DOI: 10.3389/fmolb.2022.959737.

De novo endocytic clathrin coats develop curvature at early stages of their formation.

Willy N, Ferguson J, Akatay A, Huber S, Djakbarova U, Silahli S Dev Cell. 2021; 56(22):3146-3159.e5.

PMID: 34774130 PMC: 11414472. DOI: 10.1016/j.devcel.2021.10.019.

CALM supports clathrin-coated vesicle completion upon membrane tension increase.

Willy N, Colombo F, Huber S, Smith A, Norton E, Kural C Proc Natl Acad Sci U S A. 2021; 118(25).

PMID: 34155137 PMC: 8237669. DOI: 10.1073/pnas.2010438118.

Dynamic interplay between cell membrane tension and clathrin-mediated endocytosis.

Djakbarova U, Madraki Y, Chan E, Kural C Biol Cell. 2021; 113(8):344-373.

PMID: 33788963 PMC: 8898183. DOI: 10.1111/boc.202000110.

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