Experimental toolbox for quantitative evaluation of clathrin-mediated endocytosis in the plant model Arabidopsis.

Abstract:

:Clathrin-mediated endocytosis (CME) is a crucial cellular process implicated in many aspects of plant growth, development, intra- and intercellular signaling, nutrient uptake and pathogen defense. Despite these significant roles, little is known about the precise molecular details of how CME functions in planta To facilitate the direct quantitative study of plant CME, we review current routinely used methods and present refined, standardized quantitative imaging protocols that allow the detailed characterization of CME at multiple scales in plant tissues. These protocols include: (1) an efficient electron microscopy protocol for the imaging of Arabidopsis CME vesicles in situ, thus providing a method for the detailed characterization of the ultrastructure of clathrin-coated vesicles; (2) a detailed protocol and analysis for quantitative live-cell fluorescence microscopy to precisely examine the temporal interplay of endocytosis components during single CME events; (3) a semi-automated analysis to allow the quantitative characterization of global internalization of cargos in whole plant tissues; and (4) an overview and validation of useful genetic and pharmacological tools to interrogate the molecular mechanisms and function of CME in intact plant samples.This article has an associated First Person interview with the first author of the paper.

journal_name

J Cell Sci

journal_title

Journal of cell science

authors

Johnson A,Gnyliukh N,Kaufmann WA,Narasimhan M,Vert G,Bednarek SY,Friml J

doi

10.1242/jcs.248062

subject

Has Abstract

pub_date

2020-08-06 00:00:00

issue

15

eissn

0021-9533

issn

1477-9137

pii

jcs.248062

journal_volume

133

pub_type

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