Image-based Quantification of Macropinocytosis Using Dextran Uptake into Cultured Cells

Anh H Le; Laura M Machesky

doi:10.21769/BioProtoc.4367

Image-based Quantification of Macropinocytosis Using Dextran Uptake into Cultured Cells

Bio Protoc. 2022 Apr 5;12(7):e4367. doi: 10.21769/BioProtoc.4367.

Authors

Anh H Le^{1

2}, Laura M Machesky^{1

2}

Affiliations

¹ Cancer Research UK Beatson Institute, Bearsden, Glasgow, UK.
² Institute of Cancer Sciences, University of Glasgow, Bearsden, Glasgow, UK.

Abstract

Macropinocytosis is an evolutionarily conserved process, which is characterized by the formation of membrane ruffles and the uptake of extracellular fluid. We recently demonstrated a role for CYFIP-related Rac1 Interactor (CYRI) proteins in macropinocytosis. High-molecular weight dextran (70kDa or higher) has generally been used as a marker for macropinocytosis because it is too large to fit in smaller endocytic vesicles, such as those of clathrin or caveolin-mediated endocytosis. Through the use of an image-based dextran uptake assay, we showed that cells lacking CYRI proteins internalise less dextran compared to their wild-type counterparts. Here, we will describe a step-by-step experimentation procedure to detect internalised dextran in cultured cells, and an image pipeline to analyse the acquired images, using the open-access software ImageJ/Fiji. This protocol is detailed yet simple and easily adaptable to different treatment conditions, and the analysis can also be automated for improved processing speed.

Keywords: Actin; CYRI-A; Cell migration; Dextran; Fam49A; Macropinocytosis; Macropinosomes.

Abstract

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