Single Cell Fluorescence Ratio Image Analysis for Studying ESCRT Function in Receptor Trafficking

Jalal M Kazan; Gergely L Lukacs; Pirjo M Apaja; Arnim Pause

doi:10.1007/978-1-4939-9492-2_7

Single Cell Fluorescence Ratio Image Analysis for Studying ESCRT Function in Receptor Trafficking

Methods Mol Biol. 2019:1998:93-103. doi: 10.1007/978-1-4939-9492-2_7.

Authors

Jalal M Kazan^{1

2}, Gergely L Lukacs³, Pirjo M Apaja^{4

5}, Arnim Pause^{6

7}

Affiliations

¹ Department of Biochemistry, McGill University, Montréal, QC, Canada.
² Goodman Cancer Research Centre, McGill University, Montréal, QC, Canada.
³ Department of Physiology, McGill University, Montréal, QC, Canada.
⁴ Department of Physiology, McGill University, Montréal, QC, Canada. Pirjo.Apaja@sahmri.com.
⁵ Nutrition and Metabolism Theme, HCN and EMBL Australia, South Australian Health and Medical Research Institute, Adelaide, SA, Australia. Pirjo.Apaja@sahmri.com.
⁶ Department of Biochemistry, McGill University, Montréal, QC, Canada. Arnim.Pause@mcgill.ca.
⁷ Goodman Cancer Research Centre, McGill University, Montréal, QC, Canada. Arnim.Pause@mcgill.ca.

PMID: 31250296
DOI: 10.1007/978-1-4939-9492-2_7

Abstract

The endosomal sorting complexes required for transport (ESCRT) comprise a major trafficking pathway for plasma membrane proteins and are fundamental for ubiquitin-dependent cargo endocytosis. Here, we describe a method for studying the effect of ESCRT complexes on endo-lysosomal membrane trafficking and their role in receptor integrin α5β1 downregulation. Single cell fluorescence ratio image analysis (FRIA), using appropriate fluorescence probes, enables the measurement of dynamics of integrin α5β1 containing vesicles and represents a live cell-based method for studying the role of ESCRTs.

Keywords: ESCRT; FRIA; HD-PTP; Integrin α5β1; Lysosomes; Recycling; UBAP1; Vesicular pH.

MeSH terms

Endosomal Sorting Complexes Required for Transport / chemistry
Endosomal Sorting Complexes Required for Transport / metabolism*
Fluorescent Dyes / chemistry
HeLa Cells
Humans
Image Processing, Computer-Assisted / methods
Integrin alpha5beta1 / chemistry
Integrin alpha5beta1 / metabolism*
Intracellular Membranes / chemistry
Intracellular Membranes / metabolism
Intravital Microscopy / methods*
Lysosomes / chemistry
Lysosomes / metabolism
Molecular Imaging / methods
Molecular Probes / chemistry
Single-Cell Analysis / methods*

Substances

Endosomal Sorting Complexes Required for Transport
Fluorescent Dyes
Integrin alpha5beta1
Molecular Probes