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Monitoring Chemokine Receptor Trafficking by Confocal Immunofluorescence Microscopy. Methods Enzymol 2016;570:281-92

Date

02/29/2016

Pubmed ID

26921951

Pubmed Central ID

PMC5201001

DOI

10.1016/bs.mie.2015.10.004

Scopus ID

2-s2.0-84959135724   5 Citations

Abstract

Here, we describe a protocol to detect chemokine receptor CXCR4 by confocal immunofluorescence microscopy in HeLa cells treated with its chemokine ligand CXCL12. Typically, ligand-activated chemokine receptors undergo a multistep process of desensitization and/or internalization from the plasma membrane in order to terminate signaling. Once internalized to endosomes, chemokine receptors readily enter the recycling pathway and return to the cell surface, giving rise to resensitization of signaling. The chemokine receptor CXCR4, when activated by CXCL12 is also internalized to endosomes, but in contrast to many chemokine receptors it is mainly sorted to the degradative pathway, contributing to a loss in the cellular complement of CXCR4 and long-term downregulation of signaling. The trafficking of CXCR4 from early endosomes to lysosomes can be easily detected by confocal immunofluorescence microscopy by immunostaining fixed cells for the receptor and with markers of these vesicular compartments. This approach is advantageous because it can be used to identify factors that regulate the trafficking of CXCR4 from early endosomes to lysosomes. The protocol described here focuses on CXCR4, but it can be easily adapted to other chemokine receptors.

Author List

Marchese A

Author

Adriano Marchese PhD Professor in the Biochemistry department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Chemokine CXCL12
Endosomes
Fluorescent Antibody Technique
HeLa Cells
Humans
Lysosomes
Microscopy, Confocal
Protein Transport
Receptors, CXCR4
Receptors, Chemokine