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Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

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Revealing CD38 cellular localization using a cell permeable, mechanism-based fluorescent small-molecule probe.

Jonathan H Shrimp¹, Jing Hu, Min Dong

¹Department of Chemistry and Chemical Biology, Cornell University , Ithaca, New York 14853, United States.

Journal of the American Chemical Society

|March 26, 2014

Summary

A new fluorescent probe reveals Nicotinamide adenine dinucleotide (NAD) metabolizing enzyme CD38 is primarily on the cell surface, not inside cells. This finding impacts understanding of CD38

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High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Published on: July 25, 2014

Area of Science:

Biochemistry
Cell Biology
Enzymology

Background:

Nicotinamide adenine dinucleotide (NAD) is a crucial signaling molecule involved in numerous biological pathways.
CD38 is a mammalian enzyme that metabolizes NAD, converting it to adenosine diphosphate ribose (ADPR) and cyclic adenosine diphosphate ribose (cADPR).
The cellular localization of CD38, whether on the plasma membrane or intracellularly, is critical for understanding its physiological function and potential impact on NAD levels.

Purpose of the Study:

To develop and apply a novel cell-permeable, fluorescent small molecule probe (SR101-F-araNMN) for activity-based labeling of CD38 in live cells.
To accurately validate the intracellular localization of CD38, addressing conflicting previous reports.
To elucidate the predominant cellular localization of CD38 and its implications for NAD metabolism.

Main Methods:

Synthesis of a cell-permeable, fluorescent small molecule probe, SR101-F-araNMN, designed for covalent labeling of enzymatically active CD38.
Application of the fluorescent probe to live cell imaging in Raji, RA-treated HL-60, and K562 cell lines.
Validation of CD38 localization using the activity-based fluorescent probe with minimal perturbation to live cells.

Main Results:

The fluorescent probe SR101-F-araNMN successfully labeled enzymatically active CD38 in live cells.
CD38 was predominantly localized on the plasma membrane of Raji and RA-treated HL-60 cells.
No CD38 expression was detected in K562 cells, contradicting previous reports of solely intracellular CD38.

Conclusions:

The developed fluorescent activity-based probes provide a reliable method for determining CD38 localization across different cell types.
The findings suggest that CD38 primarily functions to hydrolyze extracellular NAD, rather than intracellular NAD.
This study enhances the understanding of CD38's physiological role in NAD metabolism and cellular signaling.