Recent Publications (Original)

Willa Wen-You Yim , Hayashi Yamamoto & Noboru Mizushima

A pulse-chasable reporter processing assay for mammalian autophagic flux with HaloTag

Elife. 2022 Aug 8;11:e78923. doi: 10.7554/eLife.78923. Online ahead of print.

Based on our finding that HaloTag becomes resistant to proteolysis upon ligand binding, we developed the HaloTag processing assay. Pulse-labeled HaloTag reporters are processed in lysosomes, releasing proteolysis-resistant labeled Halo that persist in lysosomes. Free labeled Halo amount = autophagic flux.

This method is

• Lysosomal inhibitor-free
• External control-free
• Adaptable to monitor most autophagy pathways or autophagic degradation of a protein of interest
• Quantitative

In our study, we demonstrate the HaloTag processing assay for  

• General (LC3-dependent) autophagy – Halo-LC3, Halo-GFP-LC3, Halo-GFP-LC3-RFP
• Selective autophagy of ER or mitochondria – Halo-GFP-KDEL, pSu9-Halo-GFP
• Bulk non-selective (LC3-independent) autophagy – Halo-GFP

The plasmids can be found on Addgene (https://www.addgene.org/browse/article/28225176/).