HaloTag as a substrate-based macroautophagy reporter.

ABSTRACT

Macroautophagy is a conserved cellular degradation pathway that, upon upregulation, confers resilience toward various stress conditions, including protection against proteotoxicity associated with neurodegenerative diseases, leading to cell survival. Monitoring autophagy regulation in living cells is important to understand its role in physiology and pathology, which remains challenging. Here, we report that when HaloTag is expressed within a cell of interest and reacts with tetramethylrhodamine (TMR; its ligand attached to a fluorophore), the rate of fluorescent TMR-HaloTag conjugate accumulation in autophagosomes and lysosomes, observed by fluorescence microscopy, reflects the rate of autophagy. Notably, we found that TMR-HaloTag conjugates were mainly degraded by the proteasome (~95%) under basal conditions, while lysosomal degradation (~10% upon pharmacological autophagy activation) was slow and incomplete, forming a degraded product that remained fluorescent within a SDS-PAGE gel, in agreement with previous reports that HaloTag is resistant to lysosomal degradation when fused to proteins of interest. Autophagy activation is distinguished from autophagy inhibition by the increased production of the degraded TMR-HaloTag band relative to the full-length TMR-HaloTag band as assessed by SDS-PAGE and by a faster rate of TMR-HaloTag conjugate lysosomal puncta accumulation as observed by fluorescence microscopy. Pharmacological proteasome inhibition leads to accumulation of TMR-HaloTag in lysosomes, indicating possible cross talk between autophagy and proteasomal degradation.