HaloTag as a substrate-based macroautophagy reporter

• 成果类型: Article
• 署名作者: Xiao, Qiang; Cruz, Gabrielle; Botham, Rachel; Fox, Susan G.; Yu, Anan; Allen, Seth; Morimoto, Richard I.; Kelly, Jeffery W.
• 署名单位: Scripps Research Institute; Scripps Research Institute; State University of New York (SUNY) System; SUNY Fredonia; Northwestern University
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-14655
• DOI: 10.1073/pnas.2322500121
• 发表日期: 2024-08-06
• 关键词: mammalian target; autophagic flux; highly potent; protein; inhibitor; TECHNOLOGY; platform; cells; lc3

摘要

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 (similar to 95%) under basal conditions, while lysosomal degradation (similar to 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 puncta accumulation as observed by fluorescence microscopy. Pharmacological proteasome talk between autophagy and proteasomal degradation.