Despite proteotoxic stress and heat shock being implicated in diverse pathologies, currently no methodology to inflict defined, subcellular thermal damage exists. Here, we present such a single-cell method compatible with laser-scanning microscopes, adopting the plasmon resonance principle. Dose-defined heat causes protein damage in subcellular compartments, rapid heat-shock chaperone recruitment, and ensuing engagement of the ubiquitin-proteasome system, providing unprecedented insights into the spatiotemporal response to thermal damage relevant for degenerative diseases, with broad applicability in biomedicine. Using this versatile method, we discover that HSP70 chaperone and its interactors are recruited to sites of thermally damaged proteins within seconds, and we report here mechanistically important determinants of such HSP70 recruitment. Finally, we demonstrate a so-far unsuspected involvement of p97(VCP) translocase in the processing of heat-damaged proteins. Overall, we report an approach to inflict targeted thermal protein damage and its application to elucidate cellular stress-response pathways that are emerging as promising therapeutic targets.

• MeSH
• Single-Cell Analysis methods   MeSH
• Metal Nanoparticles chemistry   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Surface Plasmon Resonance MeSH
• Proteasome Endopeptidase Complex metabolism   MeSH
• Valosin Containing Protein genetics   metabolism   MeSH
• HSP70 Heat-Shock Proteins metabolism   MeSH
• Heat-Shock Response * MeSH
• Silver chemistry   MeSH
• Ubiquitin metabolism   MeSH
• Hot Temperature adverse effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Proteasome Endopeptidase Complex MeSH
• Valosin Containing Protein MeSH
• HSP70 Heat-Shock Proteins MeSH
• Silver MeSH
• Ubiquitin MeSH
• VCP protein, human MeSH Browser