Investigating Biomolecular Condensates and Heat Shock Proteins in Cellular Responses to Sublethal Heat Shock and Fever

Environmental temperature dictates biology. Animals use thermal environments to guide vital behaviors such as migration, circadian rhythms, growth, and feeding. Heat is also an important influence on human health. About 1 in 100 deaths globally stem from heat-related causes, and beyond lethal heatstroke, more mild heat < 40°C can alter and dysregulate human physiology down to the cellular level, particularly in immune cells. Such sublethal heat shocks occur in hyperthermia and heat illness, as well as frequently in fever: a systemic heat shock which regulates the immune system during infection. Yet even in the well-known context of fever, we lack understanding of how human cells sense sublethal heat shock. The cell biology of extreme heat shock > 40°C is well-characterized, but far less is understood about sublethal, fever-range temperatures < 40°C. However, we do know that certain immune cells upregulate heat shock protein expression in response to fever. The induction of heat shock proteins, or the heat shock response, occurs in eukaryotes when heat activates transcription factor Hsf1, via titration of its repressor (heat shock protein Hsp70) away from Hsf1. This titration is caused by the generation of new, heat shock-induced substrates for Hsp70 to bind. These substrates, i.e., the upstream sensors of heat, are unidentified in sublethal heat shock. We hypothesize biomolecular condensates are these substrates which help cells sense sublethal heat shock. Condensation, or reorganization of proteins and RNA into larger foci, occurs in response to environmental stimuli across species from yeast to humans. Our group showed recently that heat-induced condensates are Hsp70 substrates in yeast. We hypothesize that sublethal heat shock-induced condensates are Hsp70 substrates in humans, enabling cells to sense and respond to such fever-range temperatures. It is not known what proteins condense in human cells at these temperatures, nor if such condensates might be Hsp70 substrates. Moreover, in any species, we lack molecular-scale understanding of how condensates and Hsp70 interact. We are poised to unlock exactly this knowledge using a complement of biochemical, microscopic, and molecular-level approaches. First, we will uncover protein condensation in human cell lines at fever-range temperature, using the established sedimentation-mass spectrometry method of our group. Second, we will observe directly how condensates and Hsp70 interact at the molecular scale, using single-molecule microscopy. Together, these aims will help us elucidate fundamentally how cells sense and respond to sublethal heat shock.