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Abstract
When a cell experiences environmental change, such as heat shock, its molecular landscape transforms. Many proteins reassemble into larger bodies called condensates. Also, specific classes of proteins, called heat shock proteins or molecular chaperones, are produced. An important role of chaperones is to disperse protein condensates as cells adapt to environmental change. However, much remains unknown about condensates themselves, how their dispersal happens, and why only specific types of chaperones support dispersal. In this thesis, I address two main questions: what do condensates and their dispersal look like at the molecular scale; and why do two similar chaperones, Sis1 and Ydj1, have opposing effects on dispersal? To answer the first question, we used single-molecule microscopy to track individual, dye-labeled poly(A)-binding protein (Pab1) condensates formed during physiological heat shock. We find that condensates are heterogeneous but small entities, which typically contain ~100’s of proteins. We observe that condensate dispersal by chaperones is heterogeneous and often incomplete, and we see unexpected effects upon absence of ATP and addition of the chaperone Sse1. These results are among the first direct visualizations of chaperone-mediated condensate dispersal and help build a more complete picture of this molecular process. To answer the second question, we use biochemical and single-molecule approaches to interrogate Sis1 and Ydj1’s divergent behaviors. While Sis1 is essential to dispersal, Ydj1 fails at and even impedes dispersal—yet both chaperones are J-domain proteins that fulfill similar functions: substrate binding, substrate handoff to the chaperone Hsp70, and stimulation of Hsp70 ATP hydrolysis. We observe that both Sis1 and Ydj1 bind condensates, suggesting that the difference lies in coordination with Hsp70. Unexpectedly, mutation of Hsp70’s EEVD tail, which should disrupt interaction with Sis1, does not completely prevent dispersal. We further investigate Sis1 dimerization and Hsp70 ATP hydrolysis as contributors to Sis1 function. These and other results guide us to a more nuanced understanding of how Sis1, Ydj1, and J-domain proteins work in the condensate dispersal context. Other projects I have pursued include investigating human chaperones and human cell responses to fever temperature; visualizing differences in the condensation of Pab1 from yeast species adapted to different thermal environments; and helping develop a new technology which could advance future single-molecule studies of chaperones and condensates. Together, this thesis provides new insight into the molecular events that transpire when cells respond to heat shock and environmental change and offers promising opportunities for future research.