Project Summary Biomolecular condensates are micrometer-scale membraneless structures that self-assemble in living cells. They are ubiquitous across diverse organisms and exist in protein-only conglomerates or protein associated with nucleic acids. Biomolecular condensate formation is critical to normal cellular processes and the mis-regulation of condensate assembly or disassembly drives various pathologies. We recently discovered that the pathological fusion protein EML4-ALK spontaneously forms micrometer-scale condensates in the cytoplasm while lacking conventional condensate-forming domains or sequence motifs. This condensate elicits a novel mode of cell signaling by acting as a physical platform that enriches signaling proteins. The recruitment of client proteins to elevate local protein concentrations implies a general strategy through which multi-protein condensates achieve biological function. A major gap in the field is that very little is known about condensate-promoting motifs beyond a few conventional motifs, or about how proteins work together in condensates to achieve their biological function. We propose a research program that systematically elucidates novel motifs and biophysical principles in protein- only condensates. This will be accomplished by leveraging innovative approaches such as CRISPR imaging, optogenetics manipulation, and custom-written analysis and quantification algorithms to pursue two interrelated research themes. The first theme is identification of alternative mechanisms that enable protein condensate formation by: discovering novel condensate-promoting motifs by interrogating fusion proteins known or suspected to form condensates; determining the essentiality and modularity of such motifs; and mapping motif- function relationships in condensate-mediated processes. The second theme is to uncover physical principles that regulate composition and dynamics in multi-protein condensates. Although the molecular details may differ, the sequence space and principles demonstrated here are broadly applicable to a diverse range of proteins and cellular processes. The long-term objective of our research program is to build an expanded biophysical foundation to understand biomolecular condensate assembly and functions across cellular homeostasis and pathology. Such knowledge brings new opportunities to modulate cellular processes through independent physical approaches instead of traditional ways of interfering with biochemical reactions, and provide a biophysical framework to prevent, diagnose, and treat condensate-driven diseases.