The role of protein phase separation in the circadian clock

Cornell University, Ithaca NY

Investigators

Abstract

PROJECT SUMMARY Circadian clocks confer organismal fitness by anticipating environmental changes brought on by the day/night cycle. At the molecular level, the clock is an autoregulatory transcription-translation feedback loop (TTFL) composed of protein assemblies whose components contain a substantial amount of conformational disorder. While the basic molecular mechanism of the TTFL is understood, the key molecular interactions that underlie the timing of the clock, particularly how disordered regions contribute to clock protein interactions, are unknown. Aberrant clock behaviors are extensive and associated with a wide variety of diseases including neurological, metabolic and immune disorders. As intrinsically disordered proteins are implicated in liquid-liquid phase separation (LLPS), we and others have proposed that phase separation plays a role in the TTFL oscillator. Formation and dissolution of clock protein coacervates may contribute to their spatiotemporal organization and regulate their activities to control clock timing. The filamentous fungus Neurospora crassa contains a core clock mechanism analogous to that of higher organisms and has hence long served as a tractable model system. Here, I focus on the positive arm of the N. crassa clock that is comprised of two proteins, WC1 and WC2, that form the heteromeric White-Collar Complex (WCC). We will investigate the forces that underlie WC1 and WC2 phase separation and, in turn, determine how phase separation influences the structural dynamics and functions of these proteins, including timing and light entrainment. Aim 1 will explore the sequence features, solution conditions, and post-translational modifications that control WC1 and WC2 LLPS and how LLPS, in turn, regulates interactions and blue-light sensing. I will induce LLPS of full-length, truncated and phospho-mimetic constructs under various solution conditions and assess how interactions between the WCC and other clock components change as a function of LLPS. I will also study the effect of LLPS on photoactivation of WC1. Aim 2 will define the molecular interactions between White-Collar proteins and other clock components by determining WC1 and WC2 stoichiometry and dynamics and mapping out interaction interfaces and affinities using diffraction, light scattering, cross-linking mass spectrometry and optical analytical techniques. Additionally, we will obtain structural details of the ordered regions with the WCC using cryo-electron microscopy. These results will provide insight into how intrinsic disorder drives WC1 and WC2 LLPS and how a phase separated compartment influences biomolecular interactions, enzymatic activities and light-sensing capabilities. My work will inform on how aberrations that disrupt condensate formation may lead to clock dysfunction and disease. This project, carried out over three years at Cornell University and in collaboration with clock biologists, will advance my technical skills in molecular biology and biophysical chemistry and grow my expertise as a professional scientist who can lead complex projects in either academic or industrial contexts.