RESUMEN
Biomolecular condensates are membraneless compartments that impart spatial and temporal organization to cells. Condensates can undergo maturation, transitioning from dynamic liquid-like states into solid-like states associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Huntington's disease. Despite their important roles, many aspects of condensate biology remain incompletely understood, requiring tools for acutely manipulating condensate-relevant processes within cells. Here we used the BCL6 BTB domain and its ligands BI-3802 and BI-3812 to create a chemical genetic platform, BTBolig, allowing inducible condensate formation and dissolution. We also developed optogenetic and chemical methods for controlled induction of condensate maturation, where we surprisingly observed recruitment of chaperones into the condensate core and formation of dynamic biphasic condensates. Our work provides insights into the interaction of condensates with proteostasis pathways and introduces a suite of chemical-genetic approaches to probe the role of biomolecular condensates in health and disease.
Asunto(s)
Condensados Biomoleculares , Enfermedad de Huntington , Humanos , Enfermedad de Huntington/genética , Optogenética , ProteostasisRESUMEN
Synthetic regulation of gene expression provides a powerful approach to reprogram molecular and cellular processes and test the function of specific genes and gene products. In the last decade, optogenetic systems that allow light-dependent gene regulation have become valuable tools, providing tight spatiotemporal control of protein levels. Here we discuss and build on recent optogenetic approaches for regulating gene expression in mammalian cells using cryptochrome 2 (CRY2), a photoreceptor protein from Arabidopsis. We provide detailed protocols for using light to manipulate activity of a CRY2-based engineered photoactivatable Cre DNA recombinase, and to induce or disrupt transcription factor function. In addition, we provide instructions and software for building an inexpensive Rasberry-Pi-based programable LED device for optogenetic experiments, delivering pulsed light with customized control of illumination duration, frequency, and intensity.
Asunto(s)
Proteínas de Arabidopsis/genética , Criptocromos/genética , Regulación de la Expresión Génica/efectos de la radiación , Optogenética/métodos , Factores de Transcripción/metabolismo , Proteínas de Arabidopsis/metabolismo , Criptocromos/metabolismo , Genes Reporteros/genética , Células HEK293 , Humanos , Integrasas/metabolismo , Luz , Luciferasas/genética , Luciferasas/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Optogenética/instrumentación , Programas InformáticosRESUMEN
Dynamic membraneless compartments formed by protein condensates have multifunctional roles in cellular biology. Tools that inducibly trigger condensate formation have been useful for exploring their cellular function, however, there are few tools that provide inducible control over condensate disruption. To address this need we developed DisCo (Disassembly of Condensates), which relies on the use of chemical dimerizers to inducibly recruit a ligand to the condensate-forming protein, triggering condensate dissociation. We demonstrate use of DisCo to disrupt condensates of FUS, associated with amyotrophic lateral sclerosis, and to prevent formation of polyglutamine-containing huntingtin condensates, associated with Huntington's disease. In addition, we combined DisCo with a tool to induce condensates with light, CRY2olig, achieving bidirectional control of condensate formation and disassembly using orthogonal inputs of light and rapamycin. Our results demonstrate a method to manipulate condensate states that will have broad utility, enabling better understanding of the biological role of condensates in health and disease.
Asunto(s)
Proteínas Fluorescentes Verdes/química , Ensayos Analíticos de Alto Rendimiento/métodos , Multimerización de Proteína , Proteínas/química , Animales , Células COS , Chlorocebus aethiops , Transferencia Resonante de Energía de Fluorescencia , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Microscopía Fluorescente/métodos , Proteínas/genética , Proteínas/metabolismo , Proteína FUS de Unión a ARN/química , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismoRESUMEN
Optogenetic tools allow for use of light as an external input to control cellular processes. When applied to regulate the function of transcription factors, optogenetic approaches provide a tunable, reversible, and bidirectional method to control gene expression. Herein, we present a detailed method to induce gene expression in mammalian cells using the light dependent dimerization of cryptochrome 2 (CRY2) and CIB1 to complement a split transcription factor. We also describe a protocol to disrupt gene expression with light by fusing a dimeric transcription factor to CRY2. When combined with a light-induced degron attached to the gene product, this method allows for rapid modulation of target protein abundance.