Spatiotemporal control of intracellular phase transitions using light-activated optoDroplets

By Yongdae Shin, Joel D. Berry, Nicole L. Pannucci, M. Haataja, Jared E. Toettcher, C. Brangwynne. Cell, 2016

861 citations recorded by the source corpus.

Summary Phase transitions driven by intrinsically disordered protein regions (IDRs) have emerged as a ubiquitous mechanism for assembling liquid-like RNA/protein (RNP) bodies and other membrane-less organelles. However, a lack of tools to control intracellular phase transitions limits our ability to understand their role in cell physiology and disease. Here, we introduce an optogenetic platform, which uses light to activate IDR-mediated phase transitions in living cells. We use this “optoDroplet” system to study condensed phases driven by the IDRs of various RNP body proteins, including FUS, DDX4, and HNRNPA1. Above a concentration threshold, these constructs undergo light-activated phase separation, forming spatiotemporally-definable liquid optoDroplets. FUS optoDroplet assembly is fully reversible even after multiple activation cycles. However, cells driven deep within the phase boundary form solid-like gels, which undergo aging into irreversible aggregates. This system can thus elucidate not only physiological phase transitions, but also their link to pathological aggregates.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/ff6f0754a88b3c20ed7675afb5fb42216e23b81f

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Citations in the corpus

Sequence determinants of protein phase behavior from a coarse-grained model (2017, background): “Folded domains 29 along with IDRs have also been shown to modulate LLPS properties [34].”
Liquid phase condensation in cell physiology and disease (2017, background): “One recent study has probed phase transitions in living cells by using optogenetic protein constructs fused to various IDRs, including FUS, DDX4, and hnRNPA1 (96).”
Liquid Nuclear Condensates Mechanically Sense and Restructure the Genome. (2018, positive): “All miRFP670-Cry2 fragments used here are identical to the one described previously (Shin et al., 2017) except for a fluorescent reporter swapped from mCh to miRFP670.”
Coactivator condensation at super-enhancers links phase separation and gene control (2018, methodology): “To investigate whether the coactivator IDRs facilitate phase separation in cells, we used a previously developed assay to manipulate local protein concentrations within the cell; this optoIDR assay tests IDR-dependent, light-inducible droplet formation in vivo (44).”
Competing protein-RNA interaction networks control multiphase intracellular organization (2020, positive): “…shifts are absent for self-associating FUS IDR Corelet condensates (Figures S4D and S4E), which do not recruit SG proteins (Figure S2A) and are thus not SGs; this is consistent with previous studies using an orthogonal Cry2-based optoDroplet approach (Shin et al., 2017; Zhang et al., 2019).”
Intrinsically disordered regions contribute promiscuous interactions to RNP granule assembly (2017, background): “For example, even very high expression levels of hnRNPA1-Cry2 or DDX4-Cry2 fusion proteins do not phase separate in cells, unless the Cry2 protein is first triggered to assemble through specific light activated interactions (See Figure 2B&C of Shin et al., 2016).”
Tau protein liquid–liquid phase separation can initiate tau aggregation (2018, neutral): “…40 aa) C-terminal tail, and a long ( (cid:1) 250 aa) flexible N-terminal half of tau, which projects from the surface of microtubules in the MT-bound state (Goode et al , 1997), and forms a polyelectrolyte brush around fibrillary aggregates of tau (Sillen et al , 2005; Wegmann et al , 2013).”