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.

To maximize a desired product, metabolic engineers typically express enzymes to high, constant levels. Yet permanent pathway activation can have undesirable consequences including competition with essential pathways and accumulation of toxic intermediates. Faced with similar challenges, natural metabolic systems compartmentalize enzymes into organelles or post-translationally induce activity under certain conditions. Here, we report that optogenetic control can be used to extend compartmentalization and dynamic control to engineered metabolisms in yeast. We describe a suite of optogenetic tools to trigger assembly and disassembly of metabolically-active enzyme clusters. Using the deoxyviolacein biosynthesis pathway as a model system, we find that light-switchable clustering can enhance product formation by six-fold and product specificity by 18-fold by decreasing the concentration of intermediate metabolites and reducing flux through competing pathways. Inducible compartmentalization of enzymes into synthetic organelles can thus be used to control engineered metabolic pathways, limit intermediates and favor the formation of desired products.

SUMMARY Animal development is characterized by signaling events that occur at precise locations and times within the embryo, yet determining when and where such precision is needed for proper embryogenesis has been a longstanding challenge. Here we address this question for Erk signaling, a key developmental patterning cue. We describe an optogenetic system for activating Erk with high spatiotemporal precision in vivo. Implementing this system in Drosophila, we find that embryogenesis is remarkably robust to ectopic Erk signaling, except from 1 to 4 hours post fertilization when perturbing the spatial extent of Erk pathway activation leads to dramatic disruptions of patterning and morphogenesis. Later in development, the effects of ectopic signaling are buffered, at least in part by combinatorial mechanisms. Our approach can be used to systematically probe the differential contributions of the Erk pathway and concurrent signals, leading to a more quantitative understanding of developmental signaling.

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• p210 BCR/ABL interacts with b-catenin in the bone marrow transplantation model for chronic myelogenous leukemia.• Loss of the interaction results in an altered disease phenotype, suggesting a role for b-catenin in chronic phase disease.We have identified a ubiquitin-binding domain within the NH 2 -terminal sequences of p210 BCR/ABL and determined that the binding site co-localizes with the binding site for b-catenin. The domain does not support the auto-or trans-kinase activity of p210 BCR/ABL or its ability to interact with GRB2 and activate ERK1/2 signaling. Expression of p210 BCR/ ABL, but not a b-catenin-binding mutant, in hematopoietic cells is associated with the accumulation of p-b-catenin (Tyr654) and increased TCF/LEF-mediated transcription. In a bone marrow transplantation model, the interaction between b-catenin and p-b-catenin (Tyr654) is detectable in mice transplanted with p210 BCR/ABL, but not the mutant. Whereas mice transplanted with p210 BCR/ABL exhibit myeloid disease with expansion of monocytes and neutrophils, mice transplanted with the mutant predominantly exhibit expansion of neutrophils, polycythemia, and increased lifespan. The increased disease latency is associated with expansion of megakaryocyte-erythrocyte progenitors, a decrease in common myeloid progenitors, and reduced b-catenin signaling in the bone marrow of the diseased mice. These observations support a model in which p210 BCR/ABL may influence lineage-specific leukemic expansion by directly binding and phosphorylating b-catenin and altering its transcriptional activity. They further suggest that the interaction may play a role in chronic phase disease progression. (Blood. 2013;122(12):2114-2124

Patients with chronic myelogenous leukemia (CML) typically carry a balanced reciprocal translocation between chromosomes 9 and 22, resulting in the production of an in-frame fusion protein known as p210 BCR/ABL. Expression of p210 BCR/ABL in myeloid cells is associated with a variety of transformed cellular phenotypes including changes in nucleotide excision repair (NER). Consistent with this, previous studies have demonstrated that p210 BCR/ABL interacts directly with xeroderma pigmentosum group B (XPB), a protein necessary for both transcription and NER. In the current study we have mapped the docking site for XPB within the RhoGEF domain of BCR, and have constructed a p210 BCR/ABL mutant that is deficient in XPB binding. The mutant has normal kinase activity, and retains Grb2 binding ability, but can no longer phosphorylate XPB. When expressed in 32Dc13 cells, the mutant still confers IL-3 independent growth, but abrogates the resistance to ultraviolet C (UVC)-induced apoptosis seen in wild-type p210 BCR/ABL expressing cells. In a bone marrow transplantation model for CML, mice that express the mutant have significantly extended lifespans. Whereas p210 BCR/ABL transplanted mice became moribund within 28–35 days of transplantation, mutant mice survived for an average of 78 days. Necropsies performed at death revealed splenomegaly and clear evidence of myeloproliferation in the mutant mice, however tissue infiltration and lung hemorrhage was less extensive than in the p210 BCR/ABL expressing mice. These results suggest that the interaction between p210 BCR/ABL and XPB regulates disease progression, and may represent a novel target for therapeutic intervention.

Previous studies have demonstrated that p210 BCR/ABL1 interacts directly with the xeroderma pigmentosum group B (XPB) protein, and that XPB is phosphorylated on tyrosine in cells that express p210 BCR/ABL1. In the current study, we have constructed a p210 BCR/ABL1 mutant that can no longer bind to XPB. The mutant has normal kinase activity and interacts with GRB2, but can no longer phosphorylate XPB. Loss of XPB binding is associated with reduced expression of c-MYC and reduced transforming potential in ex-vivo clonogenicity assays, but does not affect nucleotide excision repair in lymphoid or myeloid cells. When examined in a bone marrow transplantation (BMT) model for chronic myelogenous leukemia, mice that express the mutant exhibit attenuated myeloproliferation and lymphoproliferation when compared with mice that express unmodified p210 BCR/ABL1. Thus, the mutant-transplanted mice show predominantly neutrophilic expansion and altered progenitor expansion, and have significantly extended lifespans. This was confirmed in a BMT model for B-cell acute lymphoblastic leukemia, wherein the majority of the mutant-transplanted mice remain disease free. These results suggest that the interaction between p210 BCR/ABL1 and XPB can contribute to disease progression by influencing the lineage commitment of lymphoid and myeloid progenitors.