Rapid and reversible dissolution of biomolecular condensates using light-controlled recruitment of a solubility tag

Brumbaugh-Reed, Ellen H.; Gao, Yang; Aoki, Kazuhiro; Toettcher, Jared E.

doi:10.1038/s41467-024-50858-0

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Published: 07 August 2024

Rapid and reversible dissolution of biomolecular condensates using light-controlled recruitment of a solubility tag

Nature Communications volume 15, Article number: 6717 (2024) Cite this article

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Abstract

Biomolecular condensates are broadly implicated in both normal cellular regulation and disease. Consequently, several chemical biology and optogenetic approaches have been developed to induce phase separation of a protein of interest. However, few tools are available to perform the converse function – dissolving a condensate of interest on demand. Such a tool would aid in testing whether the condensate plays specific functional roles. Here we show that light-gated recruitment of a solubilizing domain, maltose-binding protein (MBP), results in rapid and controlled dissolution of condensates formed from proteins of interest. Our optogenetic MBP-based dissolution strategy (OptoMBP) is rapid, reversible, and can be spatially controlled with subcellular precision. We also provide a proof-of-principle application of OptoMBP by disrupting condensation of the oncogenic fusion protein FUS-CHOP and reverting FUS-CHOP driven transcriptional changes. We envision that the OptoMBP system could be broadly useful for disrupting constitutive protein condensates to probe their biological functions.

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Introduction

Many proteins are organized into biomolecular condensates in either normal or diseased cell states^1,2,3,4. Biomolecular condensates form as a result of many weak, multivalent interactions, leading to co-assembly of proteins and/or nucleic acids into a dense phase containing a high concentration of the condensed components surrounded by a dilute phase⁵. Often, protein phase separation is facilitated by proteins’ intrinsically disordered regions (IDRs): amino acid sequences which lack a fixed secondary structure⁶ and associate with one another through electrostatic, cation-pi, pi-stacking, and hydrophobic interactions⁷. Biomolecular condensates are thought to play important roles in a diverse array of biological processes such as stress responses⁸, immune regulation^9,10, genome organization¹¹, and transcription^12,13. Condensates are thought to carry out biological functions by acting as organizational hubs for biochemical processes, either by colocalizing interacting partners to increase reaction rates or by sequestering components to inhibit their ability to interact¹⁴. Furthermore, aberrant phase transitions are indicated in a number of pathologies ranging from cancer to neurodegenerative disorders^14,15,16.

Due to the importance of phase separation in the regulation of biological processes, a wide variety of optogenetic and chemogenetic strategies have been developed to trigger phase separation of engineered proteins. For example, the optoDroplet system consists of an IDR fused to the blue-light sensitive Cry2^PHR protein domain¹⁷. Upon light stimulation, Cry2 oligomerizes and nucleates condensates which grow through IDR-driven interactions. Several optogenetic tools rely on a similar molecular logic, either through constitutive fusion of an IDR to a light-switchable oligomerization domain (e.g., PixELLs)¹⁸ or through light-dependent association between an IDR and a constitutive oligomerization domain (e.g., Corelets and CasDrop)^19,20. These tools can function as light-activated or light-inhibited phase separation systems depending on whether the underlying optogenetic system’s oligomerization state increases (e.g., OptoDroplets) or decreases (e.g., PixELLs) in response to illumination. Alternative architectures have also been explored, such as light-induced binding between proteins containing multiple discrete folded interaction domains (e.g., the iPOLYMER system)²¹.

In contrast, comparatively few optogenetic tools are available to drive a naturally phase-separated protein from a clustered to a dispersed state. One historically important technique to dissolve biomolecular condensates relies on the addition of the organic compound 1,6-hexanediol to inhibit weak hydrophobic interactions^11,22,23,24. However, the compound’s indiscriminate activity means it cannot be used to specifically dissolve a condensate of interest, and recent studies suggest that it may also interfere with many other cellular processes (e.g., enzyme activity)^25,26. Insertion of a protease cut-site can be used to remove IDRs from a protein upon protease addition, resulting in dissolution of the protein clusters²⁷. However, cleavage and removal of IDR sequences may disrupt other IDR-encoded biological functions (e.g., short linear motifs for binding, localization, or post-translational modification)²⁸. Some approaches take advantage of a different conceptual strategy: recruitment of a “solubilizing” domain to alter a condensate’s propensity to phase separate. For example, the DisCo (Disassembly of Condensates)²⁹ system takes advantage of rapamycin-inducible dimerization³⁰ between the FK506 binding protein (FKBP) and FKBP/rapamycin binding domain (FRB) to dissolve condensates, and a promising recent extension of this approach harnessed the small ubiquitin-like modifier (SUMO) protein to further promote solubilization³¹. Photocleavage of the MBP solubility tag away from an IDR sequence is also very effective at triggering phase separation of the IDR^27,32. However, the irreversibility of these chemical³³ and photocleavable tools preclude reversible or spatially precise control.

Thus, there is still an unmet need for tools to precisely control biomolecular condensate dissolution in the complex environment of the cell. Ideally, such a tool should rapidly disperse a specific endogenous condensate of interest without cleaving or otherwise altering the sequences of its constituents. The tool should also be reversible and spatially controlled, enabling dissolution of condensates at desired times and subcellular locations. This level of precision could be used to study particular condensates within a cell, such as those specifically associated with a single transcriptionally-active gene locus³⁴. Finally, such a strategy should ideally be broadly applicable to target any endogenous condensate-forming protein of interest.

Here, we report progress toward these goals by developing an optogenetic strategy for IDR-based condensate dissolution. Our approach centers on light-based recruitment of maltose binding protein (MBP), a widely-used purification tag that has been used to increase solubility of a broad range of proteins during bacterial expression and purification, including proteins which undergo phase separation, in vitro and in vivo^{27,32,35,36,37}. We show that in addition to its utility as a fusion tag, MBP can be recruited to a target protein to rapidly and reversibly to alter its partitioning between soluble and condensed phases. Condensates can also be manipulated with precise spatial control, for example, to target the dissolution of individual condensates within the nucleus. We further show that light-based MBP recruitment leads to dissolution of condensates in several cases, including all three FET family members (FUS, EWS, and TAF15), the LAF-1 RGG domain, and the RNA-binding protein Oskar, suggesting that this approach may be of broad utility for interrogating the biological consequences of protein phase separation. Finally, we apply our OptoMBP approach to probe an exemplary functional context: transcription induced by condensates of the oncogenic transcription factor FUS-CHOP. We show that light-based dissolution is sufficient to partially revert aberrant FUS-CHOP gene expression within 6 hours, suggesting a direct role of FUS-CHOP phase separation in gene expression. Our approach opens the door to rapid, reversible, and precise control over protein organization in both native biological processes and disease states.

Results

Recruiting a solubility tag to condensates can drive dissolution

The propensity of a protein to undergo phase separation can be regulated by many factors including concentration^38,39,40, temperature^41,42, salt concentration²⁷, pH⁴³, post-translational modifications^44,45, and interactions with additional proteins^46,47. We reasoned that temperature, salt concentration, and pH are nonspecific, post-translational modifications can have many consequences beyond tuning phase separation, and that it is difficult to alter total protein concentration rapidly and with high subcellular precision. We thus focused on tuning phase separation through binding to candidate “solubilizing” protein domains.

As a first step toward this goal, we established a model condensate that could be expressed and screened for binding-induced dissolution. Our model condensate was based on the intrinsically disordered region (IDR) of Fused in Sarcoma (FUS)^48,49,50, a sequence that has been studied extensively as part of prior optogenetic and chemogenetic tools for manipulating biomolecular condensates^17,18,19. At typical cellular concentrations, a single copy of the FUS^IDR remains dispersed unless it is linked to additional oligomerization domains¹⁷. We hypothesized that a protein containing multiple FUS^IDR sequences would condense without the need for additional oligomerization domains, forming a simple IDR-driven constitutive condensate in cells. Indeed, a construct containing three FUS^IDR repeats, the FusionRed fluorescent protein, an improved light-inducible dimer (iLID) optogenetic recruitment domain⁵¹, and an FRB chemogenetic recruitment domain³⁰ (3xFUS^IDR-FusionRed-FRB-iLID; Fig. 1A) constitutively formed protein droplets in the nucleus when transfected into HEK293T cells (Fig. 1B). This construct is compatible with both optogenetic and chemogenetic dimerization systems through the interactions between iLID-SspB and FRB-FKBP, respectively^30,51. In the iLID-SspB system, blue light induces a conformational change in the iLID protein that reveals a binding site for SspB; the system reverts to the unbound state in the dark with a characteristic timescale of ~30 sec in the dark. The FRB-FKBP chemical dimerization system functions in an analogous manner: the addition of the small molecule rapamycin or one of its analogs induces the formation of a tight complex between FRB, FKBP, and the drug.

**Fig. 1: Screen for light-controlled dissolution of FUS^IDR based condensates.**

We next set out to determine whether optogenetic recruitment of a solubilizer domain could drive dissolution of 3xFUS^IDR condensates. We chose MBP as a lead candidate (size: 41 Da; pI = 5.2; see Fig. S1A for surface electrostatics and aromatic residues), a protein commonly used to increase the solubility of proteins purified from bacterial expression systems⁵². The mechanism of MBP-induced condensate or aggregate solubilization is still an active area of research^53,54, but occurs both in cells and in vitro using purified proteins³², suggesting that the effect is intrinsic to the MBP-target fusion protein and does not require the action of additional chaperones or cellular factors. MBP has also been shown to prevent condensate formation during protein purification of a wide variety of IDRs^35,36,37. We also tested Glutathione S-transferase (GST), an affinity tag commonly used in protein purification that has also been suggested to promote solubility⁵⁵, and mCherry, which was previously suggested to inhibit protein phase separation²⁹. Finally, we also tested single and double repeats of mEGFP to probe if condensation dissolution might be a function of cargo protein size. Each candidate domain was fused to SspB, which forms one-to-one heterodimers with iLID upon blue light illumination, along with the mIFP monomeric infrared fluorescent protein⁵⁶ (henceforth iRFP) for visualization. We used non-fluorescent mutants of mCherry⁵⁷ and mEGFP⁵⁸ in our candidate optogenetic dissolution systems so as not to interfere with the detection of other fluorescent proteins in the assay.

We co-transfected HEK293T cells with our 3xFUS^IDR construct and a candidate SspB- and iRFP-tagged solubilizer domain and imaged cells before and after treatment with 3 min of 450 nm blue light to dimerize SspB and iLID (Fig. 1B). Qualitatively, we observed no illumination-induced changes in 3xFUS^IDR condensates in cells lacking a solubilizer construct or expressing GST, mEGFP, or mEGFP-mEGFP. GST colocalized to FUS^IDR condensates even in the absence of blue light exposure (Fig. 1B), indicative of a constitutive, light-independent interaction between these constructs that did not interfere with FUS^IDR condensate formation. We observed weak light-induced dissolution upon mCherry recruitment. Notably, only MBP recruitment appeared to drive complete dissociation of 3xFUS^IDR droplets upon illumination.

To quantify the results of these experiments, we selected cells for analysis with comparable expression levels of FUS^IDR and the solubilizer protein (Fig. S1B, C) and quantified the changes in the coefficient of variation (CV) and local enrichment (enrichment) of FusionRed pixel intensity in the cell nucleus upon light illumination (Fig. 1C, D). Cells with a high degree of FUS^IDR condensate formation have a high CV in pixel intensity, as they harbor both bright pixels within condensates and dark pixels between them. We estimated the FusionRed enrichment in condensates by dividing the maximum FusionRed pixel intensity by the mean pixel intensity in the nucleus. Both CV and enrichment are expected to decrease following condensate dissolution. We only observed a significant change in both metrics for MBP and mCherry recruitment, with a larger magnitude of effect for MBP (Fig. 1C, D), results that are consistent with our qualitative observations (Fig. 1B). Notably, the solubilizing effects of mCherry and MBP cannot be solely explained by their cargo sizes, as measured by each protein’s computed radius of gyration⁵⁹ (Fig. S1D). Because each domain was recruited using the same optogenetic binding motif, we can conclude that the extent of dissolution is primarily determined by properties other than cargo size, concentration, or binding affinity.

We performed additional analyses to further characterize MBP-driven condensate dissolution. We benchmarked our results to a gold standard by comparing the extent of MBP-induced dissolution to 1,6-hexanediol-induced dissolution, a chemical that has been frequently used to disperse a variety of condensates^11,22,23,24. We observed similar changes in FusionRed CV and enrichment upon both hexanediol treatment and light-induced MBP recruitment, confirming our qualitative observation that both treatments appeared to drive complete dissolution of the 3xFUS^IDR condensates (Fig. S1E-G). We also implemented a chemogenetic variant of the MBP recruitment system that takes advantage of the FRB domain on our 3xFUS^IDR construct for rapamycin-based protein dimerization³⁰ (Fig. 1E). We transfected the 3xFUS^IDR construct with either the SspB-tagged or FKBP-tagged MBP construct and selected cells with similar expression levels for analysis (Fig. S1H-I), which revealed comparable dissolution of 3xFUS^IDR condensates in response to either 3’ of illumination or 30’ of treatment with the rapamycin analog (rapalog) AP21967 (Fig. 1F–H), and increased dissolution upon MBP-FKBP recruitment compared to an FKBP-iRFP construct lacking MBP (Fig. S1J, K), despite FKBP-mediated solubilization consistent with prior results using the DisCo system²⁹. Overall, we found that the kinetics of MBP recruitment and 3xFUS^IDR dissolution were slower upon rapalog treatment than with light, suggesting that rapalog incorporation into these intracellular droplets limited its kinetics (Fig. S2). Together, these results suggest that inducible MBP recruitment acts as a potent dissolver of FUS^IDR condensates. Based on its superior performance in these early screens, we focused on light-based MBP recruitment for subsequent experiments, which we termed the OptoMBP system.

OptoMBP-driven dissolution is rapid, reversible, and spatially precise

We next set out to map the spatial and temporal precision of OptoMBP-induced condensate dissolution. To do so, we used a 450 nm LED light source which was coupled to a spinning disk confocal microscope using a digital micromirror device (see Methods). This system can be used to produce instantaneous, quantitative control over the light intensity and restrict illumination to user-defined 2-D spatial regions^60,61. To characterize the dynamics of disassembly, we imaged 3xFUS^IDR and MBP localization in living cells at high time resolution after blue light illumination (Fig. 2A). We quantified the localization of both constructs over time using the change in the coefficient of variation of per-nucleus pixel intensities as reported previously¹⁸. Quantification across multiple cells revealed that MBP was recruited into FUS droplets within ~5 sec after blue light illumination (Fig. 2B), matching previously-measured kinetics of optogenetic recruitment in living cells^60,61. The resulting 3xFUS^IDR/MBP condensates subsequently dispersed with a characteristic timescale of ~13 sec, comparable to the kinetics observed in the PixELL system¹⁸, where light is used to acutely disrupt interactions between two engineered proteins (FUS^IDR-PixD and FUS^IDR-PixE) to drive their disassembly (Fig. 2C). These results indicate that MBP-induced dissolution is exceptionally rapid, proceeding as quickly as acute disruption of intramolecular interactions within the condensate.

**Fig. 2: Spatiotemporal control of condensate dissolution.**

We next tested whether MBP-induced dissolution of 3xFUS^IDR condensates was also reversible after removal of the light stimulus, enabling multiple cycles of condensate assembly and disassembly. We switched HEK293T cells expressing 3xFUS^IDR and the OptoMBP system between illuminated and dark conditions and measured condensation over time. This experiment revealed that cycles of illumination could drive repeated dissolution and reassembly of 3xFUS^IDR droplets with similar kinetics in each cycle (Fig. 2D, E; Fig. S3A; Supplementary Movie 1). Droplet formation in the dark was slower than droplet dissolution in the light, occurring with a characteristic timescale of ~110 seconds (Fig. 2F). This is consistent with prior observations of minutes-timescale FUS^IDR condensation in cells¹⁷ and is likely driven by the timescale of condensate nucleation. Overall, our kinetic data demonstrate that OptoMBP based dissolution of FUS^IDR-based condensates is both rapid and reversible in living cells.

An additional benefit of optogenetic control is the ability to apply inputs with high spatial precision to nucleate or dissolve droplets at particular positions within the cell^17,18. To test whether the OptoMBP system is capable of subcellular spatial control, we first illuminated half of an OptoMBP-expressing cell and monitored the spatial distribution of 3xFUS^IDR condensates. We observed localized dissolution that was predominantly confined to the illuminated area, even after minutes of continuous illumination (Fig. 2G, H; Fig. S3B; Supplementary Movie 2). We also tested whether the spatial asymmetry persisted after removal of the light stimulus as predicted by prior work¹⁸. Indeed, condensates in the previously illuminated region remained smaller than droplets in the non-illuminated area several minutes after recovery, confirming that local disassembly produces long-term spatial asymmetries in condensate location (Fig. S3C).

To further probe the limits of spatial control, we tested whether it might be possible to dissolve a single FUS droplet within the nucleus, a scale that is beyond the reach of prior optogenetic dissolution approaches¹⁸. Such a finely targeted stimulus could be useful for perturbing an individual transcriptional locus or other localized condensate-associated processes. We conducted this experiment in NIH3T3 cells stably expressing an optogenetically targetable FUS-CHOP oncoprotein (see detailed description below), because the larger nucleus and distance between droplets facilitated illumination of only a single droplet. Indeed, we observed robust dissolution of illuminated single droplets with only minimal effects on droplets in distal regions of the nucleus (Fig. 2I; Fig. S3D; Supplementary Movie 3).

Quantification of localized dissolution in both 3xFUS^IDR expressing HEK293T cells (Fig. 2H) and FUS-CHOP expressing NIH3T3 cells (Fig. 2J) revealed a notably slower dissolution timescale of 30 sec – 3 min compared to our previous global illumination experiments (Fig. 2C). We reasoned that condensates could be maintained for longer time periods either due to (1) slower photoconversion at the lower illumination intensities required for locally precise patterning, or (2) of mixing between lit-state and dark-state 3xFUS^IDR molecules between the illuminated and non-illuminated regions leading to more gradual complete photoactivation. To discriminate between these hypotheses, we performed kinetic measurements in cells which were globally illuminated at standard intensity (~200 mW/cm²), at low intensity (~30 mW/cm²), or at low intensity and under partial-cell illumination (Fig. S3E, F). We found that kinetics were only dramatically changed by the transition from whole-cell (τ = 0.32 + /− 0.05 min) to partial-cell illumination (τ = 1.74 + /− 0.27 min), indicating that protein transport in and out of the illumination region plays an important role in the timescale of light-induced condensate disassembly. Taken together, our results demonstrate that the OptoMBP system can be used to perform rapid and spatially precise condensate dissolution in live cells.

Dissolution depends sensitively on solubilizer concentration

How does the efficacy of the OptoMBP system depend on the expression levels of MBP and the target protein (in this case, the 3xFUS^IDR)? Intracellular biomolecular condensates are complex and may involve interactions between many components: co-condensing proteins and RNAs⁴⁰, chaperones that regulate condensate assembly and disassembly⁶², or cooperative interactions between multiple monomers (e.g. to nucleate a new condensate), and we reasoned that OptoMBP-based dissolution may also exhibit a complex dose response if MBP acts cooperatively or recruits additional effectors. We thus set out to quantify light-induced condensate dissolution across a range of FUS and MBP expression levels.

We engineered a NIH3T3 mouse fibroblast cell line in which both the 3xFUS^IDR-FusionRed- FRB-iLID and MBP-SspB-iRFP constructs were stably expressed using lentiviral vectors (Fig. 1A). Lentiviral transduction can drive a wide range of protein expression levels depending on integration site, and we sorted cells to cover a ~ 10-fold range of expression of both components of the system (Fig. S4A). We then imaged single cells to measure their initial FusionRed and iRFP expression as well as the extent of FUS^IDR condensation before and after blue light illumination. For dark-incubated cells we observed a sharp threshold in cellular FUS^IDR expression above which nuclear condensates were observed and corresponded to high enrichment scores (maximum divided by mean nuclear pixel intensity) (Fig. 3A, purple dots). However, while the presence or absence of condensates was well predicted by 3xFUS^IDR concentration, we observed that the dilute phase 3xFUS^IDR concentration did not plateau at a well-defined saturation concentration, as would be expected for a simple single-component condensate, but instead continued increasing with total nuclear 3xFUS^IDR levels, a phenomenon that has been previously described for multi-component biomolecular condensates⁴⁰ (Fig. S4B). We also observed variable enrichment scores between cells even at the same overall cellular 3xFUS^IDR concentration (Fig. 3A). Overall, these data are consistent with 3xFUS^IDR condensation existing as a complex process that likely involves interaction with additional components whose levels may vary between cells⁶³.

**Fig. 3: Dissolution efficiency is dependent on MBP concentration.**

We next set out to measure how the 3xFUS^IDR dissolution depended on the concentration of our OptoMBP solubilizer construct. We separately analyzed dissolution in cells with low (<3500 aribtrary fluorescent units; arb. units), medium (3500-4500 arb. units), and high (>4500 arb. units) MBP-SspB-iRFP expression (Fig. 3B). Optogenetic activation of cells expressing high levels of MBP-SspB-iRFP produced rapid dissolution of 3xFUS^IDR condensates in nearly all cells (Fig. 3B, high [MBP]), whereas cells harboring medium or low MBP expression dissolved condensates in progressively fewer cells (Fig. 3B, med [MBP], low [MBP]). These data confirm that MBP expression level is a crucial parameter in determining the extent of optogenetic condensate dissolution. We further tested whether light-induced dissolution behaved stoichiometrically – that is, whether cells with higher 3xFUS^IDR expression required proportionally higher MBP expression to drive dissolution. To do so, we analyzed single-cell expression of both 3xFUS^IDR and MBP as well as 3xFUS^IDR condensation in the dark and light (Fig. S4C). Unexpectedly, we did not observe a clear relationship between 3xFUS^IDR expression level and the concentration of MBP required for light-induced dissolution. Instead, our data revealed a threshold in MBP expression (~4500 arbitrary units; arb. units) above which nearly all 3xFUS^IDR condensates were dissolved under blue light, regardless of their 3xFUS^IDR expression level. At lower MBP expression levels, illumination was still able to dissolve 3xFUS^IDR condensates in a lower but still substantial fraction of cells (~30%; Fig. 3B). MBP-induced condensate dissolution thus appears to act nonlinearly, with a large increase in condensate dissolution observed over a narrow range of concentrations. We also note that fewer cells expressing high levels of MBP showed very high enrichment scores prior to illumination compared to cells with low MBP levels (Fig. 3B, top row), which could result from baseline dark-state binding⁵¹ between solubilizer and condensate partially limiting 3xFUS^IDR condensation in these cells, or from other effects of high intracellular MBP expression.

Taken together, these data underscore the complex nature of condensate formation and dissolution in vivo, wherein multiple interacting components might define parameters such as the dilute-phase concentration of each component and the threshold concentration above which condensates are observed³⁸. They also point to important performance characteristics of the OptoMBP system. Above a threshold concentration of MBP, the OptoMBP system appears to be effective at reversing 3xFUS^IDR phase separation at a wide range of expression levels. The sharp dependence with MBP concentration but weak dependence on 3xFUS^IDR concentration suggests that a critical threshold of MBP must be surpassed to drive a robust solubilizing response. These findings may help to constrain models of the biophysical basis for MBP-induced solubilization, a process that is still poorly understood^54,64.

OptoMBP-based dissolution generalizes to several condensates

MBP has been reported to solubilize a wide range of target proteins in fusion constructs⁵², so we hypothesized that OptoMBP recruitment may also dissolve condensates other than 3xFUS^IDR. We first tested other members of the FUS-EWS-TAF15 (FET) protein family, all of which form nuclear condensates, bind and regulate RNA⁶⁵, and contain disordered domains⁶⁶ rich in glycine, glutamine, serine, and tyrosine (Fig. 4A). We first created EWS and TAF15 constructs consisting of three repeats of their disordered domain, architectures that are comparable to our 3xFUS^IDR construct. As with 3xFUS^IDR, 3xEWS^IDR and 3xTAF15^IDR formed condensates when transfected into HEK293T cells (Fig. S5A), and optogenetic MBP recruitment resulted in robust dissolution in each case (Fig. 4B). Analysis showed that CV and enrichment decreased after illumination for all three constructs (Fig. 4C-D), with no change in condensates observed in response to recruitment of a control SspB-iRFP lacking MBP (Fig. S5B-C).

**Fig. 4: MBP-based condensate dissolution extends to FET family proteins.**

We next tested whether similar results might be obtained from the full-length FET family members containing both the disordered and the RNA-binding domains. We constructed fluorescent, iLID-tagged variants of all three FET family protein members and observed visible condensates after transient transfection, although we found that these condensates were less spherical and, for FUS and TAF15, showed lower enrichment in the condensed phase compared to their IDR-only counterparts (Fig. S5A). We observed dissolution in each case upon OptoMBP recruitment (Fig. 4E), and analysis of the changes in CV and enrichment for all FET family constructs (Fig. 4F, G) confirmed our qualitative observation that MBP recruitment results in dissolution compared to SspB-iRFP alone (Fig. S5B, C).

We further probed whether MBP-based dissolution might generalize outside of the FET family to other condensate-forming proteins of interest. We tested three repeats of the disordered RGG domain from LAF-1 which has been shown to be solubilized by MBP fusion^27,32; the full-length RNA binding protein Oskar (Supplementary Movie 4), which is required to form germ granules during Drosophila development⁶⁷; mRNA decapping protein 1 A (DCP1A)⁴⁰, which participates in mRNA degradation; and PML IV, a major component of PML-nuclear bodies⁶⁸. All four proteins formed condensates in either the nucleus (Oskar, PML IV) or cytosol (3xRGG, DCP1A) when fused to FusionRed and the iLID optogenetic recruitment domains and transfected into HEK293T cells (Fig. S5D). Moreover, all four proteins recruited MBP-SspB when illuminated with blue light (Fig. S5D, early). However, condensate dissolution was variable across this broader set of proteins. We observed robust dissolution for the 3xRGG construct and Oskar, but DCP1A and PML IV were unaffected by light-induced MBP recruitment (Fig. S5D, late). Together, these results show that OptoMBP-based dissolution generalizes outside of the FET family to additional types of condensate-forming proteins in both the nucleus and cytoplasm. However, it is not universally applicable, with some condensates showing little to no dissolution upon MBP recruitment.

Oncogenic condensate dissolution drives transcriptional changes

Finally, we set out to apply the OptoMBP system to perturb condensate-dependent biological functions. A growing body of evidence demonstrates that condensates can represent functionally relevant states. For example, co-condensation of enzymes may enhance reaction rates⁹ or coordinate processes at length scales much longer than individual proteins⁶⁹. Gene expression is thought to be controlled by condensation of transcription factors and other transcriptional machinery^12,34,70, but directly testing this model by triggering condensation on demand can drive complex and unexpected outcomes. For example, while the morphogen transcription factor Bicoid has been shown to cluster at sites of active transcription⁷¹, light-induced clustering of the Bicoid-Cry2 optogenetic fusion protein paradoxically acts as a dominant-negative transcription factor that blocks endogenous Bicoid-induced gene expression⁷², potentially due to improper clustering between these proteins at sites other than the appropriate target enhancers. We reasoned that an alternative approach to manipulating transcription-associated condensates might be to dissolve endogenously formed condensates using the OptoMBP system, which can potentially act with high temporal and spatial resolution.

We set out to monitor the effects of OptoMBP-induced dissolution on model transcriptional condensates formed of the oncogenic fusion protein FUS-CHOP, which is associated with liposarcoma^73,74,75. The FUS-CHOP oncoprotein is produced by a chromosomal translocation event wherein the disordered, N-terminal region of FUS is fused to the transcription factor CHOP (CCAAT/enhancer-binding protein homologous protein, also known as DDIT3 - DNA damage inducible transcript 3). Previous work has shown that FUS-CHOP forms biomolecular condensates and a plausible model for its action involves improper transcriptional activation triggered by FUS condensation at DNA loci targeted by the CHOP DNA binding domain. However, the role of condensate formation on the transcriptional activity of the FUS-CHOP oncoprotein is not well understood^16,76.

We developed an NIH3T3 cell line containing a doxycycline-inducible FUS-CHOP construct fused to FusionRed fluorescent protein and the iLID light-activatable domain (Fig. 5A), which we termed FUS-CHOP/OptoMBP cells. We chose NIH3T3 cells because of their ease of manipulation and prior use in studying the transcriptional activity and oncogenic properties of FUS-CHOP and related fusion proteins⁷⁷. We co-expressed FUS-CHOP with the rtTA transcription factor and constitutively expressed OptoMBP light-induced dissociation system (MBP-SspB-iRFP) (Fig. 5A). We observed nuclear FUS-CHOP-FusionRed-iLID condensates within 24 h after doxycycline induction, and, as in prior FUS-related contexts, these condensates could be rapidly and completely dissolved by light-induced MBP recruitment (Fig. 5B, C; Fig. 2I, J, Supplementary Movies 3 and 5).

**Fig. 5: Dissolution of the oncogenic fusion protein FUS-CHOP results in reversion transcriptional changes.**

We next set out to test how optogenetic FUS-CHOP dissolution might alter transcription driven by this oncoprotein. We plated FUS-CHOP/OptoMBP cells and treated them with doxycycline to induce FUS-CHOP expression for 24 h, with mock-treated cells used as a control (Fig. 5D). We then continued incubating cells in the dark or under 450 nm blue LED light (at an intensity of ~1 mW/cm², comparable to previously published transcriptional assays^70,78) for an additional 6 hours to provide sufficient time to dissolve FUS-CHOP condensates and resolve any resulting changes in gene expression. Finally, we conducted bulk RNASeq to assess transcriptional changes in three replicates of each of the four conditions (Fig. 5E,F).

We first assessed the transcriptional consequences of FUS-CHOP expression by identifying differentially expressed genes between doxycycline-treated and untreated conditions in the dark (Fig. S6A). Differential expression analysis revealed widespread expression changes with 283 genes increasing more than 2-fold after doxycycline induction and 570 genes decreasing more than 2-fold. We compared these data to a previously published FUS-CHOP RNASeq data set in a human fibrosarcoma cell line, HT1080⁷⁹; looking at the top 100 hits in HT1080 published genes that also displayed at least a 2-fold change in expression in our study, ~80% displayed the same direction of change in both datasets (Fig. S6B).

We next assessed gene expression differences between dark and blue light in the absence of doxycycline to control for any illumination-induced changes in gene expression that were not related to FUS-CHOP or our optogenetic system (Fig. S6C). Light-triggered gene expression that is independent of optogenetic tool expression has been previously documented in neuronal contexts, albeit at higher illumination intensities (>100 mW/cm²) than our system requires⁸⁰. To minimize confounding effects of illumination on our analysis, we implemented stringent p-value (<0.05) and fold-change (>25%) cutoffs to exclude light-controlled genes from subsequent analysis. This analysis yielded 253 genes whose expression differed upon blue light illumination in the absence of FUS-CHOP. We also performed gene set enrichment analysis (GSEA) comparing non-illuminated and illuminated samples in the absence of doxycycline. This analysis revealed enrichment for genes involved in the unfolded protein response, particularly heat shock response factors (Fig. S6D).

Finally, we determined how the signature of FUS-CHOP gene expression was altered in response to light-induced dissolution by the OptoMBP system. We hypothesized that genes whose expression is altered by FUS-CHOP induction might revert towards their original expression level in response to dissolution by OptoMBP. To test this hypothesis, we compared the changes in the expression of FUS-CHOP-responsive genes to randomly chosen gene sets drawn from the same distribution of expression levels in our dataset, as well as to the overall changes across all genes (Fig. S6E–G). We found that light stimulation disproportionately reverted gene expression from a FUS-CHOP perturbed state back toward basal expression. Approximately 15% of FUS-CHOP-upregulated genes were reduced by at least 25% by light, compared to 5% of randomly chosen genes; only 2% of FUS-CHOP-upregulated genes showed a further increase upon illumination (Fig. 5G). Similar proportions were also observed for genes whose expression was decreased upon FUS-CHOP expression and restored to higher levels upon illumination (Fig. 5H). These data indicate that even a short 6 h light stimulus is sufficient to partially revert FUS-CHOP-induced changes in gene expression.

To further characterize the genes affected by light-induced dissolution, we performed gene set enrichment analysis (GSEA) on FUS-CHOP-expressing cells before and after illumination (Fig. 5I). We found that this gene set was enriched for various gene categories: NFκB, pro-inflammatory, and p53 gene signatures, as well as de-enrichment for Myc targets, E2F targets, and G2/M associated gene expression. These gene sets also closely matched a prior study examining the consequences of FUS-CHOP silencing in a liposarcoma cell line⁸¹ (Fig. 5I; bold labels), suggesting that dissolution of transcriptional condensates functionally resembles overall loss of the transcription factor. Taken together, our results suggest that FUS-CHOP broadly alters gene expression within 24 h, and that these effects can be partially reversed by specifically targeting FUS-CHOP phase separation with OptoMBP. These data are consistent with a direct role for FUS-CHOP condensation in promoting gene expression, as disassembly of FUS-CHOP condensates began to revert gene expression changes even under continued induction of oncoprotein expression.

Discussion

In this study, we develop and characterize the OptoMBP system, an optogenetic tool to dissolve constitutively formed biomolecular condensates. Optogenetic tools offer many advantages including the potential for rapid reversibility, fine spatial control, and a high degree of specificity⁸². We found that light-based recruitment of the classic solubilizer maltose-binding protein (MBP) was highly effective at dissolving a variety of protein condensates: those formed from FET family members, the Oskar RNA binding protein, the LAF-1 RGG domain, and the FUS-CHOP oncoprotein. Light-based MBP recruitment can be quite potent, even matching the efficacy of 1,6-hexanediol, a broad-spectrum compound widely used for condensate disruption. Moreover, optogenetics confers additional advantages in reversibility and spatial control. One can envision future studies where MBP is used to dissolve transcription factor condensates at a single transcriptionally active locus of interest to specifically alter their presence or composition and monitor real-time effects on gene expression or genome spatial organization. It is also possible that tools such as OptoMBP could be extended to other forms of protein condensates and aggregates, such as the pathological aggregates associated with neurodegeneration. To that end, we note that prior work using a rapamycin-based solubilizer suggests that chemically induced solubilizer recruitment can interfere with the formation of huntingtin aggregates²⁹.

Our results also raise an open question: what is the mechanism by which MBP achieves its solubilizing effects? One can imagine several possibilities: MBP might be of sufficient size to sterically block favorable interactions within the condensate; it could preferentially bind the condensate-forming protein in the dilute phase, thereby destabilizing condensates through polyphasic linkage⁸³; the high solubility of MBP may alter the saturation concentration of the complex; or MBP may bind to the same sequences that would normally be used to form multivalent interactions. In general, we do not observe a correlation between cargo size and dissolution propensity, suggesting that the first of these mechanisms (steric hindrance) is unlikely to be the cause of MBP solubilization, although the other proposed mechanisms cannot be excluded. Surprisingly, we observe a relatively sharp threshold in MBP concentration at which condensate dissolution is observed, and this threshold does not appear to vary strongly with the concentration of the target protein, suggesting that a simple stoichiometric model of MBP-target binding may not be sufficient to explain the effect. Despite extensive study, the mechanism by which MBP solubilizes target proteins in direct fusion constructs is not fully understood^53,54,64. The mechanisms underlying phase separation of different protein sequences are also still an active are of study^84,85,86, and a complete accounting for these intermolecular interactions might help to explain whether condensates will be susceptible or resistant to MBP-based dissolution. One could imagine OptoMBP serving as a platform to study the mechanism of MBP solubilization, and other promising solubilizing domains such as SUMO may also be instrumental in the development of more general condensate-dissolution tools³¹. There is still much to be done to decipher the code that links condensate and solubilizer.

Our study establishes that the OptoMBP system is exceptionally effective at dissolving condensates formed of FET family proteins. This includes isolated FET intrinsically disordered domains, full-length family members that retain RNA binding domains, and an oncogenic fusion protein containing the FUS IDR and a DNA binding domain. In the latter case, we were further able to examine the link between phase separation of a transcription regulator (FUS-CHOP) and downstream gene expression. We found that OptoMBP-based dissolution partially reversed FUS-CHOP-driven gene expression changes within 6 h of illumination, mimicking the gene signature obtained by directly silencing FUS-CHOP⁸¹. While prior studies established that fusion to the FUS domain controls both phase separation and oncogenicity^76,77, our results hint at a causal link between FUS-CHOP condensate state and its function as a transcription factor, in line with other recent studies using distinct tools and transcription factor targets^31,70. Of course, our OptoMBP experiments only provide one piece of evidence, and it remains possible that MBP recruitment modulates transcription in additional ways that are independent from its condensate-dissolving function. Nevertheless, we anticipate that OptoMBP and related approaches will be useful to better understand the role of transcription factor condensates in gene expression contexts where condensation may be as likely to interfere with gene expression as it is to promote it^72,87.

In sum, the OptoMBP system provides a step towards programmable, optogenetic dissolution of biomolecular condensates. In future work, it will be essential to better define the mechanism of MBP-induced solubilization to be able to predict which condensates will be dissolved by OptoMBP and to rationally design improved variants that expand its reach to additional targets. It will also be important to develop systems that enable MBP recruitment to endogenous proteins, not just those which are tagged with a dimerization domain. The recent development of light-controlled nanobody and monobody binders could play a role by enabling light-gated binding to endogenous targets^88,89. Nevertheless, the OptoMBP system adds functionality to the toolbox of approaches to manipulate biomolecular condensates and regulate their biological functions.

Methods

Plasmid construction

Constructs utilized in this study were inserted in a pHR or pPB vector that included a SFFV or TetOn promoter and WPRE terminator. Constructs were cloned with InFusion (Clontech) using backbone PCR. The MBP and GST genes were a gift from Prof. Daniel Hammer, University of Pennsylvania. RGG, DCP1A, and PMLIV genes were a gift from David Sanders and Prof. Cliff Brangwynne, Princeton University. The Oskar gene was a gift from Prof. Elizabeth Gavis, Princeton University. EWS, TAF15, and CHOP genes were generated by gene synthesis (Integrated DNA Technologies). FUS, FUS-CHOP, and Oskar OptoMBP plasmids are available from Addgene (plasmid #223285-223289).

Cell transient transfection

For microscopy experiments, HEK293T cells were plated on a glass-bottom 96-well plate (Cellvis) at around 30% confluency. The next day, these cells were transfected using lipofectamine 3000 (ThermoFisher) following the manufacturer’s instructions. For experiments involving two constructs, equal amounts of both plasmids were co-transfected. Cells were imaged 24 hours after transfection.

Lentivirus production and transduction

Lenti-X cells were plated at approximately 30% confluency in a 6 well plate. A pHR expression plasmid and lentiviral packaging plasmids were co-transfected using the Fugene HD transfection reagent (Promega). Fifty two hours after transfection, the supernatant was collected and passed through a 0.45 μm filter. Cells to be infected with lentivirus were plated at 30% confluence in a 6 well plate and 500 μL of virus solution, 50 μL of 1 M HEPES, and 1 μL of 5 μg/mL polybrene (Sigma Aldrich) were added. Cells were then expanded for sorting.

Stable cell line generation

NIH3T3 and HEK293T cells were obtained from the Lim lab (UCSF) and validated by STR profiling (ATCC). To generate stable NIH3T3 cell lines co-expressing 3xFUS^IDR-FusionRed-iLID and MBP-SspB-iRFP, we produced lentivirus vectors for both genes. NIH3T3 cells infected with MBP-SspB-iRFP lentivirus and iRFP positive cells were isolated using Fluorescence-activated Cell Sorting (FACS). These MBP-expressing cells were then infected with 3xFUS^IDR-FusionRed-iLID lentivirus. iRFP and FusionRed double positive cells were then isolated with FACS. To generate FUS-CHOP cell lines, we first generated a cell line expressing rtTA. NIH3T3 cells were infected with BFP-P2A-rtTA lentivirus and BFP positive cells were isolated. To these cells, PiggyBAC transposon plasmids containing both MBP-SspB-iRFP-NLS and TetOn-FUS-CHOP-FusionRed-iLID genes were cotransfected with the PiggyBAC transposase. Cells were then sorted for expression of both BFP and iRFP. Sorting was performed on a SH800S Cell Sorter (Sony Biotechnology) with 405, 488, 561, 638 nm lasers and 450/50, 525/50, 600/60, 665/30, 720/60 and 758/60 nm emission filters.

Cell imaging

Confocal microscopy was performed on a Nikon Eclipse Ti microscope equipped with a Yokogawa CSU-X1 spinning disk, Agilent 4 color laser line module (405/488/561/640 nm), fluorescence emission filters (470/40, 535/50, 570/25 and 710/75), an iXon DU897 EMCCD camera, and Mightex Polygon 400 digital micromirror device (DMD). Live cells were maintained at 5% CO₂ and 37 °C during imaging and imaged using a 60X oil immersion objective. For dissolution experiments images were taken with the 561 nm and 640 nm laser lines.

Chemical and small molecule treatment

To dimerize FRB and FKBP, rapalog AP21967 (Takara) was added to a final concentration of 500 nM. To induce expression of FUS-CHOP, doxycycline was added to a final concentration of 1 μg/mL. To fully dissolve droplets, 1,6-hexanediol was dissolved in DMEM and added to a final concentration of 10%.

Blue light activation

For all microscopy experiments, optogenetic stimulation was carried out using a 450 nm LED light source (X-Cite XLED) was applied with an LED and delivered to the region being imaged through a digital micromirror device (DMD; Mightex Polygon 400) for 1 sec per acquisition cycle (with 10 sec between acquisitions). For subcellular activation (Fig. 2), the DMD region of interest was adjusted to encompass about half of the cell being imaged or encompass a single condensate in the cell nucleus. The DMD was set to 50% dithering and LED to 50% power for all illumination experiments except the “low intensity” conditions for subcellular illumination and Fig. S3, where the DMD was set to 40% dithering and LED to 10% power. Quantification of illumination at the sample using a light meter (ThorLabs PM200) revealed 450 nm intensities of ~200 mW/cm² (standard illumination) and 30 mW/cm² (low intensity).

Data quantification and analysis

Microscopy images were quantified using ImageJ (NIH) and MATLAB R2022a (MathWorks). In ImageJ, the mean, max, and standard deviation of the red channel pixel intensity in the nucleus (FUS, oskar, PML IV) or cytoplasm (RGG, DCP1A) was measured. ΔCV was defined as (σ_f/μ_f)-(σ_i/μ_i) and Δenrichment was defined as (max_f/μ_f)-(max_i/μ_i). Plots were created using RStudio (Posit) or MATLAB R2022a. Significance was determined using the Mann–Whitney U test: n.s.: p > 0.05; *: p < 0.05; **: p < 0.01; ***: p < 0.001.

RNASeq

Total RNA was isolated from our FUS-CHOP NIH3T3 cell line using the RNeasy mini kit (Qiagen). RNA-Seq directional library preparation and sequencing on Illumina NovaSeq 6000 was performed by the Princeton Genomics Core Facility. Using Galaxy⁹⁰ version 24.1, reads were mapped using the Spliced Transcripts Alignment to a Reference (STAR) algorithm⁹¹ version 2.7 and then counted using the featureCounts algorithm⁹² version 2.0. Differential expression analysis was performed using DESeq2⁹³ version 1.44 in RStudio. Volcano plots were generated with the EnhancedVolcano R package using and heatmaps were generated using the pheatmap R package version 1.0.12. Gene set enrichment analysis (GSEA) was performed in RStudio with FGSEA⁹⁴ version 1.25 using the mouse-ortholog hallmark gene sets⁹⁵.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

Source data for all figures are provided in the Supplementary Information/Source Data file. The RNA sequencing FASTQ files generated in this study have been deposited in the Sequence Read Archive (SRA) under accession numbers SAMN39855040-51 [https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1074263]. Raw microscopy images are available under restricted access due to the difficulty of hosting this data format; access can be obtained by contacting the corresponding author. Source data are provided with this paper.

Code availability

All code used to analyze data is available on the laboratory’s Github repository (https://github.com/toettchlab/BrumbaughReed2024).

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Acknowledgements

We thank all members of the Toettcher and Aoki laboratories for helpful discussions and expertise. We thank the Princeton Genomics Core Facility for RNA sample library preparation/sequencing and guidance. This work was supported by the International Research Collaboration Center at the National Institute of Basic Biology, Japan (to E.H.B.R.) and NIH grant U01DK127429 and a Vallee Scholar award (to J.E.T.).

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Authors and Affiliations

Department of Molecular Biology, Princeton University, Princeton, NJ, 08544, USA
Ellen H. Brumbaugh-Reed, Yang Gao & Jared E. Toettcher
Omenn-Darling Bioengineering Institute, Princeton University, Princeton, NJ, 08544, USA
Ellen H. Brumbaugh-Reed & Jared E. Toettcher
International Research Collaboration Center (IRCC), National Institutes of Natural Sciences, Tokyo, 105-0001, Japan
Ellen H. Brumbaugh-Reed & Kazuhiro Aoki
Quantitative Biology Research Group, Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, Okazaki, Aichi, 444-8787, Japan
Kazuhiro Aoki
Division of Quantitative Biology, National Institute for Basic Biology, National Institutes of Natural Sciences, Okazaki, Aichi, 444-8787, Japan
Kazuhiro Aoki
Department of Basic Biology, School of Life Science, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Aichi, 444-8787, Japan
Kazuhiro Aoki
Laboratory of Cell Cycle Regulation Graduate School of Biostudies, Kyoto University, Kyoto, Kyoto, 606-8315, Japan
Kazuhiro Aoki

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Ellen H. Brumbaugh-Reed
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Yang Gao
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Kazuhiro Aoki
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Jared E. Toettcher
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Contributions

E.H.B.R., J.E.T. conceived of the study. E.H.B.R. and Y.G. performed all experiments. E.H.B.R. and J.E.T. analyzed the data. K.A. and J.E.T. supervised the research. E.H.B.R. and J.E.T. drafted the manuscript, which all authors edited.

Corresponding author

Correspondence to Jared E. Toettcher.

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Competing interests

J.E.T. is a scientific advisor for Prolific Machines and Nereid Therapeutics. J.E.T., K.A., and E.H.B.R. are co-inventors on a patent application (Dissolving biomolecular condensates using optical or chemical recruitment of soluble proteins; patent application no. 63309934). Y.G. reports no competing interests.

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Brumbaugh-Reed, E.H., Gao, Y., Aoki, K. et al. Rapid and reversible dissolution of biomolecular condensates using light-controlled recruitment of a solubility tag. Nat Commun 15, 6717 (2024). https://doi.org/10.1038/s41467-024-50858-0

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Received: 25 January 2024
Accepted: 23 July 2024
Published: 07 August 2024
Version of record: 07 August 2024
DOI: https://doi.org/10.1038/s41467-024-50858-0

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