Many proteins contain disordered regions of low-sequence complexity, which cause aging-associated diseases because they are prone to aggregate. Here, we study FUS, a prion-like protein containing intrinsically disordered domains associated with the neurodegenerative disease ALS. We show that, in cells, FUS forms liquid compartments at sites of DNA damage and in the cytoplasm upon stress. We confirm this by reconstituting liquid FUS compartments in vitro. Using an in vitro “aging” experiment, we demonstrate that liquid droplets of FUS protein convert with time from a liquid to an aggregated state, and this conversion is accelerated by patient-derived mutations. We conclude that the physiological role of FUS requires forming dynamic liquid-like compartments. We propose that liquid-like compartments carry the trade-off between functionality and risk of aggregation and that aberrant phase transitions within liquid-like compartments lie at the heart of ALS and, presumably, other age-related diseases.

Here, we show that both in vivo and at physiological concentrations in vitro FUS forms liquid-like droplets. We further demonstrate that the liquid-like state can convert into a solid state and that this conversion is exacerbated by disease-associated mutations in the prion-like domain. Our findings suggest that aberrant phase transitions may be at the heart of many neurodegenerative diseases.

One prototypical prion-like protein involved in the compartmentalization of cells is the RNA-binding protein FUS. It is enriched in the nucleus and involved in transcription, DNA repair, and RNA biogenesis (). Mutations in FUS are associated with amyotrophic lateral sclerosis (ALS) and rare forms of frontotemporal lobar degeneration (FTLD) (). Recent reports show that the prion-like LC domains of FUS can polymerize into fibrous amyloid-like assemblies in a cell-free system (). Once assembled, these structures exhibit the macroscopic behavior of hydrogels. However, it has been difficult to understand the relationship between amyloid-like hydrogels that form in vitro and the in vivo function of the protein, because there has been little work on the dynamics of FUS in living cells and the relationship between this dynamic behavior and the onset of disease.

Domains of low-sequence complexity (LC domains) have been implicated in the formation of membrane-less compartments (). LC domains are also present in yeast prion proteins, which have the ability to interconvert into fibers rather than a liquid state (). Thus, proteins harboring these domains have been called “prion-like.” Prion-like LC domains are particularly abundant in RNA- and DNA-binding proteins and have been conserved across evolution (). However, mutations in prion-like proteins also cause devastating protein misfolding diseases, and these diseases are typically accompanied by the formation of solid aggregates (). Thus, determining how prion-like proteins organize cellular compartments will not only advance our understanding of compartment formation but will also provide important insight into a diverse set of aging-associated pathologies.

Cells have a problem: How do they organize their complex biochemistry in time and space? Eukaryotic cells have addressed this problem by using functionally distinct compartments, many of which are bound by membranes. In these cases, it is easy to understand how the biochemistry is constrained in one place: the membrane prevents the diffusion of molecules in the absence of specific transport systems. However, many, if not most, cellular compartments are not membrane enclosed (). Examples include germ (P) granules (), processing (P/GW) bodies (), stress granules (), nucleoli (), Cajal bodies () and likely signaling compartments (). These structures are highly dynamic, and the components within them are in constant exchange with the surrounding cytoplasm or nucleoplasm. Recently, an increasing number of these non-membrane-bound compartments have been shown to behave like condensed liquid phases of the cytoplasm or nucleoplasm (). It is thought that these structures form by liquid-liquid demixing, often upon a specific triggering event.

Many ALS-associated mutations map to the C-terminal nuclear localization sequence (NLS) of FUS. Mutations in the NLS increase the cytosolic fraction of FUS (). We confirmed this result by making a BAC cell line containing a deletion of the NLS ( Figure S6 D). Because phase transitions are extremely sensitive to protein concentration, we hypothesized that these NLS mutations could accelerate the conversion of FUS to a fibrous state by increasing the concentration of FUS in the cytoplasm. Indeed, we found that increasing concentrations of FUS had a higher propensity to convert from a droplet to a fibrous state ( Figures S6 E and S6F). Therefore, we conclude that the aggregation of FUS is concentration dependent and that ALS-associated mutations in FUS may promote the formation of aggregates as seen in patient cells by accelerating the conversion from a liquid droplet to a fibrous state.

To quantify the biophysical properties of the FUS fibers, we performed photobleaching experiments on structures from wild-type and G156E FUS. At t (time) = 0, both wild-type and G156E droplets recovered quickly after photobleaching ( Figure 6 C). However, by t = 8 hr, the G156E structures no longer recovered, suggesting that they interconverted from a liquid to an aggregated state. Even within one fibrous structure, the fibers projecting out turned over more slowly than the body ( Figure S6 B). This shows that, over time, a population of FUS protein converts from a liquid to a solid state. We confirmed this by performing cryoelectron microscopy (cryo-EM) studies of the droplet and fiber state. We found that FUS droplets were amorphous and lacked ordered structures ( Figure S6 C). The fiber state of FUS, instead, was characterized by many fibrillar assemblies with a diameter of around 9 nm, and many of these fibrils were laterally aligned ( Figure S6 C). These structures are reminiscent of the amyloid-like aggregates identified in previous work (). Thus, we conclude that the liquid droplet state is metastable and transitions into a thermodynamically more stable aggregated state with time.

Therefore, we can conclude that, with time, a solution of FUS containing liquid droplets will convert into fibrous structures. Although wild-type and mutant FUS convert from droplets to fibers, the mutant consistently converts more quickly than the wild-type. When we looked more carefully at the time points containing aggregates, we saw that some of the droplets had short fibers ( Figure 6 A), while others had much longer fibers emanating from them. We call these structures with small fibers “sea urchins,” because they are reminiscent of similarly named transition structures seen in crystallization studies (), and longer fibers “starbursts” ( Figure 6 B; Movie S7 ). Time-lapse imaging of the sea urchins revealed that the fibers protruding from the surface continuously grew until the structure resembled that of a commonly observed starburst ( Movie S7 ). One intriguing possibility is that this structure is the transition structure of a droplet converting into fibers. Such a process is consistent with the droplets acting as centers of fiber nucleation and growth.

Next, we investigated the aging morphology of FUS droplets by fluorescence microscopy. Figure 6 A shows a typical experiment. For wild-type FUS, the droplets increased in size with time so that, by 8 hr, large droplets were observed in all fields. For G156E FUS, the droplets also increased in size. However, by 8 hr, hardly any G156E droplets remained. Rather, only fibrous structures could be seen ( Figure 6 A). For wild-type FUS, mainly droplets were seen by 8 hr, although some fibers were now detectable as well. By 12 hr, most of the wild-type FUS protein had converted into fibers, but a few droplets could still be observed. Next, we investigated another patient-derived mutant of FUS with a mutation adjacent to the prion-like domain (R244C) and found that R244C also accelerated the conversion of FUS droplets to a fibrous state ( Figure S6 A).

(F) Time course showing the morphological changes of the in vitro droplets in “aging” assays containing 4, 8, or 16 μM of purified wild-type FUS-GFP over 480 min. Also shown is a Coomassie-stained SDS-PAGE gel loaded with 2 μl of each assay reaction.

(E) WT FUS-GFP construct lacking the Nuclear Localization Signal (NLS) domain (FUSΔNLS-GFP) was designed (schematic shown). Representative images of the morphological changes of the droplets formed from purified WT FUS-GFP (WT) or FUS-ΔNLS-GFP (ΔNLS) over 180 min (10 μM FUS protein each). Also shown is a Coomassie-stained SDS-PAGE gel loaded with 2 μl of each assay reaction.

(D) HeLa cells expressing FUS-GFP (left) or FUSΔNLS-GFP (middle) from BAC transgenes. Cell and nucleus boundaries are shown in black or red, respectively. Wild-type FUS is primarily nuclear, whereas FUSΔNLS is enriched in the cytoplasm. On the right, a corresponding immunoblot of HeLa cells without a BAC transgene (WT) or with a BAC transgene for expression of FUS-GFP and FUSΔNLS-GFP. The expected molecular weight of endogenous FUS is 75 kDa. The asterisk ( ∗ ) and triangle (Δ) mark transgenic FUS-GFP and FUSΔNLS-GFP, respectively.

(C) Cryo-transmission electron microscopy of liquid-like FUS droplets and FUS fibers after aging. WT FUS-GFP droplets (top panel) and fibers after aging (bottom panel) were applied to Cryo-TEM grid with holey carbon support (2-μm holes) and plunge frozen at RT. The positions of the higher magnification images (middle) are depicted as a red squares in the low magnification images (left). Top left: overview micrograph showing the holey carbon support with repetitive 2-μm holes. A large FUS droplet (∼6-μm diameter) is shown in the center of the image. Top middle: higher magnification of the droplet edge showing a continuous homogenous density within the droplet. Top right: zoom into the drop edge showing the density is homogenous and amorphous. Inset: Fourier transform of an area within the droplet (middle image) confirming the lack of ordered structure. Bottom left: Low magnification overview showing elongated aggregates on top of the holey carbon support. Bottom middle: higher magnification of an aggregate density within the carbon hole. The aggregates are composed of entangled thin fibers. Bottom right: zoom into an area with separate fibrils. Intensity line scans across one fibril at two positions (depicted in orange and blue) are presented in the inset: the fibers are ∼9 nm in diameter with a clear low-density area in the center of the fibril density, indicating possible composition of two proto-filaments.

(B) Fluorescent recovery curves of the bulk phase (photobleached in the green box) and emanating fibers (photobleached in the red box) from an 8-hr in vitro aging experiment. Error bars represent SD over three independent measurements of bulk phase and fibers, each.

(A) Top: A schematic of the FUS protein with the positions of ALS patient-derived mutations (G156E and R244C) that were tested in the “aging” assay. Bottom: Representative images of the droplets formed from purified WT, G156E, or R244C FUS-GFP over 360 min of “aging” (10 μM FUS protein each). A Coomassie-stained SDS-PAGE gel loaded with 2 μl of each assay reaction is shown on the right.

(A) Representative images of the morphological changes in in vitro droplets of wild-type (WT) or G156E FUS-GFP during an “aging” experiment over 8 hr.

Next, we set up an “aging” experiment in which we added dextran to FUS solutions and monitored the behavior of the droplets with time. Using an optical tweezer, we first showed that one FUS droplet held by one laser beam can fuse with several other droplets ( Movie S5 ). To quantify this behavior, we used two laser beams to control fusion events ( Figure 5 C; Movie S6 ). The results of a typical experiment are shown in Figure 5 D. Between 0 and 2 hr after droplet formation, wild-type FUS droplets fused very quickly, often within hundreds of milliseconds ( Movie S6 ). Similar behavior was seen for G156E FUS. However, the relaxation time of G156E FUS was significantly longer than for wild-type FUS ( Figure 5 E), and the spread of values became much broader, with some droplets taking as long as 15 s to fuse. More strikingly, as the droplets aged in a test tube, we noticed that an increasing fraction of the G156E FUS droplets no longer fused ( Figure 5 F; Movie S6 ) so that, after 8 hr, we could detect no fusion events for G156E FUS, while 81% of the wild-type FUS droplets still fused. By 12 hr, wild-type droplets also stopped fusing. This indicates that there is indeed a change in the biophysical properties of FUS droplets with time. It further suggests that the properties of the G156E FUS droplets are changing more quickly than those of the wild-type FUS droplets. This is consistent with the observation that G156E has a higher propensity to aggregate as observed by. Concomitant with the decline in fusion ability, we also noticed fibrous structures arising in the G156E FUS droplets ( Figure S5 B; Movie S7 ). Therefore, we conclude that FUS droplets age and undergo drastic changes in their biophysical properties and morphology.

FUS can form pathological protein aggregates, and specific mutations in FUS have been identified in patients suffering from neurodegenerative diseases (). Previous studies have shown that a patient-derived mutation in the prion-like domain of FUS (G156E) has an increased tendency to form aggregates (). To investigate the link between the compartment-forming abilities of FUS and disease, we expressed G156E FUS-GFP in insect cells and purified the protein using the same protocol as used previously for wild-type FUS ( Figures 5 A and 5B ). When 10 μM wild-type FUS and G156E FUS were incubated in 500 mM salt, only diffuse fluorescence was seen over many hours ( Figure S5 A). Upon dilution of G156E FUS to a concentration of 10 μM in the presence of dextran, we found that it formed liquid-like structures, similar to those observed for wild-type FUS. We were also unable to observe any obvious differences in the properties of G156E and wild-type droplets (data not shown). In addition, we expressed G156E FUS-GFP from a BAC transgene in HeLa cells, but we could not detect any obvious differences in FUS compartments formed in wild-type cells (data not shown).

(A) 10 μM WT FUS-GFP (WT) and G156E FUS-GFP (G156E) are stable and remain diffuse in the in vitro aging assay conditions in absence of dextran over 8 hr. Upon addition of 10% dextran at 8 hr, FUS forms droplets similar to the 0 hr time point.

(E) A boxplot of fusion times of wild-type (WT) and G156E FUS-GFP droplet pairs scaled by the mean drop size at different time points using optical trap. The line within the boxplot represents the median and the outer edges of the box are the 25th and 75th percentiles. The whiskers extend to the most extreme points not considered outliers. Outliers are calculated as the points that are smaller than q 1 − 1.5 ( q 3 − q 1 ) and larger than q 3 − 1.5 ( q 3 + q 1 ) , where q 1 and q 3 are the 25th percentile and 75th percentile, respectively. N/A indicates that fusion times could not be determined because fusion events were no longer detectable. s, seconds; h, hour.

(C) Diagram illustrating the method to control in vitro fusion experiments using two optical traps demonstrated by the signal of two FUS droplets. The green bar indicates the time of a fusion event, which can be inferred from the increase in the combined normalized laser signal. AU, arbitrary units.

Previous studies identified a poly(ADP) ribose (PAR)-binding domain in the C-terminal region of FUS and showed that FUS is rapidly recruited to DNA damage sites in a PAR-dependent manner (). Indeed, we found that PAR polymerase 1 (PARP1) arrives within seconds of DNA damage, and FUS was detectable immediately after arrival of PARP1 ( Figure 4 A). To investigate whether PAR is required for FUS accumulation, we interfered with PAR formation by adding an inhibitor of PARP1/2 to cells shortly before laser-mediated irradiation (). Indeed, inhibition of PARP1/2 prevented the recruitment of FUS ( Figures 4 B and 4C). Conversely, inhibition of a PAR-degrading enzyme (PARG) prolonged the presence of FUS at DNA lesions ( Figures 4 B and 4D). This suggests that PAR polymers are required for the recruitment as well as the prolonged presence of FUS in DNA damage-associated compartments. To study whether PAR affects FUS droplet formation in vitro, we first adjusted the FUS concentration in our cell-free assay to 0.4 μM and lowered the concentration of dextran. At this concentration, we observed no spontaneously formed FUS droplets. However, when purified PAR was added, FUS droplet formation was strongly enhanced ( Figures S4 A–S4C). In summary, these data provide evidence for the intrinsic ability of multivalent PAR chains to nucleate the formation of FUS droplets. Therefore, we conclude that local PAR synthesis and the cooperative PAR-binding activity of FUS are required to drive the formation of a non-membrane-bound compartment for DNA repair.

(C) Quantification of the total number and volume of droplets formed with 400 nM purified FUS in the absence (No PAR) and presence of 1 μM PAR in vitro. Error bars represent SD over three replicates.

(B) A representative snapshot of the automated quantification of the total number of FUS-GFP droplets shown in (A) after background correction and thresholding. The droplets labeled in green were quantified, whereas the droplets in red were discarded. The results are presented in (C).

(D) Percentage of cells with FUS-GFP at damage sites in 1 or 45 min after DNA damage in control and PARG-inhibitor-treated conditions (n = 10 per condition). Error bars represent SD. The p value at 1 min = 0.5185; the p value at 45 min = 0.0011.

(B) FUS-GFP-expressing cells at 1 min and 45 min after DNA damage induction by laser micro-irradiation in control, PARP1-, or PARG-inhibitor (inhib.)-treated conditions. Sites of laser micro-irradiation are shown by red lines. Dark puncta are sites of strong FUS-GFP concentration.

(A) HeLa cells expressing FUS-mCherry and PARP1-GFP are imaged after DNA damage induction by laser micro-irradiation. Normalized fluorescence intensity of FUS-mCherry (green) or PARP1-GFP (blue) at DNA damage sites is plotted over time after DNA damage induction.

Therefore, FUS structures have all the hallmarks of liquid droplets both in vivo and in vitro: they are spheres, they fuse, they deform under shear stress, and they rearrange their contents within seconds. This indicates that under physiological conditions, FUS does not assemble into solid-like aggregates or gels but rather forms dynamic droplets that exhibit all the properties of a true liquid. It further confirms that FUS has an intrinsic ability to phase separate and form liquid droplets, suggesting that FUS may play a central role in forming liquid compartments at sites of DNA damage and during stress.

Previous studies had identified the N-terminal prion-like domain as critical for the formation of hydrogels (). Therefore, we generated a deletion mutant of FUS lacking this N-terminal domain (FUSΔLC) and purified it from insect cells using the same protocol as for wild-type FUS. When we mixed 10 μM of wild-type or FUSΔLC protein with 10% dextran, only wild-type protein formed droplets, whereas truncated FUS remained diffuse ( Figure S3 J). This indicates that the prion-like domain is essential for forming liquid droplets, presumably because of its sticky nature and ability to undergo many weak interactions.

Because of the discrepancy with our in vivo data, which shows that FUS coalesces into liquid compartments in living cells, we looked for conditions that promote FUS assembly into compartments at physiological concentrations. A 10% solution of either dextran or polyethylene glycol (PEG) induced assembly of FUS into round micrometer-sized structures ( Figures 3 C and S3 C–S3G). To characterize whether these FUS assemblies have liquid-like properties, we performed a series of biophysical experiments. First, we investigated whether FUS molecules can freely move around within the droplets. Indeed, the FUS signal rapidly rearranged from the unbleached region to the bleached region of a half-bleached droplet ( Figures 3 D–3F and S3 H; Movie S4 ), consistent with a high internal mobility. The ability of a droplet to deform or of two droplets to fuse also helps distinguish liquids from solid gels (). Therefore, we next asked whether FUS droplets are deformable by shear force. Indeed, under such conditions, FUS droplets changed their shape, as would be expected for a dynamic liquid with no memory of its previous state ( Figure 3 G; Movie S4 ). Furthermore, we found that when two FUS droplets touched each other, they rapidly fused and relaxed into one larger droplet within seconds ( Figures 3 H and S3 I; Movie S4 ). Such rapid relaxation times are expected for liquid droplets with low viscosity.

Our data so far indicate that FUS is a component of liquid-like compartments in vivo that form by demixing from the cytoplasm. To investigate whether FUS is able to phase separate on its own, we studied the behavior of recombinant GFP-tagged FUS expressed in insect cells (see Experimental Procedures for details) ( Figures 3 A and 3B ; Figures S3 A and S3B). For these experiments, we chose a FUS-GFP concentration of 10 μM, which is slightly higher than the measured physiological concentration ( Figure S1 A) but easier to work with in our imaging-based assays. At this concentration, we found that FUS was diffusely distributed ( Figure 3 C). At a concentration of 500 μM, about 100-fold higher than the physiological concentration, FUS formed a gel-like state ( Figure 3 C; Movie S3 ), as previously reported ().

(J) WT FUS-GFP construct lacking the Low Complexity (LC) domain (FUSΔLC-GFP) was designed (schematic shown). 10 μM of purified wild-type WT FUS-GFP (WT) forms droplets in presence of 10% dextran, whereas purified FUS lacking the low complexity domain (FUSΔLC) does not form droplets under the same conditions. A corresponding Coomassie-stained SDS-PAGE gel showing WT FUS-GFP (∼82 kDa) and FUSΔLC-GFP (ΔLC, ∼53 kDa).

(H) Quantification of the fluorescence intensity recovery after half-bleach and full-bleach of in-vitro-formed FUS-GFP droplets. The bar graph on the right shows the slow and fast timescales obtained from exponential fits of the half-bleach experiments, signifying a fast internal rearrangement of the molecules within the droplets and slow diffusion of molecules from solution into the droplet across the phase boundary. The timescale of a full-bleach experiment is shown for comparison.

(G) Concentration series of purified FUS-GFP droplets in the presence of 10% dextran. Note that the size and number of the droplets increase with the FUS concentration. Studied by fluorescence microscopy.

(B) Still image of purified FUS-GFP protein injected into live cells expressing the stress granule component G3BP2-mCherry. Both cells in view were injected and stressed with arsenate treatment for 1 hr before microinjection. For clarity, the cell boundary and nucleus of one cell are outlined in green and red, respectively. The red arrow points to a representative single stress granule showing co-localization with injected FUS-GFP.

(F) Internal rearrangement of fluorescent FUS-GFP molecules within a half-bleached FUS-GFP droplet in (D and E) is shown by the quantification of the fluorescence intensity in the bleached (blue line) and unbleached (green line) regions of the droplet. Intensity changes in a bleached or an unbleached pixel region over time was plotted. Please note that the continuous upward slope of both the bleached and unbleached region is due to exchange of molecules from solution.

(A) Recombinant human FUS was tagged with GFP at the C terminus, separated by a 13-amino-acid linker. The protein was purified from insect cells, and the His tag was cleaved off using Prescission Protease.

Therefore, FUS assemblies have all the hallmarks of a liquid state: they turn over quickly; are spherical; and when they fuse, they relax into one spherical assembly (). Taken together, these experiments show that FUS assemblies are liquid droplets, which probably form by liquid-liquid demixing in the cytoplasm or nucleoplasm.

To investigate the shape of stress-induced FUS structures, we observed FUS-GFP-expressing cells with a digital scanned light-sheet microscope (DSLM) with structured illumination, which allows improved 3D imaging of dynamic subcellular objects (). Using this imaging technology, we found that FUS granules are spherical ( Figure 2 C). The calculated sphericity was close to that of a perfect sphere for both heat-induced and arsenate-induced cytoplasmic FUS granules ( Figure 2 D). We cannot measure the sphericity of the nuclear structures because they are too small. We then used DSLM microscopy to perform a time-resolved analysis of individual FUS granules in the cytoplasm. We found that FUS granules underwent frequent fusion events and, as soon as they interacted, rapidly relaxed into a spherical shape ( Figures 2 E and S2 B; Movie S2 ). The relaxation time of these granules was on the order of a few minutes ( Figure 2 F). Using the relaxation time and the FRAP (fluorescence recovery after photobleaching) times, we approximated viscosity values, as previously described (). We estimate viscosities around 10- to 100-fold of water (10–100 mPa⋅s). We were unable to see fusion events for nuclear FUS compartments, presumably because they are fixed in place on the DNA.

The constant mixing of components within a liquid can be tested by a technique known as “half-bleach” (). In this method, roughly half a structure is bleached, and then the distribution of the fluorescence within the photomanipulated structure is determined over time (see Experimental Procedures for details). The spatiotemporal analysis of such half-bleach events showed that FUS was redistributed rapidly within stress granules and nuclear FUS assemblies, from the unbleached area to the bleached area ( Figures 2 A and 2B ; Figure S2 A; Movie S2 ). Thus, we conclude that FUS molecules can diffuse freely in stress granules and nuclear assemblies, in agreement with a liquid material state.

(A) A montage of fluorescence recovery of FUS-GFP in a nuclear granule of a HeLa cell after half-bleach. Cells were treated with hypertonic solution to slow down FRAP recovery to a detectable timescale.

(F) The shape changes of an FUS-GFP droplet over the time course of a fusion event measured as aspect ratio (ratio between the long axis and the short axis of a stress granule over time with an exponential fit). As fused granules relax to a spherical shape, the aspect ratio reaches 1.

(B) Internal rearrangement of fluorescent FUS-GFP molecules within a half-bleached FUS-GFP stress granule in (A) is shown by quantification of fluorescence in bleached (orange line) versus unbleached regions (blue lines) over time. Intensity changes in a bleached or an unbleached pixel region over time was plotted.

The dynamic nature of FUS compartments is reminiscent of other RNA protein compartments such as P granules and nucleoli, which form by liquid-liquid demixing in the cytoplasm or nucleoplasm. Three characteristics define a liquid-like compartment. First, the components should undergo rapid internal rearrangement. Second, the compartments should be roughly spherical due to surface tension. Third, two droplets should fuse and relax into one droplet. Therefore, we tested whether FUS compartments have these liquid-like characteristics.

Our data so far support previous work showing that FUS localizes to multiple different compartments, depending on the type of stress the cell is experiencing. Our data further suggest that these compartments are extremely dynamic. In the nucleus, FUS localizes to active sites of transcription and rapidly assembles on sites of DNA damage. Upon heat shock, FUS shuttles out to the cytoplasm, where it forms a compartment in a concentration-dependent manner. In all these compartments, FUS turns over within hundreds of milliseconds to around 1 s.

FUS has also previously been implicated in DNA repair (). Indeed, we found that FUS accumulated at DNA lesions within a second of laser-mediated irradiation ( Figures 1 D, S1 C, and S1D; Movie S1 ). Next, we exposed the FUS cell line to heat stress to induce previously reported stress-associated compartments in the cytoplasm (). Indeed, FUS accumulated in the cytoplasm in heat-stressed cells and coalesced into stress granules ( Figures 1 D and S1 E; Movie S1 ). Formation and dissolution of these stress granules was coupled to changes in cytoplasmic and nuclear FUS levels ( Figures 1 E, 1F, and S1 E; Movie S1 ), suggesting that the coalescence of these compartments occurs over a certain concentration of soluble components. To further investigate the dynamics of FUS protein in these compartments, we photobleached FUS-containing cellular structures and followed the recovery of the fluorescence over time. Indeed, for all three structures examined, we observed a rapid exchange between assembled and soluble FUS, with a half-time of recovery ranging from hundreds of milliseconds to 1 s ( Figures 1 G and S1 F).

Using FUS-GFP HeLa cells, we found that, in unstressed cells, FUS predominately localized to the nucleus ( Figure 1 B), in agreement with previous reports. We also noticed that FUS formed small foci in the nucleoplasm ( Figure 1 B). A similar distribution was observed in mouse ES cells ( Figure S1 C). Cells treated with actinomycin D, a potent inhibitor of RNA polymerase II, showed no nuclear assemblies ( Figures 1 B and 1C), confirming previous observations that FUS is involved in transcription regulation and splicing (). This suggests that, under normal conditions, FUS assembles into compartments that may be associated with transcription or splicing.

Previous reports have implicated FUS in the formation of stress-inducible compartments, such as DNA damage sites and stress granules (). These studies often used transient transfection protocols and overexpression plasmids to study the subcellular localization of FUS. We used an approach based on BAC (bacterial artificial chromosome) transgeneOmics (). BACs have the advantage that they allow the expression of transgenes from their native genomic environment, including most, if not all, regulatory elements. First, we generated a BAC transgene for the expression of GFP-tagged FUS and introduced it into HeLa and embryonic stem (ES) cells to generate stable cell lines ( Figure 1 A). Next, we used mass spectrometry to determine the physiological concentration of FUS in HeLa cells ( Figure S1 A). We found that the concentration is around 2 μM, with a technical precision between replicates of 2.24 ± 0.7 μM and an estimated accuracy of 2- to 3-fold (). This makes FUS one of the top 5% of proteins in terms of protein abundance. Because FUS is 2- to 4-fold enriched in the nucleus under normal conditions ( Figure S1 B), the local concentration in the nucleus may be between 4 and 8 μM.

(F) Recovery half-times of fluorescence intensity after photobleaching of FUS-GFP in the cytoplasmic stress granules (Heat) and DNA damage sites (n = 10 per condition) of mES cells. The line within the boxplot represents the median, and the outer edges of the box are the 25th and 75th percentiles. The whiskers extend to the minimum and maximum values. p value = 0.0003.

(C) Inverted black and white images of FUS-GFP-expressing mouse embryonic stem (mES) cells in Control, DNA damage, and Heat shock conditions. Strong FUS-GFP localization is shown as dark puncta at sites of DNA damage (marked with red lines) and stress granules (marked with red arrows). FUS compartments are magnified in the inset.

(G) Recovery half-times (Characteristic time of recovery) of FUS-GFP fluorescence after photobleaching in the nuclear puncta, stress granules, and DNA damage sites (n = 10 per condition). For nuclear puncta versus stress granule, p = 0.1016; for nuclear puncta versus DNA damage sites, p = 0.0004; for stress granule versus DNA damage site, p = 0.0089. The line within the boxplot represents the median, and the outer edges of the box are the 25th and 75th percentiles. The whiskers extend to the minimum and maximum values.

(E) FUS-GFP fluorescence in the nucleus, cytoplasm, and stress granules (Granule) were measured after the induction of stress (time point 0) by time-lapse imaging. The representative graph reports the fluorescence intensity in each compartment.

(D) FUS-GFP-expressing cells in control, DNA-damage, and heat-stress conditions. Sites of strong FUS-GFP localization are shown by dark puncta at the irradiation-induced DNA damage sites (marked with red lines) and stress granules (marked with red arrows). The insets show approximately 13-fold magnifications of the FUS compartments.

(A) Immunoblot of Kyoto HeLa cells without a BAC transgene (WT) or with a BAC transgene for expression of FUS-GFP (FUS-GFP). FUS-GFP cells with FUS small interfering RNA (siRNA) treatment (FUS siRNA). The expected molecular weight (MW) of endogenous FUS is 75 kDa. The asterisk marks transgenic FUS-GFP. The FUS-GFP expression level (estimated by band intensity) is approximately 60% of the endogenous FUS. The total amount of FUS expressed in FUS-GFP cells (endogenous plus FUS-GFP) is about 1.75-fold higher than the FUS expression in wild-type cells. α-Tub, α-tubulin.

Discussion

In this paper, we show that, both in vivo and at physiological protein concentrations in vitro, the prion-like protein FUS forms liquid compartments. The evidence for this is 3-fold. First, the individual FUS molecules rapidly rearrange within the compartment. Second, the compartments formed by FUS are spherical. Finally, two FUS compartments can fuse and relax into one sphere. FUS rapidly shuttles between liquid compartments in the nucleus and the cytoplasm depending on the type of stress. Importantly, we show that, with time, a population of FUS droplets converts from a liquid state to an aggregated state, which is reminiscent of the pathological state seen in ALS patients with mutations in the FUS protein. This conversion from liquid to solid is accelerated either by mutations in the prion-like domain that induce the early onset of ALS or by raising the protein concentration, which mimics mutations in the NLS. Therefore, our data suggest that aberrant phase transitions may be at the heart of ALS and potentially other related diseases.

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Lindquist S.L. Nucleated conformational conversion and the replication of conformational information by a prion determinant. The droplets with fibers protruding from them are remarkably similar to the transition states in protein crystallization studies. For instance, when lysozyme or hemoglobin is crystalized, intermediate liquid droplets can be seen with small fibers emanating from them (). These states are also reminiscent of previously reported states that precede the formation of amyloid in in vitro aggregation reactions of the yeast prion Sup35 (). An alternative idea would be that there are two competing reactions in a test tube: liquid compartment formation and fibrous aggregation. Compartment formation is much quicker, but the soluble protein could slowly aggregate in the background, thus depleting the pool of monomeric FUS and leading to disassembly of the FUS droplets over time. We think this idea is less compelling, because we observe a decrease in droplet fusion with time, a change in droplet morphology, and also the droplets increase in size during incubation (compare panels 0 hr and 4 hr in Figure 6 A). However, most likely, both conversion of liquid droplets into fibers structures and a fibrous aggregation reaction in the solution are taking place at the same time.

Jucker and Walker, 2013 Jucker M.

Walker L.C. Self-propagation of pathogenic protein aggregates in neurodegenerative diseases. One problem is that we still do not know how pathological aggregates arise in living cells. Do they form in liquid compartments, or do they form in the bulk cytoplasm or nucleoplasm? We speculate that the liquid-solid transformation of FUS initially takes place in subcellular compartments but that later stages of the disease are characterized by aggregation reactions occurring in a compartment-independent manner. This is because amyloid-like aggregates are highly infectious, and once they have formed, they can seed further aggregation reactions in the same or in neighboring cells ().

Taylor and Dillin, 2011 Taylor R.C.

Dillin A. Aging as an event of proteostasis collapse. We were surprised to see that two distinct point mutations induced such a strong effect in vitro, whereas the disease would only manifest in a living organism after many years. However, in vivo, there will be many factors that are working against aberrant conformations, such as molecular chaperones and ATP-consuming degradation machines. As these control mechanisms weaken with age (), there presumably is a decline in the ability of a cell to counteract the formation of aberrant conformational states in compartment-forming proteins. Indeed, in vitro, we see both the wild-type and mutant FUS droplets changing biophysical properties, although the mutant changes more quickly than the wild-type protein. Therefore, it may be difficult to recapitulate the mutant state of the protein in living cells, because disease formation is a gradual process and young cells have very active quality control machinery in place. Indeed, our preliminary results suggest that when G156E FUS is expressed from its own promoter in HeLa cells, it has similar dynamics as wild-type FUS. Future work in differentiated neuronal systems will be required to identify the specific mechanisms that fail on a pathway to disease.

Kato et al., 2012 Kato M.

Han T.W.

Xie S.

Shi K.

Du X.

Wu L.C.

Mirzaei H.

Goldsmith E.J.

Longgood J.

Pei J.

et al. Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels. Figure 7 Diagram Illustrating the Molecular Mechanisms Underlying the Formation of FUS Compartments and Their Conversion into an Aggregated State Show full caption Left: FUS compartment formation upon DNA damage is driven by PAR polymerase and local PAR formation. PAR polymerase and other proteins (X) are modified with PAR chains in the process. PAR formation leads to FUS recruitment and initiates phase separation and compartment formation through LC domain interactions. Other proteins, such as EWS and TAF15 are likely recruited, thus forming a compartment for DNA damage repair. Right: FUS compartments form through phase separation from a concentrated solution of FUS, a reaction probably driven by weak interactions between prion-like LC domains. Liquid FUS droplets convert with time into an aggregated state, which presumably is associated with disease. LC domains are indicated in red; RB, RNA- and PAR-binding domains, indicated in blue. Recent work has shown that at high concentrations, the prion-like LC domain of FUS forms amyloid-like fibers and that this leads to hydrogel formation in a test tube (). These experiments were operating at ∼100 times the physiological cellular concentration and, therefore, are unlikely to represent the physiological state of the protein, which we think is a liquid state. However, it seems likely that these amyloid structures are similar or identical to the fibrous aggregates that we see in our in vitro aging experiments. Therefore, taken together, we can propose the following model as to how FUS enters into a disease state ( Figure 7 ): normally, FUS forms liquid compartments in cells. During transcription, FUS assembles into liquid compartments at active genes; during DNA damage, FUS assembles into liquid compartments at sites of DNA damage; under stress conditions, FUS rapidly shuttles to the cytoplasm and forms stress granules. The liquid nature of these compartments allows rapid diffusion necessary for chemical reactions on biological timescales. However, liquid formation comes with a cost: high local concentrations of a conformationally promiscuous protein. As cells age and the activity of the quality control machinery declines, and exacerbated by mutations that increase the aggregation propensities, FUS will convert into an aggregated state. Therefore, if a cell uses a dynamic liquid state to perform a physiological function, it will have to fight its whole life against the thermodynamic drive toward aggregate formation and disease.

Nomura et al., 2014 Nomura T.

Watanabe S.

Kaneko K.

Yamanaka K.

Nukina N.

Furukawa Y. Intranuclear aggregation of mutant FUS/TLS as a molecular pathomechanism of amyotrophic lateral sclerosis. We would like to reiterate that we do not think that fibrous aggregates represent the physiological function of FUS. Rather, the physiological function of the FUS protein is to act as a liquid, probably with little fixed structure. However, we currently have no information as to the molecular mechanism that drives the formation of a liquid-like state, apart from the fact that it requires the prion-like LC domain ( Figure 7 ). The prion-like domain of FUS is intrinsically disordered, and the liquid state of FUS probably arises from its ability to sample many conformational states. Anything that increases the strength of interaction between prion-like domains could lead to formation of aggregates rather than liquids. Indeed, a mutant that we selected, G156E, maps to the prion-like domain and has been shown in vitro to exacerbate the aggregation potential of the protein (). This will presumably involve a number of conformational states, starting off with an increase in viscosity of the liquid and ending in the commonly seen aging-associated aggregates. Our EM data show that, in vitro, the liquid-like droplets are amorphous, without any obvious structure. Therefore, liquid formation itself does not require formation of obvious oligomers or fibers. However, it is possible, in vivo, that small oligomers are constantly forming and being disassembled by the quality control machinery. Future studies using cryo-EM will be required to investigate the structure of FUS compartments in vivo.

Our data do not address the issue of why aggregates are associated with disease. There are two possibilities: the aggregates themselves are toxic, or the proteins themselves, by being locked in a less dynamic state, are no longer able to perform their physiological function. In the case of FUS, this function is to help protect cells against stress, both in the nucleus and the cytoplasm. However, the intermediate biophysical states discussed earlier could make cells more prone to stress, because the decrease in dynamics may impair the formation of stress compartments or the shuttling between the nucleus and the cytoplasm. It is possible that chemicals that slightly increase the fluidity of compartments could help ameliorate the development of the disease.