stress granules ( sgs ) are large cytoplasmic rna - protein assemblies that form during cellular stress , and play a pivotal role in the integrated stress response ( kedersha et  al . , 2013 ) .
following translational arrest , polysomes disassemble leading to a sudden increase in free mrna levels in the cytoplasm . it is believed that this free mrna subsequently nucleates the formation of sgs by binding of a large set of rna - binding proteins ( rbps ) ( jain et  al . , 2016 ) .
interestingly , several of these sg proteins have been linked to the neurodegenerative disorders amyotrophic lateral sclerosis ( als ) and frontotemporal lobar degeneration ( ftld ) , in which neuronal cytoplasmic fibrillar aggregates of rbps , e.g. , tdp-43 and fus , appear ( boeynaems et  al .
it is thought that these inclusion bodies play a key role in the disease pathogenesis ( ramaswami et  al . , 2013 ) .
moreover , mutations in several of these sg  proteins are also the genetic cause of the disease in a subset of familial cases ( renton et  al . , 2014 ) .
hence , sgs have been suggested as a stepping stone towards pathological aggregation in als and ftld ( aguzzi and altmeyer , 2016 , king et  al . , 2012 ,
based on previous observations from other rna granules and nucleoli ( brangwynne et  al . , 2009 , brangwynne et  al . , 2011 ) , recent studies have shown that the physical basis of sg formation is liquid - liquid phase separation ( llps ) ( burke et  al . , 2015 , conicella et  al . , 2016 , lin et  al . , 2015 ,
, 2015 , murakami et  al . , 2015 , patel et  al . , 2015 ) .
depending on in  vitro conditions , recombinant tdp-43 , fus , and other hnrnps spontaneously demix from an aqueous solution and form liquid - like protein - rich droplets .
it was shown that the intrinsically disordered prion - like domains ( prlds ) of these proteins by themselves are sufficient in inducing llps .
interestingly , these liquid - like droplets can mature over time to more fibrillar states , and this process is accelerated by disease - causing mutations ( conicella et  al . , 2016 , molliex et  al . , 2015 , murakami et  al . , 2015 , patel et  al . , 2015 ) .
other reports have shown that these prlds can also form reversible hydrogels , which have a high  sheet content ( kato et  al . , 2012 ) .
it was recently suggested that cellular sgs consist of a stable core surrounded by a liquid - like shell , and atp - dependent remodeling seems necessary for maintaining sg fluidity ( jain et  al . , 2016 , kedersha et  al . , 2016 ) .
these data point at the importance of a tight control of sg dynamics in preventing pathological aggregation . as mentioned above
, prlds can undergo llps or form hydrogels in  vitro ( burke et  al .
, 2015 , kato et  al . , 2012 , lin et  al . , 2015 ,
, 2015 , murakami et  al . , 2015 , patel et  al . , 2015 ) .
this type of low complexity domain ( lcd ) shows a strong sequence similarity to yeast prions in that they are enriched in polar uncharged amino acids ( usually glutamine / asparagine ) and glycine ( king et  al . , 2012 ) .
degenerate tyrosine - containing repeats in the prlds of fus and other hnrnps , which can mediate hydrophobic and  interactions , seem to be crucial in promoting hydrogel formation ( kato et  al . , 2012 ) .
the llps of these prlds indeed is inhibited by aliphatic alcohols , which interfere with hydrophobic interactions ( molliex et  al . ,
phase separation based on hydrophobic interactions is also likely to underlie nuclear speckle and nuclear pore mesh formation ( frey et  al . , 2006 ,
moreover , we find that pathogenic arginine - rich dipeptide repeats in c9orf72 als / ftld also undergo llps in  vitro and in cells , hereby uncovering a potential new toxic pathway in the disease .
sg proteins also contain other domains that might be non - redundant in phase separation .
besides canonical rna - binding domains , e.g. , rrm , these proteins are also enriched in disordered arginine - rich sequences , e.g. , rgg boxes ( aguzzi and altmeyer , 2016 ) ( figure  1a ) , which can bind rna or poly(adp - ribose ) and seed llps or aggregation of prlds ( altmeyer et  al . , 2015 , burke et  al . , 2015 , lin et  al . , 2015 , molliex et  al . , 2015 , patel et  al . , 2015 , schwartz et  al . , 2013 ) .
while globular rrms have a clear rna - binding function , these disordered arginine - rich motifs are more promiscuous and also serve as protein binding modules ( dormann et  al . , 2012 , thandapani et  al . ,
2013 ) . given the strong conservation of both disorder and arginine content in rbps ( varadi et  al . , 2015 ) , we hypothesize that arginine - rich domains could play a role in llps in physiological and pathological processes .
in fact , 40 amino - acid - long arg / gly - rich peptides from the rgg boxes of fus , hnrnpa1 , and fmrp spontaneously phase separate in the presence of a molecular crowder ( figures 1b1d , s1a , and s1b ) .
this emerging basic mechanism of llps puts particular emphasis on recent observations from c9orf72 hexanucleotide repeat expansions , which are the most common genetic cause of als and ftld ( dejesus - hernandez et  al .
a potential toxic mechanism is the generation of five species of dipeptide repeats ( dprs ) through aberrant translation of the repeat rna ( ash et  al .
several groups have shown that two of them , namely , glycine - arginine ( gr ) and proline - arginine ( pr ) repeats , are highly toxic ( boeynaems et  al .
, 2016a , freibaum et  al . , 2015 , jovii et  al . , 2015 ,
we wondered whether these peptides exhibit a behavior similar to the rgg peptides in  vitro .
synthetic gr20 and pr20 , but not gp20 , strongly phase separated following the addition of a molecular crowder ( figures 1c and 1d ) .
interestingly , the extent of phase separation for all examined peptides is significantly correlated with arginine content ( figures 1c and s2a ) , which indicates that arginines are likely to be the mediators of phase separation for these peptides . to further investigate such arginine - mediated phase separations , we used the pr dpr as a model .
as shown for other proteins ( molliex et  al . , 2015 , nott et  al . , 2015 , patel et  al . , 2015 ) , phase separation of pr20 is temperature dependent and reversible ( figures 2a , 2b , and s1a  s1c ) .
pr20 droplets are highly circular ( figure  2c ) , suggesting that they minimize surface tension . when we applied shear stress to these droplets , we indeed observed plastic deformation with minimization of their surface area after relaxation ( figure  2d ) .
there was also a conservation of volume after the fusion of two droplets ( figure  2e ) .
all these observations indicate that pr20 droplets behave as a liquid . besides temperature , llps of pr was also sensitive to protein concentration , molecular crowding , and the length of the peptide ( figures 3a and 3b ) . in agreement with the polar nature of the peptide
, we found that llps was not perturbed by an aliphatic alcohol , but was hindered by increasing the salt concentration ( figures 3c , 3d , and s1d ) .
additionally , the fluorescence of the hydrophobic dye 8-anilino-1-naphthalenesulfonic acid ( ans ) showed maximum intensity at 495  nm and was reduced following llps of pr20 ( figure  s1f ) ( hawe et  al . , 2008 ) .
these observations argue against hydrophobic interactions and indicate the importance of electrostatic forces via arginines in llps .
phase separation of like - charged inorganic polyelectrolytes has been reported before , and due to charge repulsion , this process is only conceivable in the presence of counteranions in solution ( brangwynne et  al .
we reasoned that phosphate ions in our buffer could be a likely candidate in stabilizing pr - pr interactions . in accordance , when we incubated pr
in pure water with the molecular crowder and added different salts , the extent of phase separation depended on the charge of the anions ( figure  3e ) .
indeed , addition of polyu rna promoted llps in a dose - dependent manner even without molecular crowders being present ( figures 3f , 4a , and s1e ) ; and this was also the case for other arginine - rich peptides ( figure  s2a ) .
similar to the peg - induced llps , pr20-rna droplets were highly circular ( figure  4a ) .
they also continuously increased in size over time , as measured by dynamic light scattering ( dls ) ( figure  4b ) , mimicking observations for cellular sgs ( wheeler et  al . , 2016 ) .
due to its polyvalent nature , polyu was much more effective than phosphate and crowding agent peg in promoting llps .
besides purely electrostatic interactions , arginines can also engage in  interactions with aromatic side chains ( brangwynne et  al . , 2015 ) .
in support of the contribution of this mechanism , incubating pr20 with poly - tyrosine induced clustering of the insoluble poly - tyrosine polymers ( figures 3 g and s1h ) .
disordered linear binding motifs often undergo induced folding following ligand binding ( tompa , 2016 ) , but they can also remain predominantly disordered when bound in so - called  fuzzy  complexes ( tompa and fuxreiter , 2008 ) and when phase separated , as already observed in the case of fus lcd droplets ( burke et  al . , 2015 ) .
cd spectroscopy clearly illustrated the random coil nature of the dispersed pr20 peptide , and this was also the case for other arginine - rich peptides ( figures s2b 
however , after titration with polyu rna , the pr20 cd spectrum was lost , i.e. , there is no signal for the peptide within droplets ( figure  s2 g ) . therefore , we applied two other approaches to monitor the structural state of pr20 in droplets , both supporting its largely disordered ( fuzzy ) state .
fluorescence anisotropy of peptide tagged with a fluorophore ( figure  s2h ) is very low ( 0.02 ) , and even after the addition of polyu rna it only increases to about 0.2 , far from the value measured in the highly viscous glycerol ( 0.38 ) , the latter being close to the theoretical upper limit ( 0.4 ) for an immobile fluorophore .
global hydrogen - deuterium exchange ( hdx ) experiments ( figures s2i and s2j ) show full accessibility of the free peptide , which is compatible with its disordered state .
following the addition of polyu rna , accessibility drops only slightly ( to 80% of its initial value ) , indicating that the peptide is still largely accessible , and suggesting that it stays disordered after llps . to check whether the pr - rna droplets could have any internal fibrillar structure , we performed cryo - transmission electron microscopy ( tem ) , and could not discern any visible structures in the droplets ( figure  4c ) .
further , we performed fluorescence recovery after photobleaching ( frap ) analyses with the fluorescently tagged pr20 ( figures 4d and 4e ) , and found in full - droplet bleaching experiments that demixed pr readily interchanges with pr in the dispersed phase ( figure  4d ) .
aging of the pr - rna droplets had no effect on intradroplet fluorescence recovery ( figure  4e ) .
this result is in contrast to findings for prld llps , which undergo a liquid - to - solid transition likely through  sheet formation ( conicella et  al . , 2016 ,
we took soluble hela cell lysate , to which we added increasing concentrations of pr30 .
the pr30 peptide spontaneously demixed , and could be separated from the solution by gentle centrifugation .
the resultant pellet was subsequently washed and analyzed by gel electrophoresis ( figure  5a ) .
unexpectedly , this pellet was largely resistant to washing steps , suggesting that pr induced the precipitation of different cellular proteins to the insoluble fraction .
the demixing of pr was also largely independent of the presence of rna in the lysate , as addition of rnase only had a modest effect on the extent of protein precipitation ( figure  s3 ) . to see whether weak interactions could still be involved in the process
, we performed mild crosslinking with paraformaldehyde as recently used to identify both the stable core and liquid - like shell of cellular sgs ( jain et  al . , 2016 ) .
the pr fraction seemed to be enriched for a specific set of proteins as was evident from the band pattern .
mass spectroscopy ( ms ) analysis on both crosslinked and uncrosslinked samples identified 874 proteins that were detected and quantified in both conditions .
we performed gene ontology ( go ) enrichment analyses to identify overrepresented biological processes and cellular compartments in our hit list .
significantly overrepresented terms centered on rna and protein metabolism ( go biological process ; figure  5b ) and different cellular liquid - like compartments ( go cellular compartment ; figure  s3 ) . indeed , when comparing the protein content of the pr30 precipitate with those of stress granules and nucleoli , we found a strong overlap with both cellular liquid compartments ( figure  5c ) , while this was not the case for an unrelated organelle ( figure  s4 ) .
the pr30 precipitate was also enriched for metastable aggregation - prone proteins ( figure  5d ) .
given that 60% of identified proteins have been implicated in rna metabolism ( figure  s4 ) , we evaluated overrepresentation of rna binding domains .
these included globular rrm or dead / h box domains and disordered rgg boxes ( figure  5e ) .
besides rgg boxes , r motifs were also highly abundant ( figure  5f ) , once more illustrating the importance of arginine - rich binding modules in llps .
lastly , we found that the  pr30 precipitate is enriched for disordered ( figure  5 g ) and aggregation - prone supersaturated proteins ( figure  5h ) ( ciryam et  al . , 2013 ) .
quantitative analysis revealed that 190 proteins were more abundant after crosslinking ( figure  s5 ) , which suggests that weak interactions between these proteins and the pr30-induced insoluble fraction could be stabilized by our mild crosslinking protocol .
interestingly , this set of cross - linking - sensitive proteins was enriched for different protein families involved in stress granule biology , such as hnrnps ( molliex et  al . , 2015 ) and aminoacyl transferases ( david et  al . , 2011 )
although both 40s and 60s ribosomal proteins were highly abundant in our protein set identified by ms , only 40s , but not 60s , constituents were overrepresented in the crosslinking - sensitive fraction .
cellular sgs also contain 40s , but exclude 60s subunits ( kedersha et  al . ,
our in  vitro cell - free findings , we overexpressed a codon - optimized pr100 construct in hela cells ( figure  6a ) . as previously reported , pr100 predominantly localized to the nucleolus ( kwon et  al .
, 2014 , tao et  al . , 2015 , wen et  al . , 2014 , yamakawa et  al . , 2015 ) . yet
, the evidence whether arginine - rich dprs affect sgs has remained ambiguous ( tao et  al .
we found cytoplasmic pr100 granules positive for sg markers after overexpression of pr100 ( figure  6a ) , and this process was dose and length dependent ( figure  s6 ) .
pr100 expression was more efficient at inducing sgs than pa100 expression , showing that these sgs do not just originate from the transfection protocol ( figure  6b ) .
pr100-induced sgs were  also responsive to known sg modifying compounds , i.e. , arsenite and puromycin treatment enhanced sg formation , whereas cyclohexamide reduced their number ( figure  6c ) . by using a mutant mef cell line carrying a non - phosphorylatable form of eif2
, we found that pr100 requires the integrated stress  response for sg induction ( figure  6d ) .
also the presence of g3bp1/2 seemed to be required for this , based on the use of knockout cell lines ( figure  6e ) ( kedersha et  al .
when studying these pr100-induced sgs in more detail , we observed that they are slightly less dynamic than arsenite - induced sgs , based on frap analysis of a g3bp1-gfp fusion protein ( figure  6f ) .
moreover , the protein content of pr100 sgs was strongly altered compared to arsenite sgs .
while there was no difference in the sg enrichment of a general sg marker ( yb-1 ) , both a late sg marker ( ddx6 ) and als - related proteins carrying a prld ( ataxin-2 and tdp-43 ) were significantly enriched in pr100 sgs compared to arsenite sgs ( figure  6 g ) .
the current hypothesis for the formation of aggregates in als / ftld patients is a liquid - to - solid phase transition or perturbed clearance of sgs .
although not observed in a living system so far , in  vitro droplets of fus and hnrnps have been shown to mature to a solid state over the course of hours ( molliex et  al . , 2015 , patel et  al . , 2015 ) .
this likely corresponds to a transition of their prlds from a disordered fuzzy state to a rigid  sheet structure ( burke et  al .
, 2015 , molliex et  al . , 2015 , patel et  al . , 2015 ) .
since we found that ( 1 ) pr repeats could engage in  interactions with tyrosines , ( 2 ) pr induced the precipitation of several of these proteins in  vitro , and ( 3 ) such proteins were enriched in pr100 sgs , we wondered whether pr could affect the dynamics and maturation of prld droplets .
addition of pr30 , but not gp30 , to fus lcd droplets interfered with their spontaneous fusion dynamics ( figure  7a ) .
additionally , fus lcd phase separation was also enhanced by addition of pr30 ( figures 7a and 7b ) . in agreement with the reduced dynamics of the droplets , kinetic thioflavin - t fluorescence analysis of fus lcd droplets supplemented with pr30 , indicated an increase in  sheet content over time ( figures 7c and s7 ) .
in this study , we have investigated whether highly repetitive and disordered arginine - rich peptides generated from expanded c9orf72 repeats in als / ftld could play a role in llps .
this phenomenon is of intense interest since it could explain the biogenesis and physical underpinnings of sgs , which are the prime suspect of seeding for pathological protein aggregation in als and ftld ( king et  al . , 2012 , ramaswami et  al . , 2013 ) . until now ,
llps of prld containing rbps has been mostly attributed to hydrophobic interactions . however , arginine - rich domains are  highly enriched in cellular liquid - like compartments , such as stress granules and the nucleolus , and both protein disorder  and arginine - content are highly conserved in rbps ( varadi et  al . , 2015 ) .
this conservation suggests that these arginine - rich domains must be under selective pressure , highlighting their  functionality and suggesting that a similar gain - of - function may underlie the pathological role of c9orf72 dprs in als .
in fact , rgg boxes have been shown to affect prld aggregation and llps by nucleating on rna or poly(adp - ribose ) ( altmeyer et  al . , 2015 , lin et  al . , 2015 ,
, 2015 , patel et  al . , 2015 , schwartz et  al . , 2013 ) .
we hence hypothesized that arginine - rich dprs could play a more direct role in phase separation through their disorder ( zhang et  al . ,
indeed , we found that arginine - rich dprs derived from the c9orf72 repeat expansion undergo llps , a process mimicked by short synthetic peptides corresponding to the rgg boxes of three sg proteins , which also spontaneously phase separated  in  vitro .
this phase transition was directly correlated with the  arginine content of the peptides . as shown previously for inorganic polymers ,
such like - charged phase separation was dependent on counterions ( tiraferri et  al . , 2015 ) .
recently , two other studies have also reported a similar behavior for artificial arginine - rich peptides ( aumiller and keating , 2016 ) or peptides derived from nucleolar proteins ( mitrea et  al . , 2016 ) .
intriguingly , when we added cellular proteins to pr , this resulted in the formation of an insoluble pellet , rather than liquid droplets .
however , crosslinking experiments showed that specific weak protein - protein interactions did occur in our test tube experiment .
this is consistent with recent reports indicating that cellular sgs likely consist of a stable core surrounded by a liquid shell , rather than a homogeneous liquid droplet ( jain et  al .
the pr30 interactome using ms showed that pr was able to interact with a large , yet specific , set of proteins enriched for rna - binding domains , arginine - rich motifs and protein disorder .
numerous hits were known constituents of endogenous liquid organelles , and , interestingly , several have been directly implicated in als / ftld .
hence , we suggest that dprs may play a role in disease by upsetting the internal functional balance of membraneless organelles ( brangwynne et  al . , 2015 ) .
the data of our in  vitro precipitation experiment are in agreement with three recent studies showing that intracellular pr and gr peptides can bind with a large set of proteins involved in rna- and sg metabolism ( lee et  al . , 2016 , lin et  al . , 2016 , lopez - gonzalez et  al . , 2016
when overexpressed in cells , pr was indeed able to seed sg assembly , and this sg induction was dependent on phosphorylation of eif2 and the presence of g3bp proteins .
although sg induction by pr was shown recently by others ( lee et  al . , 2016 , yamakawa et  al . ,
2015 ) , the mechanism through which this occurred remained unknown and of intense interest from a  therapeutic perspective . in this light , eif2 modulation
has already been shown to ameliorate toxicity in different tdp-43 als models ( kim et  al . , 2014 ) .
g3bp proteins have been shown to be essential in eif2-controlled sg assembly ( kedersha et  al . , 2016 ) , and the g3bp rgg domain plays a crucial role in this process , once more highlighting the importance of arginine - rich domains in sg metabolism .
pr - induced sgs displayed reduced dynamics and were enriched for als - related proteins .
moreover , pr was also able to accelerate the liquid - to - solid maturation of prld droplets , a process reminiscent of pathological aggregation ( conicella et  al . , 2016 , molliex et  al .
in line with our findings , two other reports also recently found that arginine - rich dprs can affect sgs and other higher order assemblies in similar ways ( lee et  al .
sgs are generally considered to be the stepping stone toward pathological aggregation of rbps in disease .
identifying the mechanisms behind altered sg dynamics and a deeper appreciation of their biology , hence , will provide us with invaluable clues to better understand the etiology of disease .
disease mutations are known to target several sg proteins or proteins involved in sg dynamics ( ramaswami et  al . , 2013 ) .
however , the reason why wild - type proteins mislocalize to the cytoplasm and aggregate in c9orf72 cases remained elusive .
recently , we and others have shown that nucleocytoplasmic transport defects caused by arginine - rich dprs could explain the mislocalization of these rbps ( boeynaems et  al .
, 2016a , boeynaems et  al . , 2016b , freibaum et  al . , 2015 , jovii et  al . , 2015 ) .
by focusing now on the biophysical behavior of arginine - rich dprs , we and others have found that they could also be directly implicated in the cytoplasmic aggregation of rbps .
these data suggest that dprs could initiate a pathogenic cascade in c9orf72 als / ftld , by targeting the nuclear transport - stress granule axis ( boeynaems et  al .
reagent or resourcesourceidentifierantibodiesanti - flag , mousesigma - aldrichf3165 ; rrid : ab_259529anti - flag , rabbitcell signaling#2368santi - g3bp1 , mouseabcamab56574 ; rrid : ab_941699anti - ddx6 , rabbitabcamab40684 ; rrid : ab_941268anti - yb1 , rabbitabcamab76149 ; rrid : ab_2219276anti - tdp-43 , rabbitproteintech12892 - 1-ap ; rrid : ab_2200505anti - ataxin-2 , mousebd biosciences611378 ; rrid : ab_398900anti - tia1 , goatsanta cruzsc-1751 ; rrid : ab_2201433bacterial and virus strainse . 
coli , bl21 star ( de3)thermo scientificc601003chemicals , peptides , and recombinant proteinspr20this papern / apr30this papern / agr30this papern / agp30this papern / afus rggthis papern / ahnrnpa1 rggthis papern / afmrp rggthis papern / afus lcdr .
, 20151,6-hexanediolsigma - aldrich240117polyu rnasigma - aldrichp9528poly - tyrosinesigma - aldrichp1800peg300sigma - aldrich81160anssigma - aldricha1028complete protease inhibitor cocktailsigma - aldrich11697498001arsenitesigma - aldrichs7400lipofectamine 3000thermo scientificl3000015nucbluethermo scientificr37605prolong gold antifadethermo scientificp36934critical commercial assaysalexa fluor 488 protein labeling kitthermo scientifica10235alexa fluor 568 protein labeling kitthermo scientifica10238deposited datamas spec datathis paperpride : pxd005509experimental models : cell lineshelaatccccl-2mef eif2a ssdr .
, 2015plasmid : pr100-flag - mcherrythis studyn / a further information and requests for reagents may be directed to and will be fulfilled by the lead contact , ludo van den bosch ( ludo.vandenbosch@kuleuven.vib.be ) .
paul taylor ( st . jude , usa ) ( figley et  al . , 2014 ) .
u2os g3bp1/2 ko cell line , and eif2 ss and aa mefs were a kind gift of dr .
paul anderson ( harvard , usa ) ( emara et  al . , 2012 , kedersha et  al . ,
u2os and hela cells ( atcc ) were cultured in high glucose dmem ( invitrogen ) supplemented with 10% fetal bovine serum ( greiner ) , 4  mm glutamax ( invitrogen ) , penicillin ( 100  u / ml ) , streptomycin ( 100  g / ml ) and non - essencial amino acids ( 1% ) .
flag - tagged dpr50 expression constructs were synthesized by genscript ( piscataway , usa ) .
fus lc domain was expressed in e.  coli and purified as described previously ( burke et  al . , 2015 ) .
hnrnpa2 lc domain ( residues 190 - 341 ) was expressed with a tev - cleavable n - terminal hexahistidine tag , purified from the inclusion body via histrap ( ge healthcare ) in urea containing buffers , concentrated , diluted into native buffer for tev cleavage of hexahistidine tag , resolublized by addition of solid urea , separated from his - tagged tev protease and cleaved his - tag by histrap , and concentrated to 1 - 2  mm in 8 m urea 20 mm mes ph 5.5 .
peptides were diluted to the indicated concentrations in 100 mm k2hpo4/kh2po4 buffer at ph 7 , unless otherwise indicated .
peg300 , polyu rna or poly - l - tyrosine ( sigma - aldrich ) were added at the indicated concentrations .
for fluorescence microscopy and frap analyses , pr - alexa 568 and polyu30-alexa 488 were spiked in at 200 nm and 100  nm respectively .
fus lc droplets were generated by diluting the stock solution to the desired concentration in 50 mm mes buffer at ph 5 , with the indicated nacl concentrations .
hnrnpa2 lc droplets were generated by diluting the stock solution to the desired concentration in 20 mm napi buffer with 50 mm nacl at ph 7.5 .
od600 of 60l samples was measured using truview microcuvettes in a smartspec plus spectrophotometer ( bio - rad ) .
turbidity of 100l of fus and hnrnpa2 lc droplets was measured on a spectra max m5 microplate reader ( molecular devices ) .
fus lc droplets were generated by diluting the stock solution to 250m in 50m mes buffer at ph 5 , 200m nacl .
fluorescent droplets were incubated in plastic cell counter slides ( bio - rad ) at room temperature .
every two hours three arbitrary fields were imaged on a zeiss lsm 510 meta nlo confocal microscope .
tht was added to 100l pr30  + lc droplet mixtures at a concentration of 20m .
fluorescence intensity over time was followed using a spectra max m5 microplate reader ( molecular devices ) .
lag time was estimated as the time point of a local minimum in the curve .
cd spectra of the peptides analyzed in this study were recorded in a jasco 715 spectropolarimeter equipped with a ptc 423s peltier element .
peptides were loaded into a 0.2 mm quartz cuvette at 30m concentration ( 50 mm k2hpo4/kh2po4 buffer at ph 7 ) , and spectra were recorded with 50nm / min scan speed at 25c with 9 acquisitions averaged on the fly .
spectra were background corrected with the buffer spectrum ( 50 mm k2hpo4/kh2po4 buffer at ph 7 ) , and converted into molar ellipticity .
pr droplet ( 250m pr20  + 0.04g/l polyu rna ) sample ( 3.5  l ) was applied to a 300 mesh lacey quantifoil grid and incubated for 30  s. next , excess buffer was removed by blotting the grids for 3  s using a whatman 1 filter paper and the sample was snap frozen by plunging in liquid ethane at a temperature of 180c and stored in liquid nitrogen until visualization .
next , the samples were transferred to a gatan 914 cryoholder and imaged at low dose conditions at 177c , using a jeol jem1400 tem equipped with an 11  mpxl olympus sis quemesa camera .
fluorescent droplets were incubated in plastic cell counter slides ( bio - rad ) at room temperature .
fluorescence recovery after bleaching was monitored using zen software on a  zeiss lsm 510 meta nlo confocal microscope . for intradroplet frap ,
a circular are of 1m radius was bleached in droplets with a radius between 5m and 10m .
raw data was background substracted and normalized using excell , and plotted using  prism software .
measurements were carried out in a ls55 luminescence spectrometer ( perkinelmer ) , with 200 nm pr - alexa568 n - terminally labeled protein in 50 mm k2hpo4/kh2po4 buffer at ph 7 .
after the addition of 10m of bulk pr30 , fluorescence anisotropy was measured at increasing rna concentration at 578 nm excitation and 603 nm of emission wavelengths .
20m pr30 was titrated with increasing concentration of polyu rna starting at a 0.04g/l concentration in a dynapro nanostar ( wyatt technologies ) in 50 mm k2hpo4/kh2po4 buffer at ph 7 .
the size of particles was followed for one hour after every titration step ( each titration step represented a 0.5% dilution ) .
to control sedimentation and droplet fusion sample was mixed thoroughly with a pipet randomly between time points .
ans ( 8-anilinonaphthalene-1-sulfonic acid ) fluorescence was measured for 40m pr20 or 40m gr20 peptide in the presence and absence of 0.05g/l polyu rna in a biotek mx synergy plate reader in 50 mm k2hpo4/kh2po4 buffer at ph 7 .
emission spectra were recorded between 400 nm and 600 nm at 380 nm excitation wavelength .
pr30 and polyu rna were dissolved in miliq grade water and added together to make a final concentration of 400  m pr and 4.8  mg / ml polyu rna to induce llps . for experiments on the free peptide , pr
the samples were then , either immediately or after an incubation time of 1h for the llps samples , diluted with a 20-fold excess of d2o . after the indicated time points the hdx reaction was quenched by bringing the ph of the solution to 1 using formic acid , followed by snap freezing in liquid nitrogen .
mass spectrometry analysis of deuterium incorporation was performed on a synapt g2 hdms mass spectrometer ( waters , wilmslow , uk ) by direct infusion of the undigested sample using nano - electrospray ionization with in - house prepared gold - coated borosilicate needles .
hela cells were trypsinized , pelleted and washed three times with 1x pbs ( thermo scientific ) .
cells were resuspended in k2hpo4/kh2po4 buffer with edta - free complete protease inhibitor cocktail ( roche ) and sonicated on ice .
the lysate was cleared from the insoluble fraction by centrifugation for 15min at 10,000  rpm at 4c .
the supernatans was retrieved and protein concentration was measured using micro bca assay ( thermo scientific ) . 1  mg of protein was added to 0.05  mole of pr30 to a final concentration of 100m , and left incubating for 10  min at rt .
paraformaldehyde ( sigma - aldrich ) was added to a final concentration of 0.5% , and crosslinking was left for 5  min .
500l of 2 m glycine ( sigma - aldrich ) was added to quench paraformaldehyde , and samples were left for 5  min .
the volume of the samples was increased to 1.5ml with k2hpo4/kh2po4 buffer , before gently spinning down the pr droplets at 4,000  rpm for 5  min .
pellets were subsequently washed with 1ml pbs with 0.4% triton x-100 ( sigma - aldrich ) and vortexed before spinning down .
samples for sds - page were generated identically , but procedure was downscaled four times .
pr llps pellets were resuspended in 1x reducing laemmli containing sds ( thermo scientific ) .
1/8 of pellet or input lysate were loaded onto nupage novex 4%12% bis - tris precast gels ( thermo scientific ) . after running gels were stained using coomassie brilliant blue r-250 according to manufacturer s instructions ( bio - rad ) .
uncrosslinked ( 2 replicates ) and crosslinked pellets ( 3 replicates ) were redissolved in 300  mm nacl tris buffer , sonicated and boiled to remove crosslinks .
proteins in each sample were reduced with 5  mm dtt and incubation for 30  min at 55c and then alkylated by addition of 10  mm iodoacetamide for 15  min at room temperature in the dark .
samples were further diluted to a final urea concentration of 2  m and proteins were digested with trypsin ( promega ) ( 1/100 , w / w ) overnight at 37c .
peptides were then purified on omix c18 tips ( agilent ) , dried and re - dissolved in solvent a ( 25  l 0.1% tfa in water / acetonitrile ( 98:2 , v / v ) ) of which 10  l was injected for lc - ms / ms analysis on an ultimate 3000 rslcnano system ( dionex , thermo fisher scientific ) in line connected to a q exactive hf mass spectrometer with a nanospray flex ion source ( thermo fisher scientific ) .
trapping was performed at 10  l / min for 4  min in solvent a ( on a reverse - phase column produced in - house , 100  m i.d .
x 20  mm , 5  m beads c18 reprosil - pur , dr . maisch ) followed by loading the sample on a 40  cm column packed in the needle ( produced in - house , 75  m i.d .
 400  mm , 1.9  m beads c18 reprosil - hd , dr . maisch ) .
peptides were eluted by an increase in solvent b ( 0.1% formic acid in water / acetonitrile ( 2:8 , v / v ) ) in linear gradients from 2% to 30% in 100  min , then from 30% to 56% in 40  min and finally from 56% to 99% in 5  min , all at a constant flow rate of 250 nl / min .
the mass spectrometer was operated in data - dependent mode , automatically switching between ms and ms / ms acquisition for the 16 most abundant ion peaks per ms spectrum .
full - scan ms spectra ( 375 - 1500 m / z ) were acquired at a resolution of 60,000 after accumulation to a target value of 3,000,000 with a maximum fill time of 60  ms . the 16 most intense ions above a threshold value of 22,000 were isolated ( window of 1.5 th ) for fragmentation at a normalized collision energy of 32% after filling the trap at a target value of 100,000 for maximum 45  ms .
the s - lens rf level was set at 55 and we excluded precursor ions with single and unassigned charge states .
data analysis was performed with maxquant ( version 1.5.3.30 ) ( cox and mann , 2008 ) using the andromeda search engine with default search settings including a false discovery rate set at 1% on both the peptide and protein level .
spectra were searched against the human proteins in the uniprot / swiss - prot database ( database release version of april 2016 containing 20,103 human protein sequences , www.uniprot.org ) . the mass tolerance for precursor and fragment ions were set to 4.5 and 20 ppm , respectively , during the main search .
enzyme specificity was set as c - terminal to arginine and lysine , also allowing cleavage at proline bonds with a maximum of three missed cleavages .
variable modifications were set to oxidation of methionine residues and acetylation of protein n - termini . only proteins with at least one unique or razor peptide
proteins were quantified by the maxlfq algorithm integrated in the maxquant software ( cox et  al . , 2014 ) .
further data analysis was performed with the perseus software ( version 1.5.3.0 ) after loading the protein groups file from maxquant .
proteins only identified by site and reverse database hits were removed and replicate samples of uncrosslinked and crosslinked were grouped .
proteins with less than three valid values in at least one group were removed and missing values were imputed from a normal distribution around the detection limit .
then , a t test was performed ( fdr  = 0.01 and s0  = 1 ) to compare samples uncrosslinked and crosslinked and calculate the fold change for each protein between both samples .
after removal of potential contaminants , 874 proteins were quantified in total ( table s1 ) .
cells were transiently transfected using lipofectamine 3000 ( invitrogen ) according to manufacturer s instructions .
cells were fixed 24h after transfection in 4% formaldehyde in pbs and stained according standard protocols ( including methanol fixation and permeabilization by pbs - t 0.04% ) .
following antibodies were used : anti - flag ( f3165 , sigma ) , rabbit anti - flag ( # 2368s , cell signaling ) , mouse
anti - g3bp1 ( ab56574 , abcam ) , rabbit anti - ddx6 ( ab40684 , abcam ) , rabbit anti - yb1 ( ab76149 , abcam ) , rabbit anti - tdp-43 ( 12892 - 1-ap , proteintech ) , mouse anti - ataxin-2 ( 611378 , bd biosciences ) , goat anti - tia1 ( sc-1751 , santa cruz ) . alexafluor 555 and alexafluor 488 secondary antibodies ( life technologies ) were used .
control stress granules were induced by incubating the cells for 1h with 0.5 mm naaso2 ( sigma ) .
percentage of cells carrying stress granules was assessed by g3bp1 staining in all tested cell lines .
stress granule enrichment was quantified as the ratio of fluorescence intensity in the stress granule over the intensity of the surrounding cytoplasm .
two stress granules per cell were quantified , of at least 30 cells from 3 independent experiments . for stress granule enrichment of pr100 ,
one stress granule per cell was counted , of 40 cells from 2 independent experiments .
pr100 stress granules were visualized for quantification with flag staining , arsenite stress granules were visualized by tia1 staining . for analysis of dose- and length dependency of pr cytoplasmic structures ,
cells were categorized as having diffuse cytoplasmic pr , small stress granules , large stress granules or coarse / granular / heterogeneous cytoplasmic pr staining .
data was subsequently also plotted as the percentage of cells displaying non - diffuse cytoplasmic pr staining after manual binning according to cytoplasmic pr100-flag intensity .
percentages were fitted by a one - phase exponential curve , illustrating a strong dose - dependency of cytoplasmic structure formation .
the mass spectrometry proteomics data have been deposited to the proteomexchange consortium via the pride partner repository with the dataset identifier pride : pxd005509 .
, w.r . , and p.v.d . provided ideas for the project and participated in writing the paper . s.b .
 summaryliquid - liquid phase separation ( llps ) of rna - binding proteins plays an important role in the formation of multiple membrane - less organelles involved in rna metabolism , including stress granules . 
 defects in stress granule homeostasis constitute a cornerstone of als / ftld pathogenesis . 
 polar residues ( tyrosine and glutamine ) have been previously demonstrated to be critical for phase separation of als - linked stress granule proteins . 
 we now identify an active role for arginine - rich domains in these phase separations . 
 moreover , arginine - rich dipeptide repeats ( dprs ) derived from c9orf72 hexanucleotide repeat expansions similarly undergo llps and induce phase separation of a large set of proteins involved in rna and stress granule metabolism . 
 expression of arginine - rich dprs in cells induced spontaneous stress granule assembly that required both eif2 phosphorylation and g3bp . together with recent reports showing that dprs affect nucleocytoplasmic transport , our results point to an important role for arginine - rich dprs in the pathogenesis of c9orf72 als / ftld . 
Introduction
Results
Discussion
STARMethods
Author Contributions