Conserved intrinsically disordered region of DNAJB6 dictates its surveillance of FG-Nup condensates

Conserved intrinsically disordered region of DNAJB6 dictates its surveillance of FG-Nup condensates | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Conserved intrinsically disordered region of DNAJB6 dictates its surveillance of FG-Nup condensates Tessa Bergsma , Maiara Kolbe Musskopf , Paola Gallardo , View ORCID Profile Mathieu E. Rebeaud , Jarmo Feenstra , Sidath M. Y. Fernando , Anton Steen , View ORCID Profile Harm H. Kampinga , View ORCID Profile Liesbeth M. Veenhoff doi: https://doi.org/10.1101/2025.10.20.683411 Tessa Bergsma 1 European Research Institute for the Biology of Ageing, University of Groningen, University Medical Center Groningen , 9713 AV, Groningen, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Maiara Kolbe Musskopf 2 Biomedical Sciences, University of Groningen, University Medical Center Groningen , 9713 AV, Groningen, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Paola Gallardo 1 European Research Institute for the Biology of Ageing, University of Groningen, University Medical Center Groningen , 9713 AV, Groningen, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mathieu E. Rebeaud 3 Institute of Physics, School of Basic Sciences, École Polytechnique Fédérale de Lausanne , CH-1015, Lausanne, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Mathieu E. Rebeaud Jarmo Feenstra 1 European Research Institute for the Biology of Ageing, University of Groningen, University Medical Center Groningen , 9713 AV, Groningen, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sidath M. Y. Fernando 2 Biomedical Sciences, University of Groningen, University Medical Center Groningen , 9713 AV, Groningen, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Anton Steen 1 European Research Institute for the Biology of Ageing, University of Groningen, University Medical Center Groningen , 9713 AV, Groningen, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Harm H. Kampinga 2 Biomedical Sciences, University of Groningen, University Medical Center Groningen , 9713 AV, Groningen, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Harm H. Kampinga For correspondence: h.h.kampinga{at}umcg.nl l.m.veenhoff{at}rug.nl Liesbeth M. Veenhoff 1 European Research Institute for the Biology of Ageing, University of Groningen, University Medical Center Groningen , 9713 AV, Groningen, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Liesbeth M. Veenhoff For correspondence: h.h.kampinga{at}umcg.nl l.m.veenhoff{at}rug.nl Abstract Full Text Info/History Metrics Preview PDF Abstract Molecular chaperones can prevent protein aggregation and assist proteins in reaching their structurally functional state. The molecular chaperone DNAJB6, a J-domain protein that partners with Hsp70s and nucleotide exchange factors, is very potent in preventing amyloid formation of proteins with large intrinsically disordered regions (IDRs), including several disease-associated proteins. Complementary to this, we recently demonstrated a role for DNAJB6 in surveilling IDP phase transitions, highlighting its role in nuclear pore complex assembly. Here, we further show that DNAJB6 and the closely related DNAJB2 and DNAJB8 prevent several FG-rich nucleoporins (FG-Nups) from undergoing aberrant phase-transitions. We demonstrate that this surveillance mechanism of DNAJB6 is encoded in an unusually highly conserved IDR that promotes the formation of stable, gel-like assemblies of the chaperone itself. These assemblies likely provide a stable environment that can outcompete homotypic FG-Nup interactions and instead favors dynamic, multivalent heterotypic chaperone:FG-Nup interactions. The evolutionary conservation of the DNAJB6-IDR and mutant analyses suggest that the sequence space for encoding stable gel-like assemblies is narrow and optimized to avoid self-aggregation while providing remarkable anti-amyloidogenic capacity. Introduction Intrinsically disordered proteins (IDPs) are present in all kingdoms of life, and they have gained significant attention due to their unique properties and functional capabilities ( Pancsa & Tompa, 2012 ; Xue et al , 2012 ). Unlike traditional proteins that adopt stable three-dimensional structures, IDPs lack a stable structure under physiological conditions ( Van Der Lee et al , 2014 ). This inherent flexibility allows them to partake in dynamic multivalent interactions forming homotypic or heterotypic biomolecular condensates within the cellular environment. These condensates facilitate crucial biological processes such as regulating gene expression, compartmentalizing metabolic reactions and transiently and locally concentrating molecules ( Holehouse & Kragelund, 2024 ; Farag et al , 2023 ). IDPs can adopt ensembles of rapidly interconverting structures. This behavior is dependent on the IDP sequence and is also highly context-dependent, where the context is defined by, amongst others, the concentration of the IDP, macromolecular crowding, temperature, binding partners, pH, and post-translational modifications ( Holehouse & Kragelund, 2024 ). The self-assembly of IDPs into dynamic condensates can occur through, amongst other principles, liquid-liquid phase separation (LLPS), in which a homogeneous solution of molecules demixes into distinct, coexisting liquid-like phases. Not uncommonly, liquid-like condensates transition into a gel-like phase, in which the interactions become less dynamic ( Garaizar et al , 2022 ; Gui et al , 2019 ; Kato et al , 2012 ; Murakami et al , 2015 ). Structural motifs termed Low-complexity Amyloid-like Reversible Kinked Segments (LARKS) promote phase transition into reversible semisolid hydrogels that organize in a cross-ꞵ pattern, but the paired kinked ꞵ sheets of their LARKS are less strongly bound than the paired ꞵ sheets found in amyloid fibrils ( Hughes et al , 2018 ). These motifs are commonly found in low-complexity domains (LCDs) of proteins known to partition into biomolecular condensates ( Hughes et al , 2018 ). However, under certain conditions, liquid- or gel-like condensates can further transition into an aggregated or solid-like phase, characterized by stable interactions. The resulting aggregates have been linked to various pathological conditions, including neurodegenerative diseases such as Huntington’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis, and frontotemporal dementia ( Patel et al , 2015 ; Alberti & Hyman, 2021 ). One example of a group of IDPs includes the FG-Nucleoporins (FG-Nups) of the nuclear pore complex (NPC) that are characterized by an enrichment in phenylalanine-glycine (FG) repeats. FG-Nups have a folded domain to anchor them to the NPC and an intrinsically disordered region (IDR) encoding the FG-repeats. Eleven different FG-Nups, each present in multiple copies, make up the ∼200 FG-Nups that project into the NPC’s channel in yeast cells, and similar numbers apply for the human NPC. Together with nuclear transport receptors, the FG-Nups form the selective permeability barrier, which regulates all selective nucleocytoplasmic transport of macromolecules ( Kabachinski & Schwartz, 2015 ; Fernandez-Martinez & Rout, 2021 ; Lin & Hoelz, 2019 ; Petrovic et al , 2022 ). The FG-Nups anchored inside the NPC are highly dynamic ( Kozai et al , 2023 ), but outside of the NPC context FG-Nups or fragments of FG-Nups encoding only the ID region can spontaneously undergo phase separation into liquid-, gel-, or amyloid-like states ( Yamada et al , 2010 ; Frey et al , 2006 ; Ader et al , 2010 ; Milles et al , 2013 ; Celetti et al , 2020 ; Schmidt & Görlich, 2015 ). Nup condensates can be functional and have been described as storage and assembly platforms in Drosophila oogenesis, in HeLa cells, and in Saccharomyces cerevisiae ( Hampoelz et al , 2019 ; Agote-Aran et al , 2020 ; Otto et al , 2024 ; Makio et al , 2013 ; Colombi et al , 2013 ; Thomas et al , 2023 ). FG-Nups also appear in dysfunctional condensates and aggregates in disease contexts ( Lin et al , 2021 ; Khalil et al , 2022 ; Gleixner et al , 2022 ; Grima et al , 2017 ; Kumar et al , 2023 ). Their IDRs are exemplary of an organization in “stickers and spacers,” in which the spacers tune the solubility and regulate the phase behavior of IDPs ( Mittag & Pappu, 2022 ; Das & Pappu, 2013 ). Related to the differences in amino acid sequence (e.g. the type and density of FG repeats, position and presence of charged residues) some FG-Nups form condensates more readily than others ( Yamada et al , 2010 ; Frey et al , 2006 ; Ader et al , 2010 ; Milles et al , 2013 ; Celetti et al , 2020 ; Schmidt & Görlich, 2015 ; Patel et al , 2007 ; Dekker et al , 2023 ). For example, FG-Nups with GLFG repeats have a stronger tendency to self-associate into condensates than other FG-Nups ( Yamada et al , 2010 ; Schmidt & Görlich, 2015 ; Patel et al , 2007 ; Dekker et al , 2023 ). The mixture of FG-Nups present in the NPC and in natural condensates has been proposed to be evolutionary tuned to stabilize each other, where the FG-Nup Nsp1 is a strong modulator of FG-Nup condensates, promoting a liquid-like state ( Otto et al , 2024 ). We ( Kuiper et al , 2022 ; Bergsma et al , 2025 ) and others ( Prophet et al , 2022 ) previously showed that the molecular chaperone DNAJB6 can directly interact with FG-Nups and delays the transition from more liquid-like condensates to more solid-like FG-Nup particles. We termed this phase-state surveillance. This new discovered function is relevant for NPC assembly ( Kuiper et al , 2022 ; Prophet et al , 2022 ), and adds to its well-established role in interacting with and delaying the aggregation of several other IDPs (polyQ, FUS, FTD) in vitro , in cells, and in animal models ( Kuiper et al , 2022 ; Kakkar et al , 2016 ; Månsson et al , 2014b ; Hageman et al , 2010 ; Thiruvalluvan et al , 2020 ; Li et al , 2016 ; Deshayes et al , 2019 ; Reidy et al , 2016 ; Gillis et al , 2013 ; Aprile et al , 2017 ; Bason et al , 2019 ; Österlund et al , 2020 ). DNAJB6 is a class B’ J-domain protein (JDP) and human cells express two alternatively spliced isoforms: the longer, nuclear isoform a (DNAJB6a), and the shorter isoform b (DNAJB6b), which is found both in the nucleus and the cytoplasm, and is the one referred to in this paper. In DNAJB6, the N-terminal J-domain is followed by a glycine (G) and phenylalanine (F) rich region (G/F region) and a serine (S) and threonine (T) rich region (S/T region) that is also rich in F and G residues. Previous studies showed that the S and T residues in the S/T region are crucial for DNAJB6’s ability to delay the aggregation of polyQ ( Kakkar et al , 2016 ; Gillis et al , 2013 ), while the F residues in the same region are crucial for the anti-aggregation activity towards FG-Nups ( Kuiper et al , 2022 ). Notably, the G/F and S/T regions of DNAJB6 are predominantly disordered, except for a short regulatory helix (residues 95-104) known to modulate the interaction with HSP70 ( Adupa et al , 2024 ; Abayev-Avraham et al , 2023 ). The C-terminus of DNAJB6 consists of four β-strands and was shown to be crucial to inhibit secondary nucleation in amyloidogenesis ( Österlund et al , 2023 ). DNAJB6’s ortholog DNAJB8, which is typically expressed only in testis, and DNAJB2 isoform a (hereafter referred to as DNAJB2) also belong to the class B’ JDPs with a non-canonical architecture ( Malinverni et al , 2023 ) and have a similar IDR sequence composition (located between the regulatory helix and the C-terminal β-sheets), including the presence of LARKS. Besides their similar IDR architecture, DNAJB6, DNAJB2, and DNAJB8 are all able to accumulate in nuclear envelope (NE) herniations both when expressed endogenously and when overexpressed ( Kuiper et al , 2022 ; Prophet et al , 2022 ). These herniations are stalled pre-fusion NPC assembly intermediates that accumulate MLF2 and DNAJB6 within their lumen when fusion of both NE membranes is blocked ( Kuiper et al , 2022 ; Rampello et al , 2020 ; Fischer et al ). Previous data showed that DNAJB6 and FG-Nups form partially colocalized condensates ( Kuiper et al , 2022 ; Bergsma et al , 2025 ), but it is unclear whether and how the self-association properties of both DNAJB6 and FG-Nups are important for their interaction and DNAJB6’s phase-modulatory activity towards FG-Nups. To explore this question, we examined the activity of DNAJB6 across a range of FG-Nup clients and conducted a targeted mutational and evolutionary analysis of the IDR of DNAJB6 (residues 105-188; DNAJB6-IDR). We also asked whether the ability to interact with and modulate the phase state of a range of FG-Nups is shared with similar JDPs, including DNAJB2 and DNAJB8. We find that DNAJB2, DNAJB6, and DNAJB8 interact with a range of FG-Nups delaying their aggregation. We show for DNAJB6 that this activity depends on the IDR which, unlike many other IDRs, is highly conserved in mammals. Our analysis of the stability and activity of DNAJB6-IDR mutants in vitro and in cells elucidates that the surveillance mechanism is encoded in this highly conserved, LARKS-rich disordered region which promotes the formation of stable gel-like chaperone assemblies. Results DNAJB6 can modulate a range of NupFG condensates To address the question if the activity of DNAJB6 in surveilling FG-Nups depends on the phase state of the FG-Nups, we used a panel of purified NupFG fragments. The fragments encode the ID region of the FG-Nups and we refer to them as NupFG (see methods for precise regions). We systematically analyzed the size, circularity, and intensity of large numbers of Nup60FG, Nup100FG, Nup116FG, Nup145NFG and Nup153FG particles formed in the presence and absence of DNAJB6. DNAJB6 itself also forms particles under the conditions tested ( Appendix Fig S1A ). We used two buffers, with and without the molecular crowder PEG (10%) and imaged at two timepoints, 1 hour and 24 hours ( Fig 1A ). Download figure Open in new tab Figure 1 DNAJB6 can modulate a range of NupFG condensates. (A) Representative images showing Nup60FG-5MF, Nup100FG-5MF, Nup116FG-5MF,Nup145FG-5MF and Nup153FG-5MF particles, formed in the absence or presence of 10% PEG3350 for 1h or 24h, in the absence or presence of DNAJB6-A594 (molar ratio 1:1). The total set was sorted and subdivided into three categories, based on the particle properties of the control condition and the type of mixtures formed between the different FG-Nup fragments and DNAJB6. *See Appendix Fig. 1B for rare cases where DNAJB6 and Nup100FG still co-localize. Scale bar, 1 µm. (B-D) Mean size, fluorescence intensity and circularity of particles belonging to category 1 (B), category 2 (C) and category 3 (D). (E-G ) Exemplary particle belonging to category 1-3, respectively, alongside line scan profile and median overlapping area. Scale bar, 1 µm. When comparing the properties of the NupFG particles formed in the absence and presence of DNAJB6, we discriminate three different behaviors. A first group of FG-Nups ( Fig 1A , category 1), which themselves only form few small round or irregularly shaped particles, bind to the larger DNAJB6 particles resulting in a change in the NupFGs particle properties: they become larger, less intense, and less circular and display a median overlap of 91% between the NupFG and DNAJB6 signals ( Fig 1B , E ; quantification of individual FG-Nups in Appendix Fig S1C–E ). A second group of NupFGs ( Fig 1A , category 2), which form larger particles in the absence of DNAJB6, also showed high overlap with DNAJB6 (median 90%), and these particles became smaller and more circular in the presence of DNAJB6 ( Fig 1C,F ; Appendix Fig S1C–E ). A third group of NupFGs ( Fig 1A , category 3) is rapidly condensating, forming large and intense particles on their own. In the presence of DNAJB6 these NupFG particles become smaller, less intense, and more circular ( Fig 1D,G ; Appendix Fig S1C–E ), and only show small regions of overlap with DNAJB6 (median 45%). Rather, DNAJB6 appears to wrap around these condensates, forming small contact sites at the surface, with some partial incorporation into the NupFG condensates. Combined, DNAJB6 can engage with all NupFG condensates, albeit in different manners depending on the characteristics of the NupFGs. Having observed that DNAJB6 can engage with a wide range of NupFG condensates with distinct particle properties, we aimed to test whether DNAJB6 could also engage with preformed NupFG particles. For this, we followed the dynamics of Nup100FG particles in time ( Fig 2A ). As expected, we see that with time the particles increase in intensity and size and become less circular. The addition of DNAJB6 after one hour resulted in a decrease in particle intensity and size ( Fig 2A-D ), confirming that DNAJB6 can also act on pre-formed Nup100FG particles. We tested several schemes of preforming Nup100FG or DNAJB6 particles before mixing ( Fig 2E ) and assessed the properties of the Nup100FG particles ( Fig 2F ) and the appearance of a 0,5% SDS-insoluble Nup100FG fraction which can be trapped in filter trap assays (FTA) ( Fig 2H ) ( Kuiper et al , 2022 ). Both assays revealed that Nup100FG particles showed a similar reduction in intensity and size and increase in circularity ( Fig 2G , Appendix Fig S2A-F ), and a similar reduction of Nup100FG aggregation, regardless of the timing of their mixing ( Fig 2I,J ). Download figure Open in new tab Figure 2. Preforming DNAJB6b and Nup100FG particles impacts DNAJB6 its ability to modulate FG-Nup phase transitions. (A) Representative images of Nup100FG-5MF particles formed in the presence of 10% PEG3350 and followed over time (in hours). Time of addition of DNAJB6 is indicated by the black arrow. Scale bar, 1 µm. (B-D) Mean fluorescence intensity (B), size (C) and circularity (D) of Nup100FG-5MF particles exemplified in (A), relative to the mean of the control at t0. The black-dotted lines indicate the time of addition of DNAJB6b. The bold lines plus shades show the median ± 95% CI of all measurements from two replicates. 100 particles were analysed for each time point for each of the independent replicate experiments. (E) Schematic diagram of mixing conditions for experiments depicted in (F-J). Red arrow indicates time of imaging. (F) Representative images showing Nup100FG-5MF particles in the absence or presence of DNAJB6-A594 (molar ratio 1:1). Numbers refer to conditions in (E). Scale bar, 1 µm. (G) Table showing overview of median particle size, intensity and circularity for each of the conditions exemplified in (F). A colour-gradient was applied to each of the assessed particle properties, representing the magnitude of change relative to the control condition, with lower values indicated in blue and higher values indicated in red. (H) Filter trap assay showing aggregated fraction of Nup100FG in the absence or presence of DNAJB6 (molar ratio 1:1). Numbers refer to conditions in (E). (I,J ) Quantification of the band intensities of Nup100FG on filter trap for each of the conditions exemplified in (H). Represented band intensities are relative to the average intensity of the control. Mean ± SEM (n=3). ***P<0.001, ****P<0.0001. Our systematic assessment of the condensation behavior of different NupFG fragments in combination with DNAJB6 shows that DNAJB6 interacts with all NupFG particles under all conditions, suggesting that the activity of DNAJB6 is not limited to a specific NupFG phase state. The ability of DNAJB6 and Nup100FG to efficiently engage in heterotypic interactions even when either of these proteins is preassembled further highlights their capacity to outcompete homotypic interactions and engage in heterotypic interactions. The phenylalanine residues in the S/T-rich region of DNAJB6 are essential for its ability to self-associate and modulate FG-Nup phase transitions In our previous work, we showed that the S/T-rich region of DNAJB6 is important for both its anti-amyloidogenic effects on polyQ ( Kakkar et al , 2016 ; Gillis et al , 2013 ) as well as for its action to prevent liquid-to-solid transitions of FG-Nups ( Kuiper et al , 2022 ). The canonical type B JDP, DNAJB1, that lacks such a region, can disaggregate polyQ as a trimeric complex together with HSP70 and HSP110 ( Scior et al , 2018 ; Nillegoda et al , 2015 ; Kuo et al , 2013 ), but it cannot independently act on polyQ fragments ( Månsson et al , 2014b ). Here, we show that purified DNAJB1 is also not effective at delaying the aggregation of FG-Nups ( Fig 3A,B ). However, when inserting the S/T-rich region from DNAJB6 C-terminal of the G/F-rich region of DNAJB1 (DNAJB1 DNAJB6-S/T ) ( Fig 3A,B ), it does gain anti-aggregation activity towards Nup100FG, albeit not to the same extent as wildtype DNAJB6 (DNAJB6 WT ) ( Fig 3C,D ). These results further highlight that the S/T region is critical for chaperoning FG-Nups. Download figure Open in new tab Figure 3. The S/T-rich region of DNAJB6 is critical for its function. (A) Schematic overview of the domain structure of DNAJB1, indicating the N-terminal J domain (green), followed by the G/F,G-M domain (pink), the the C-terminal domain (CTD) (grey) and the DD domain (blue). The S-T domain of DNAJB6 (yellow) is inserted immediately after the G/F-rich domain. (B) Predicted structure of DNAJB1 by Alphafold, with the arrow indicating the location where the S/T-rich domain of DNAJB6 is inserted. (C) Filter trap assay to assess aggregated fraction of Nup100FG-5MF in the absence or presence of either DNAJB6 WT , DNAJB1 WT or DNAJB1 DNAJB6-S/T . (D) Quantification of the band intensities of Nup100FG-5MF on filter trap. Represented band intensities are relative to the average intensity of the control. Mean ± SEM (n=3). **P<0.01, ***P<0.001. (E) Schematic overview of the domain structure of DNAJB6b, indicating the N-terminal J domain (green), followed by the G/F domain (pink), the S/T domain (yellow) and the C-terminal domain (CTD) (grey). The locations of the different mutations (18×S/T > A, 12×F > A) are indicated. (F) Predicted structure of DNAJB6 by Alphafold. (G) Representative images of wildtype and indicated DNAJB6 mutant (18x S/T > A, 12x F > A) particles, formed in the presence of 10% PEG3350 (1h). Scale bar, 1 µm. (H) Representative images showing Nup153FG-5MF particles in the absence or presence of either DNAJB6 WT , DNAJB6 18xS/T>A or DNAJB6 12xF>A particles, formed in the presence of 10% PEG3350 (1h) (molar ratio 1:1). Scale bar, 1 µm. (I-K) Mean fluorescence intensity (I), size (J) and circularity (K) (relative to the median of the control) of Nup153FG-5MF particles exemplified in (H) . Graphs show median ± interquartile range of ≥300 particles per condition (n=3). **P<0.01, ****P<0.0001. The S/T-rich region is enriched in S, T, and F residues that, in cellular experiments, were shown to be essential for the chaperoning capacity of DNAJB6 24,27 . To get insight in their loss of function features we purified mutants in which either 18 S and T residues were changed to alanine (DNAJB6 18xS/T>A ) or all 12 F residues were changed to alanine (DNAJB6 12xF>A ) ( Fig 3E ). We found that purified DNAJB6 18xS/T>A still self-associates, whereas DNAJB6 12xF>A completely lost this ability ( Fig 3G ). In line, DNAJB6 18xS/T>A still colocalizes with Nup153FG particles and reduces their size ( Fig 3H–K ), similar to DNAJB6 WT . In contrast, purified DNAJB6 12xF>A was unable to influence the size, intensity, or circularity of Nup153FG particles ( Fig 3H-K ), consistent with previous cellular data ( Kuiper et al , 2022 ). Consistent results were obtained for the interaction of both mutants with Nup100FG ( Appendix Fig S3A-D ). To corroborate these findings in cells, V5-tagged versions of both mutants were co-overexpressed with GFP-Nup153FG in DNAJB6 knockout cells (HEK293T DNAJB6–/– ). The size and circularity of the GFP-Nup153FG particles, as well as their detergent solubility were analyzed via microscopy and protein fractionation assays, respectively. Whereas overexpression of DNAJB6 18xS/T>A suppressed the formation of large and irregularly-shaped (possibly amyloidogenic) GFP-Nup153FG particles, overexpression of DNAJB6 12xF>A was unable to do so ( Fig 4A-C ). Similarly, DNAJB6 18xS/T>A , but not DNAJB6 12xF>A , suppressed the accumulation of GFP-Nup153FG in the SDS-insoluble fraction (P1) as efficiently as DNAJB6 WT ( Fig 4D,E ) . In line with our in vitro data, in cells DNAJB6 12xF>A also remains fully soluble, while a fraction of DNAJB6 WT is found in particles that are resistant to 0.5% NP40 (S2) ( Fig 4D,F ). So, also in cells the F residues in the S/T-rich region of DNAJB6 are essential for its ability to self-associate and modulate FG-Nup condensation and aggregation. Download figure Open in new tab Figure 4. The phenylalanine residues in the S/T-rich region of DNAJB6 are essential for its phase modulatory activity in cells and nuclear pore-associated function. (A) Representative images of HEK293T DNAJB6-/- co-transfected with GFP-Nup153FG in the absence or presence of V5-tagged DNAJB6b constructs (DNAJB6 WT , DNAJB6 18xS/T>A , or DNAJB6 12xF>A ). Scale bar, 20 μm. (B,C) Size (B) and circularity (C) of Nup153FG particles exemplified in (A) . Graphs show median ± interquartile range of combined particles from 3 independent replicates. (D) Representative western blot showing the different fractions from a protein fractionation assay performed in HEK293T DNAJB6-/- cells co-transfected with GFP-Nup153FG in the presence or absence of V5-tagged DNAJB6b constructs (DNAJB6 WT , DNAJB6 18xS/T>A , or DNAJB6 12xF>A ). (E) GFP-Nup153FG band quantification in the different fractions and conditions expressing V5-tagged DNAJB6b shown in (D) . Graphs show mean ± SEM (n=4). (F) V5-DNAJB6 band quantification in the different fractions and conditions expressing V5-tagged DNAJB6b shown in (D) . Graphs show mean ± SEM (n=4). (G) Representative images of HEK293T WT cells transfected with V5-tagged DNAJB6b constructs (DNAJB6 WT , DNAJB6 18xS/T>A , or DNAJB6 12xF>A ). Scale bar, 20 μm. (H) Quantification of MLF2 foci co-localized with DNAJB6 foci exemplified in (G) . Graphs show mean ± SEM (n=4). WCL: whole cell lysate. S1: 0.5% NP40 soluble. S2: 0.1% SDS soluble. P1: 2% SDS soluble. P2: 2% SDS insoluble. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 ****P<0.0001. To assess whether these two mutants impair DNAJB6’s function related to NPC assembly, we assessed their ability to accumulate in NE herniations. We quantified the co-localization of MLF2 (as herniation marker (Fischer et al )) and DNAJB6 foci in HEK293T WT cells overexpressing the different V5-tagged DNAJB6 constructs. The immunofluorescence experiments revealed that DNAJB6 18xS/T>A , but not DNAJB6 12xF>A , still accumulates into NE herniations ( Fig 4G,H ). Thus, DNAJB6’s ability to phase separate and delay the aggregation of FG-Nups is connected to its accumulation in NE herniations and, potentially, its function at the NPC. Combined, our data suggests that DNAJB6’s ability to interact with and delay the phase transitions of its various FG-Nup substrates depends on the phenylalanine residues in its S/T-region and its self-association ability. Specificity of DNAJB2, DNAJB6, and DNAJB8 for FG-Nups We next assessed whether the ability to interact with and modulate the phase state of a range of FG-Nups was particular to DNAJB6 or whether similar JDPs would share this ability. Bioinformatics analysis showed that the IDRs of the class B’ JDPs ( Malinverni et al , 2023 ) (DNAJB2, DNAJB6, DNAJB7, and DNAJB8) located between the regulatory helix and the C-terminal β-sheets (IDR1), are also enriched in F residues, similar to some FG-Nups ( Fig 5A ). Using the NARDINI algorithm ( Cohan et al , 2022 ) to assess the binary amino acid distribution and content in a given sequence, we find that there are minor differences in their polar and glycine residue distribution, but they all exhibit a random arrangement of aromatic residues ( Appendix Fig S4A ). Clustering analysis showed that the IDR1 from DNAJB2, DNAJB6, and DNAJB8, but not DNAJB7, closely clustered together based on their amino acid distribution and composition ( Appendix Fig S4A ), which prompted us to test whether DNAJB2 and DNAJB8 could also display phase modulatory activities towards different FG-Nups ( Fig 5B ). Download figure Open in new tab Figure 5. Specificity of DNAJB2, DNAJB6, and DNAJB8 for FG-Nups. (A) Z-scores for phenylalanine (F) fractions in the IDRs of class B’ JDPs and human FG-Nups. Dotted lines indicate the -1.96 and +1.96 boundaries for the random distribution. The IDRs from class B’ JDPs located between the regulatory helix and the C-terminal β-sheets are highlighted in bold. (B) Schematic overview of the domain structure of DNAJB6, DNAJB2 and DNAJB8, indicating the N-terminal J domain (green), followed by the G/F domain (pink), the S/T domain (yellow) and the C-terminal domain (CTD) (grey). (C-E) Filter trap assay to assess aggregated fraction of Nup100FG (C), Nup116FG (D) and Nup153FG (E) in the absence or presence of either DNAJB6, DNAJB2 or DNAJB8 at a molar ratio of either 1:10 or 1:1 (Nup100FG (3µM), Nup153FG (6µM) (1h), Nup116FG (6µM) (3h)). (F-H) Quantification of the band intensities of Nup100FG (F), Nup116FG (G) and Nup153FG (H) on filter trap. Represented band intensities are relative to the average intensity of the control. Mean ± SEM (n=3). *P<0.05, ****P<0.0001. (I-K) Representative images showing Nup100FG-5MF (I), Nup116FG-MF (J) and Nup153FG-MF particles (K), in the absence or presence of either DNAJB6b, DNAJB8 or DNAJB2a particles, formed in the presence of 10% PEG3350 (1h) (molar ratio 1:1). Scale bar, 1 µm. (L) Line scan profiles showing interaction profiles for representative images of Nup116FG in the presence of DNAJB6, DNAJB8 and DNAJB2, respectively. (M) Graph showing the median overlapping area for Nup100FG, Nup116FG, and Nup153FG with DNAJB6, DNAJB8, and DNAJB2. (N) Table showing overview of median particle size, intensity and circularity for each of the conditions exemplified in (I-K). A colour-gradient was applied to each of the assessed particle properties, representing the magnitude of change relative to the control condition, with lower values indicated in blue and higher values indicated in red. Like DNAJB6, both delay the aggregation of Nup100FG, Nup116FG and Nup153FG ( Fig 5C-H ) with some specificity: while DNAJB8 was found to be most effective against aggregation of Nup100FG at both tested ratios (NupFG:chaperone 1:0.1 and 1:1) ( Fig 5C,F ), DNAJB2 was found to be most effective against aggregation of Nup153FG ( Fig 5E,H ). Microscopy assessment showed that whilst DNAJB6 and DNAJB8 wrap around NupFG condensates forming small contact sites at the surface, as well as partially incorporate into Nup116FG and Nup153FG condensates, DNAJB2 fully partitions into all FG-Nup condensates ( Fig 5I-M , Appendix Fig S4E,F ). Combined, these findings indicate that DNAJB6, DNAJB8, and DNAJB2 all possess the capacity to self-associate and to engage with FG-Nups to modulate their phase state, but with some degree of substrate specificity. Scrambling the primary amino acid sequence of DNAJB6-IDR affects the solubility and self-association behavior of DNAJB6 In addition to the specific amino acid composition, the particular distribution of amino acids within IDPs has been demonstrated to influence both condensation properties and function ( Martin et al , 2020 ). In line with this, we aimed to determine the sequence features that may dictate the self-association behavior and phase modulatory activity of DNAJB6. We noticed that the IDR from DNAJB6 (residues 105-108; DNAJB6-IDR) displays six binary combinations from its polar, aromatic, and glycine residues, which do not organize in any specific pattern, but are rather randomly distributed ( Appendix Fig S4A ). A binary amino acid distribution is random if the z-score falls between -1.96 and +1.96, segregated if > +1.96, or uniformly distributed if < -1.96. Of note, the DNAJB6-IDR also comprises a few charged residues that concentrate in the beginning of the sequence. However, these are just below the 10% threshold from the NARDINI algorithm, and their distributions are, therefore, not computed. To probe whether maintaining such random distribution is sufficient to maintain the self-association behavior and activity of DNAJB6, we used the NARDINI algorithm to generate three scrambled sequences with a z-score matrix that is most similar to the query sequence (SCR1, SCR2, and SCR3) while less than 15% of the amino acids are kept in their original position ( Fig 6A,B ). Two additional mutants (rL1 and rL2) were generated from the disordered S/T-rich region only (residues 136-188) without controlling for their random distribution ( Fig 6A ). In DNAJB6 rL1 the residues were completely scrambled using a random sequence generator, which unintentionally introduced the clustering of polar-polar residues, while the other residues were kept randomly distributed ( Fig 6A,B ). In DNAJB6 rL2 we aimed to test the importance of the pairing of F’s and G’s, a feature that is characteristic for FG-Nups, so we deliberately split them apart ( Fig 6A,B ). As expected, DNAJB6 rL2 shows a significant segregation of aromatic and glycine residues. However, this splitting also influenced the other binary combinations involving aromatic and glycine residues, as evidenced by the segregation of polar-aromatic and aromatic-aromatic residues, and the uniform distribution of polar and glycine residues ( Fig 6B ). Download figure Open in new tab Figure 6. Scrambling the primary amino acid sequence of DNAJB6-IDR affects the solubility and self-association behavior of DNAJB6. ( A ) Primary amino acid sequences of DNAJB6 WT (residues 105-188 or 112-195) and mutants DNAJB6 rL1 , DNAJB6 rL2 , DNAJB6 SCR2 , DNAJB6 SCR3 and DNAJB6 SCR1 coloured by amino acid group. ( B ) Heatmap of z-score matrices from NARDINI algorithm showing binary amino acid distributions of the primary amino acid sequences shown in ( A ). Significantly positive (red; z-score > +1.96) values reflect that the pairs of types of amino acids have a non-random segregation from one another and from other residues, and significantly negative (blue; z-score < -1.96) values features non-random uniform dispersion of residues. ( C ) Filter-trap assay and quantification of band intensities to assess aggregated fraction of DNAJB6 WT , DNAJB6 rL1 , DNAJB6 rL2 , DNAJB6 SCR1 , DNAJB6 SCR2 , and DNAJB6 SCR3 . ( D ) Quantification of particles formed by DNAJB6 WT and mutants (DNAJB6 18xS/T>A , DNAJB6 rL1 , DNAJB6 rL2 , DNAJB6 SCR1-3 ); representative images in Appendix S5A . (n=2). The five mutants were purified, and their particles were compared to those formed by DNAJB6 WT . DNAJB6 SCR2 and DNAJB6 rL2 formed particles with similar size and sensitivity to treatment with 5% 1,6-HD, and 0,5% SDS as the wildtype protein. The particles formed by DNAJB6 SCR3 and, in particular, DNAJB6 rL1 were however larger and partly resistant to 0,5% SDS ( Fig 6C,D , Appendix Fig S5A ). Imaging also showed some SDS-resistant DNAJB6 SCR1 particles but those were small ( Appendix Fig S5A ) and likely therefore not detected in the FTA ( Fig 6C,D ). We conclude that DNAJB6 rL1 and DNAJB6 SCR3 are particularly unstable (i.e. forming a 0,5% SDS-insoluble fraction) under the conditions tested while DNAJB6 SCR1 is somewhat unstable. The observation that several of the mutants were less stable than the wildtype shows that maintaining a random amino acid distribution within the IDR of DNAJB6 does not suffice to maintain stable gel-like assemblies. Scrambling the primary amino acid sequence of DNAJB6-IDR more strongly impairs anti-aggregation of polyQ than FG-Nups Next, we compared the functionality of the well-behaving DNAJB6 SCR2 , DNAJB6 rL2 and partial unstable DNAJB6 SCR1 mutant with DNAJB6 WT . In vitro , all mutants displayed similar phase modulatory activity towards Nup100FG and Nup153FG compared to DNAJB6 WT ( Fig 7A,B , Appendix Fig S5B-G ). Yet, biochemical assays showed a partial loss of function for DNAJB6 SCR2 and DNAJB6 rL2 , and a more pronounced loss for DNAJB6 SCR1 , which was unable to reduce Nup100FG aggregation ( Fig 7C-E ). The mutants DNAJB6 SCR1 and DNAJB6 SCR2 were also assessed using the protein fractionation assay in HEK293T DNAJB6-/- cell lysates. Cells overexpressing GFP-Nup153FG only showed accumulation of the NupFG in the P1 and P2 fractions, indicative of its aggregation ( Fig 7F ). In line with our previously published work ( Kuiper et al , 2022 ), the co-overexpression of V5-DNAJB6 WT largely prevented this ( Fig 7F,G ). Expression of either DNAJB6 SCR1 or DNAJB6 SCR2 delayed the aggregation of GFP-Nup153FG to the same extent, indicating that the partial loss of function observed in vitro was not detected in cells under the conditions tested ( Fig 7F,G ). Yet, we did observe that relatively more DNAJB6 SCR1 , but not DNAJB6 SCR2 , was found in the NP40-insoluble S2 fraction, potentially reflecting its altered self-association behavior observed in vitro ( Fig 7F,H ). So, DNAJB6 SCR1 , DNAJB6 SCR2 , and DNAJB6 rL2 have altered self-association behavior with, as revealed in vitro, partially impaired functionality. Download figure Open in new tab Figure 7 Scrambling the primary amino acid sequence of DNAJB6-IDR results in altered phase modulatory activity depending on the condensation properties of the substrate. ( A ) Representative images showing Nup100FG-5MF and Nup153FG-5MF particles in the absence or presence of either DNAJB6 WT , DNAJB6 rL2 , DNAJB6 SCR1 or DNAJB6 SCR2 (1h) (molar ratio 1:1). Scale bar, 1 µm. ( B ) Table showing overview of median particle size, intensity and circularity for each of the conditions exemplified in (A). A colour-gradient was applied to each of the assessed particle properties, representing the magnitude of change relative to the control condition, with lower values indicated in blue and higher values indicated in red. ( C ) Filter trap assay to assess aggregated fraction of Nup100FG-5MF and Nup153FG in the absence or presence of either either DNAJB6 WT , DNAJB6 rL2 , DNAJB6 SCR1 or DNAJB6 SCR2 . (D-E) Quantification of the band intensities of Nup100FG-5MF (D) and Nup153FG-5MF (E) on filter trap. Represented band intensities are relative to the average intensity of the control. Mean ± SEM (n=4). ( F,I ) Representative western blots showing the different fractions from a protein fractionation assay performed in HEK293T DNAJB6-/- cells co-transfected with GFP-Nup153FG (F) or GFP-Q71 (I) and V5-tagged DNAJB6b constructs (DNAJB6 WT , DNAJB6 SCR1 or DNAJB6 SCR2 ). WCL: whole cell lysate. S1: 0.5% NP40 soluble. S2: 0.1% SDS soluble. P1: 2% SDS soluble. P2: 2% SDS insoluble. ( G,J ) GFP-Nup153FG (G) or GFP-Q71 (J) band quantification in the different fractions and conditions expressing V5-tagged DNAJB6 shown in (F,I). ( H,K ) V5-DNAJB6 band quantification in the different fractions and conditions expressing V5-tagged DNAJB6 shown in (F,I). Graphs show mean ± SEM (n=4/5). ( L ) GFP-Nup153FG and GFP-Q71 band quantification in the different fractions of the control condition shown in (F,I). Graphs show mean ± SEM (n=4). *P<0.05,**P<0.01, ***P<0.001. In the search for a substrate that might better reveal functional differences between DNAJB6 WT and the scrambled mutants in cells, we turned to testing their effect on the solubility of GFP-tagged Huntington exon 1 with a 71-polyQ stretch (GFP-Q71). This choice was instigated by our finding that DNAJB6 18xS/T>A showed unimpaired FG-Nup anti-aggregation activity ( Fig 3 , 4 ), while it has reduced polyQ anti-aggregation activity ( Kakkar et al , 2016 ). In cells, the rate of liquid-to-solid phase transitions was faster for polyQ proteins than for FG-Nups, with polyQ appearing in the 2% SDS insoluble P2 fraction of control conditions (compare Fig 7F,I , quantified in Fig 7L ). Interestingly, both the partially unstable DNAJB6 SCR1 and the stable DNAJB6 SCR2 showed impaired polyQ anti-aggregation activity compared to DNAJB6 WT , as indicated by the increased aggregate levels in the P2 fraction ( Fig 7I,J ). Like in the experiments with Nup153FG ( Fig 7H ), relatively more DNAJB6 SCR1 partitions to the S2 fraction ( Fig 7K ), underscoring that its altered stability is not related to the co-overexpressed substrate. Most importantly, these findings suggest that even if the optimal gel-like state of DNAJB6 is preserved (like in DNAJB6 SCR2 ), its phase state modulatory activity is partially impaired. Together, our data points to a unique arrangement of amino acids in the DNAJB6-IDR which is essential for the stability of the chaperone and its anti-aggregation activity. The primary amino acid sequence of DNAJB6-IDR is highly conserved among mammals Prompted by the idea that both the composition and arrangement of amino acids in the DNAJB6-IDR affect its stability and/or function, we decided to investigate the conservation of the primary amino acid sequence of this region in a curated dataset of 57 mammal species. For comparison, we included two closely related B’ class JDPs: DNAJB2 and DNAJB8, as well as an unrelated, but well-studied IDP: P53 ( Fig 8A ). The details on the selection of each protein’s IDR and folded domain are described in the methods section, and the DNAJB6-IDR is here referred to as IDR1. Multiple sequence alignments (MSAs) of the whole protein, folded domain, and IDRs were performed, and the residue conservation scores (maximum = 4.3, based on 20 amino acids) were calculated based on Shannon’s entropy, which quantifies variability at each alignment position. As expected, the folded domains showed the highest conservation scores, which were averaged to 4.0 and used as a visual reference score for highly conserved residues ( Fig 8B,C ). Interestingly, the IDR1 of DNAJB2, DNAJB6, and DNAJB8 showed significantly higher conservation compared to that of P53, and both DNAJB2 and DNAJB6 were notably more conserved in their IDR1 regions than DNAJB8 ( Appendix Fig S6C ). Given that DNAJB2 and DNAJB6 also contain a C-terminal IDR (IDR2), we included this region in our analysis for further comparison. Surprisingly, the conservation scores for IDR2 were considerably lower than those for IDR1, with DNAJB6’s IDR2 being significantly less conserved than that of DNAJB2 ( Fig 8B and Appendix Fig S6D ). Aromatic and glycine residues were among the residues with the highest conservation scores in IDR1 of DNAJB2, DNAJB6, and DNAJB8 ( Fig 8D ). Consistent with IDRs generally showing conserved amino acid composition rather than position ( Brown et al , 2010 ; Zarin et al , 2019a , 2019b ; Nguyen Ba et al , 2012 ; Langstein-Skora et al ), we find that this is also the case for the IDR1 of P53, DNAJB2, DNAJB6, and DNAJB8 ( Appendix Fig S6E-H ).These findings indicate that the IDR1 regions of DNAJB2, DNAJB6, and DNAJB8 are subject to stronger evolutionary pressures compared to a typical IDR, such as the one found in P53. For DNAJB6, this result corroborates the idea that maintaining the specific amino acid arrangement of IDR1 is crucial for achieving optimal stability and function. Download figure Open in new tab Figure 8 Evolutionary analysis of DNAJB6-IDR shows high primary amino acid sequence conservation. ( A ) Alphafold structures of DNAJB2, DNAJB6, DNAJB8, and P53 coloured by region (legend on panel C). Graph shows median with interquartile range. ( B ) Conservation scores from each region of DNAJB2, DNAJB6, DNAJB8, and P53 (E). ( C ) Average conservation score per region, per protein, from data shown in (B). Graph shows mean with 95% CI. Solid line at 4.3 indicates the maximum score based on Shannon’s entropy. Dashed line at 4.0 indicates average conservation score of all folded domains. *P<0.05, **P<0.01, ****P 4. Discussion DNAJB6 displays remarkable anti-aggregation activity towards several disease-related IDPs ( Kakkar et al , 2016 ; Månsson et al , 2014b ; Hageman et al , 2010 ; Thiruvalluvan et al , 2020 ; Deshayes et al , 2019 ; Gillis et al , 2013 ; Aprile et al , 2017 ; Österlund et al , 2020 ), and recently has also been found to surveil the phase transitions of the intrinsically disordered FG-Nups ( Kuiper et al , 2022 ; Bergsma et al , 2025 ; Prophet et al , 2022 ). Here we show that the surveillance mechanism of DNAJB6 towards multiple FG-Nups is encoded in a surprisingly highly conserved, disordered region which promotes the formation of stable gel-like chaperone assemblies. Such assemblies provide an environment that enable low-affinity, dynamic heterotypic interactions, most likely between the phenylalanines in the DNAJB6-IDR and those in the FG-Nups. We argue these heterotypic interactions outcompete the homotypic FG-FG interactions of substrates, thereby delaying their aggregation. The evolutionary conservation of the DNAJB6-IDR, and our mutant analyses suggests that the sequence space for encoding stable gel-like assemblies is limited and finely tuned to balance a high local avidity while avoiding self-aggregation of the chaperone. Of note, the size and formation mechanism of such chaperone assemblies in the physiological cellular context remains to be resolved. Our work contributes to the view that molecular chaperones, beyond their classical role in protein (re)folding, are regulators of condensate dynamics ( Akaree et al, 2025 ). Gel-like DNAJB assemblies offer a protective environment against FG-Nup aggregation Previous work showed that the ID S/T-rich region of DNAJB6 is critical for its activity towards FG-Nups and polyQ ( Kuiper et al , 2022 ; Kakkar et al , 2016 ). Here we substantiate this by showing that engineering the S/T region into DNAJB1 creates a hybrid chaperone (DNAJB1 DNAJB6-S/T ) that gains activity towards Nup100FG. Moreover, we showed that the F residues in this region are essential for its self-association into a gel-like state. This is consistent with recent findings from the Linse Group ( Carlsson et al , 2025 ) showing that this region, and not the beta-sheet in the CTD ( Cawood et al , 2022 ), is the primary driver of the self-assembly of full length DNAJB6. Our systematic analysis indicates that DNAJB6 interacts with and displays phase modulatory activity towards many FG-Nups with different condensation properties, both those that rapidly form gel-like condensates and those that are more soluble. DNAJB6 can even modify pre-assembled Nup100FG condensates making them smaller. The mechanism might involve disrupting the FG-Nup:FG-Nup intermolecular interactions, and/or acting as a physical barrier to prevent the coalescence and subsequent growth of NupFG condensates into larger structures. We additionally show that the closely related B’ JDPs, DNAJB2 and DNAJB8, also exhibit the ability to interact with and delay FG-Nup aggregation, and that their anti-aggregation efficiencies vary depending on the specific NupFG fragments involved. Despite being all LARKS-containing proteins ( Hughes et al , 2018 ), we note that the number of LARKS motifs encoded in the IDRs of DNAJB6 and DNAJB8 (19) is higher than that of DNAJB2 (9). Future research might answer if this difference explains why DNAJB6 and DNAJB8 form homotypic assemblies which only partly colocalize with NupFG condensates, while DNAJB2 forms mixed DNAJB2-NupFG condensates. Sequence-encoded stability and evolutionary tuning The dense gel-like phase of DNAJB6 presents a challenge: the high local concentration of residues capable of multivalent interactions inherently risks self-aggregation, which is incompatible with the dynamic properties required for chaperoning substrates. How is this encoded in the amino acid sequence? The DNAJB6-IDR resembles the sticker-and-spacer distribution in prion-like domains (PLDs), which are enriched in polar residues and are often interspersed by aromatic residues ( Brangwynne et al , 2015 ). It has been proposed that the strong interactions among aromatic residues (stickers) are weakened by the favorable solvation of polar residues (spacers) ( Brangwynne et al , 2015 ). In line with this, it has been shown that the uniform distribution of aromatic residues along PLDs favors solubility and LLPS over aggregation ( Martin et al , 2020 ). In fact, a proteome-wide analysis revealed that aromatic residues within human PLDs enriched in aromatic residues (> 10%) tend to be patterned in a non-random, uniform manner ( Martin et al , 2020 ). Interestingly, we find that DNAJB6-IDR is highly conserved in mammals, as well as the IDRs of the closely related B’ class JDPs DNAJB2 and DNAJB8. The high degree of primary amino acid sequence conservation of the DNAJB6-IDR is a remarkable feature considering that IDRs are generally known to conserve amino acid composition rather than position ( Brown et al , 2010 ; Zarin et al , 2019a , 2019b ; Nguyen Ba et al , 2012 ; Langstein-Skora et al ), exemplified here with p53, but demonstrated for several other IDPs ( Cohan et al , 2022 ; Zarin et al , 2019a , 2017 ; Beh et al , 2012 ; Moesa et al , 2012 ). Based on our mutational analysis, we speculate that altering the order of amino acids or the composition of the IDR may impede substrate interactions in two possible manners: either by primarily affecting DNAJB6 self-association kinetics (i.e. DNAJB6 SCR1-3 ) and/or by removing substrate interaction sites (i.e. DNAJB6 18xS/T>A and DNAJB6 12xF>A ). The latter likely not only alters its substrate affinity, and thus its ability to engage in heterotypic interactions, but may simultaneously alter its self-association properties. In line, in vitro data have shown that the association-dissociation dynamics of DNAJB6 are key to its ability to suppress Aβ fibril formation( Carlsson et al , 2024 ). Our mutational analysis further revealed that the stability of DNAJB6 is easily compromised when altering the IDR amino acid sequence (while keeping the composition) as the mutants DNAJB6 SCR3 and in particular DNAJB6 rL1 form 0,5% SDS insoluble aggregates. Possibly the density and/or distribution of LARKS motifs is altered. Even those DNAJB6 variants that we created that maintained both the amino acid composition and distribution of the wildtype IDR, are not all fully functional. While DNAJB6 18xS/T>A and DNAJB6 SCR2 most closely resemble DNAJB6 WT in terms of the characteristics of the gel-like assemblies that they form, they both reveal differences in their activity. Particularly the fast-aggregating polyQ reveals the functional consequences of the subtle changes in these mutants, possibly because this substrate, like suggested for Aβ ( Carlsson et al , 2024 ), may be especially dependent on rapid and efficient release of monomeric DNAJB6 from the gel-like assemblies, whereas the more stable, gel-like behavior of the FG-Nups may tolerate minor changes in DNAJB6 dynamics as they rather depend on the interactions with DNAJB6 gel-like assemblies. This could also explain the much lower in vitro stoichiometry ratios required for DNAJB6 to prevent polyQ or Aβ aggregation (1:10-100) ( Kakkar et al , 2016 ; Månsson et al , 2014b , 2014a ) than required for preserving FG-Nup disorder (1:1) observed here and previously ( Kuiper et al , 2022 ). Physiological relevance and broader implications We previously showed a role for DNAJB6 in the surveillance of FG-Nups in the timeframe between synthesis and assembly into NPCs, and confirmed the interaction between endogenous DNAJB6 and FG-Nups in the nucleus and cytoplasm of cells ( Kuiper et al , 2022 ). Extrapolating from our in vitro data – with unnaturally large assemblies – we speculate that in cells, these proteins interact in small, possibly gel-like DNAJB6:FG-Nup condensates. The strong self-association behavior of DNAJB6 may also be implicated in pathological contexts such as dystonia, in which DNAJB6-marked NE herniations become a sink for protein quality control components, potentially contributing to cellular dysfunction ( Prophet et al , 2022 ). In this context, we speculate that the strong capacity of DNAJB6 to form gel-like assemblies contributes to the irreversible recruitment of proteins into NE herniations. Gel-like assemblies are not unique to DNAJB6; they are also a feature of other protective cellular assemblies ( Riback et al , 2017 ; Kroschwald & Alberti, 2017 ; Protter & Parker, 2016 ; Law et al , 2023 ; Ali et al , 2023 ). Future research may reveal that such gels not always represent a transition to a non-functional state, but may more generally represent a stable protective state, combining high substrate avidity with dynamic exchange, thus preventing irreversible protein sequestration. Finally, our current and previous findings that certain chaperones evolved to maintain protein disorder ( Kuiper et al , 2022 ; Pechmann & Frydman, 2014 ; Reichmann et al , 2012 ; Macošek et al , 2021 ; Akaree et al , 2025 ) conceptually changes the view on the function of chaperones and extends it beyond their role in supporting and maintaining polypeptides folding into ordered domains. Methods Protein purification The expression and purification of yeast and human FG-Nup fragments (yNup60FG aa389-511 , yNup100FG aa1-580 , yNup116FG aa1-725 , yNup145FG aa1-219 , hNup153 aa875–1475 ) and J-domain proteins (DNAJB1, DNAJB2a, DNAJB6b, DNAJB8 and mutants DNAJB6 18xS/T>A , DNAJB6 12xF>A , DNAJB1 DNAJB6-S/T , DNAJB6 rL1 , DNAJB6 rL2 , DNAJB6 SCR1 , DNAJB6 SCR2 , DNAJB6 SCR3 ) were performed as described before ( Kuiper et al , 2022 ). In short, the pSF350 expression plasmid, containing a His6-tag at the N-terminal end and a cysteine residue at the C-terminal end was used for expression of the listed FG-Nup fragments and full-length JDPs ( Table 1 ). For cell lysis and protein purification, a 100 mM Tris-HCl, 2M guanidine-HCl, pH 8.0 buffer was used. For labelling, the FG-Nup fragments were incubated with fluorescein-5-maleimide (5MF, Thermo-Scientific, 62,245) and JDPs with Alexa fluor 594 C5-maleimide (AF594, Alexa fluor 594 C5-maleimide, Thermo-Scientific, A10256). Proteins were concentrated using Vivaspin Protein Concentrator spin columns (Vivaspin 10/30 kDa MWCO Polyethersulfone, Cytiva, 28–9322–47, 28–9322–48) and stored at −80 °C at a final concentration of 100 μM in 100 mM Tris-HCl 2M guanidine-HCl, pH 8.0 buffer, supplemented with 10% glycerol. To reduce the impact of the fluorescent labels on the protein behavior, the fluorescein-5-maleimide-labeled FG-Nup fragments and Alexa fluor 594 C5-maleimide-labelled JDPs were mixed with unlabeled proteins at the ratios indicated in Table 2 . View this table: View inline View popup Table 1 Plasmids. View this table: View inline View popup Download powerpoint Table 2 Fluorescent maleimide labelling concentrations. Sample preparation for phase separation assays To assay phase separation behavior, the purified proteins were diluted to a protein concentration of either 3 μM or 6 μM in assay buffer in low-protein-binding tubes. The assay buffer comprises 50 mM Tris-HCl, 150mM NaCl, pH8.0, supplemented with either no crowding agent or 10% w/v of polyethylene glycol 3350 (PEG3350; Sigma-Aldrich, P4338-2 KG) for varying amounts of time, as indicated in figure legends, at room temperature. To assess the effect of the different JDPs and corresponding mutants on FG-Nup particle properties, 3 μM of 5MF labeled FG-Nup fragments and AF594 labeled JDPs were mixed at a 1:1 ratio, and left to phase separate for 1 h at room temperature, unless stated otherwise in the figure legends. To assess the hexanediol (HD) and SDS sensitivity of the different JDPs, particles were left to phase separate for 1 h, followed by exposure to either 5% 1,6-HD or 0.5% SDS for 10 min. For the Nup100FG:DNAJB6 time course experiment, Nup100FG particle formation was followed over a three-and-a-half-hour timeframe, with 30 min time-intervals, in assay buffer containing 10% PEG3350, either in the absence or upon addition of DNAJB6 after 1 h of particle formation. For the experiments where we either let the FG-Nup or DNAJB6 pre-form particles prior to mixing, 3 μM of 5MF labeled FG-Nup fragments (Nup100FG or Nup153FG) and AF594 labeled DNAJB6 were either mixed from the start (1:1 ratio), or independently left to phase separate for either 30 min or 1 h at room temperature prior to mixing. After mixing, the samples were left for an additional hour, prior to microscopy or FTA assessments. Fluorescence microscopy in in vitro assays For imaging of in vitro purified proteins, 2 μl samples of phase separated protein mixtures were mounted on untreated glass-slides with coverslip. Microscopy was performed in a temperature-controlled environment at 20 °C using a DeltaVision Deconvolution Microscope (Cytiva), using an Olympus UPLS Apo 100× oil-immersion objective (NA 1.4) and InsightSSITM Solid State Illumination using the FITC 525/48 and A594 625/45 filter sets. Detection was done with a either a CoolSNAP HQ2 or EDGE sCMOS5.5 camera. Fluorescence images were acquired with 30 Z-slices of 0.2 μm. Images were deconvolved using softWoRx software (Cytiva) and processed using the open-source software FIJI/ImageJ. Image analysis For the analysis of in vitro and in cells particle properties, the PhaseMetrics plugin was used, as described in ( Bergsma et al , 2025 ). PhaseMetrics performs automatic detection and analysis of several particle properties, including particle intensity, size and circularity. A maximum intensity Z-projection is used for creating a segmented image for object detection, after which the object masks are re-directed to the sum of slices Z-projection to extract the desired measurements. To compute the overlapping area between FG-Nup and JDP particles, the object-based colocalization module was run to determine the intersected regions between the FG-Nup and JDP fluorescent signal. The accompanying “Colocalization analysis” excel macro was subsequently used to extract relevant colocalization-based measurements from the exported results table. Filter trap assays For filter trap assays (FTA), protein samples were prepared as explained above, after which samples were added to 180 μl of sample buffer supplemented with 0.5% SDS and mixed by vortex before loading onto the Bio-Dot apparatus (Bio-Rad). FTAs were performed as described before ( Kuiper et al , 2022 ). After this, membranes were blocked with 2.5% BSA in PBS-T (0.1%), incubated overnight with mouse primary antibody anti-His (1:5000, monoclonal mouse Tetra-His antibody, Qiagen, #34670), washed three times with PBS-T, incubated for 1h with secondary anti-mouse m-IgGk BP-HRP protein (Santa Cruz, sc-516102) (1:2500) and washed three times with PBS-T. Chemiluminescence was detected using enhanced chemical luminescence (ECL) reagent using the Chemidoc imaging system (BioRad). For the quantification of the FTA, band intensities were measured using FIJI and expressed relative to the average intensity of the control. Cell lines, cell culture, and transient transfections HEK293T cells (Thermo Fisher) and HEK293T DNAJB6-/- cells (see ( Thiruvalluvan et al , 2020 ) for details on cell line generation) were maintained according to standard protocols. The cells were cultured in DMEM (Gibco) supplemented with 10% FCS (Greiner Bio-One) and penicillin/streptomycin (Invitrogen). For transient transfections, cells were grown to 70–80% confluence in a 37°C incubator with 5% CO2 and seeded into 6-well plates or 8-well chambered cover glass (Cellvis; C8-1.5H-N). For microscopy experiments requiring 6-well plates, coverslips coated with 0.0001% poly-l-lysine (Sigma) were used as the cell-seeding surface. Used siRNAs, all from Dharmacon/Horizon: SMARTpool siGENOME non-targeting (D-001206-13-20), SMARTpool Accell DNAJB6 siRNA (E-013020-00-0005), ON-TARGETplus TOR1A siRNA (J-011023-05-0002), ON-TARGETplus TOR1B siRNA (J-014203-09-0002), ON-TARGETplus TOR2A siRNA (J-015292-09-0002), ON-TARGETplus TOR3A siRNA (J-017824-11-0002). Gene cloning and generation of mutants Details on the design and construction of V5-DNAJB6 WT and V5-DNAJB6 18xS/T>A constructs are available in ( Hageman et al , 2010 ) and ( Kakkar et al , 2016 ). The GFP-Nup153FG construct is described in ( Kuiper et al , 2022 ). The V5-DNAJB6 12xF>A construct was created via site-directed mutagenesis using V5-DNAJB6 WT as the template and several mutagenesis rounds with different primer pairs. V5-DNAJB6 SCR1 , V5-DNAJB6 SCR2 , and V5-DNAJB6 SCR3 mutants were made by enzyme digestion of V5-DNAJB6 WT and insertion of gBlocks (IDT) with mutations in the DNAJB6 sequence (amino acids 105-188): SCR1: TSFSFSFFFHGSFFFSGNNDFTGSRTSRFRFSGGTSTTSFFLESGFMEFSSGIGFRKGDGFPSGGS GDGSLDTAGFGPRSPSSG SCR2: SGPMGFGFSLTGGSRFTSFRFLEGSFGSPFNFFHSGRGPSGFGFRRFSSFDDSTSGFSDGAGGS GTPRSEGDFFDFNFGTFSS SCR3: SDGEDRDTPFSGFGFGRESFHGSGFFFPGTFNFPSFGSRTFTPRSFSFGDGASGLNSGSGGGGT SSGFSLSGDFRRFMFSFSF The DNAJB6 rL1 and DNAJB6 rL2 mutants were generated using the RandSeq tool from ExPASy and ordered using GeneArt. Codon usage was optimized for the entire sequence in GeneArt. In DNAJB6 rL1 the residues in the S/T-rich region were completely scrambled, and in DNAJB6 rL2 the F and G residues were deliberately separated (amino acids 105-188). rL1 (randomized sequence): GRDPFSFDFFEDPFEDFFGNRRGPRGSRSRTFGGGFLPGFSSSSGDFSFTSGTSGSSFFAMFGG TTSSHFSLGFGSNGSFGGF rL2 (randomized sequence): GRDPFSFDFFEDPFEDFFGNRRGPRGSRSRGSGSGSTFFAFPFFTFDGGSTFFLSGGHSSGGSLF MGSSGSTGSGSSGTFNFF DNAJB1 DNAJB6 S/T (DNAJB1 flanks; DNAJB6 S/T region ): GFPMGMGGFTNVNFGRSRSAQEPA GTGSFFSAFSGFPSFGSGFSSFDTGFTSFGSLGHGGLTSF SSTSFGGSGMGNF RKKQDPPVTHDLRVSL Gibson assembly was used to replace the DNAJB6 WT S/T region with the generated randomized sequences, and to insert the S/T-rich region from DNAJB6 immediately after the G/F-rich region of DNAJB1. Immunofluorescence Cells were rinsed with PBS to remove residual medium and fixed for 20 minutes using 4% paraformaldehyde (PFA) in PBS at room temperature (RT). After two washes with PBS, cells were permeabilized with 0.5% Triton X-100 for 15 minutes at RT and washed again with PBS. Subsequently, cells were washed twice with PBS+ (PBS supplemented with 0.5% bovine serum albumin and 0.15% glycine) before incubation with 80–200μl of primary antibodies diluted in PBS+ for 1 hour at RT or overnight at 4°C. Following four washes with PBS+, cells were treated with Alexa488-, Alexa594-, or Alexa633-conjugated secondary antibodies (Thermo Fisher Scientific) for 1.5–2 hours at RT in PBS+. Nuclei were stained using Hoechst 33342 dye (Life Technologies) at a 1:2,000 dilution in PBS for 5–10 minutes at RT. Finally, cells were washed twice with PBS and mounted using 80% glycerol or Mowiol® 4-88 (Carlroth) for imaging. For cells seeded in 8-well chambered cover glass, they were stored in PBS at 4°C until imaging. Image acquisition Images were acquired with Leica SP8 confocal microscope, using LAS-AFX software. Cells were imaged with 63x objective lens. The captured images were processed using Leica Software, Fiji/ImageJ, and CellProfiler. Antibodies The primary antibodies used in this study, along with their respective companies, ordering numbers, host animals, western blot dilutions, and immunofluorescence dilutions, are listed below. mAb414 (Biolegend, 902901, mouse, -/1:500), GFP/YFP (Clontech, JL-8, anti-mouse, 1:5.000/1:500), MLF2 (Santa Cruz Biotechnology, sc-393566, mouse, -/1:500), GAPDH (Fitzgerald, 10R-G109A, mouse, 1:10.000/-), DNAJB6 (Braakman lab Utrecht, rabbit, 1:1.000/-), and V5 (Invitrogen, SY30-01, rabbit, 1:5.000/1:500). The following secondary antibodies and their respective company, ordering numbers, and dilutions were used. Anti-mouse-IgG HRP (GE Healthcare, GENXA931, 1:5.000), anti-rabbit-IgG HRP (GE Healthcare, GENA934, 1:5.000), Alexa Fluor 488 donkey anti-rabbit IgG (H + L) (Thermo Fisher, A21202, 1:500), Alexa Fluor 594 donkey anti-mouse IgG (H + L) (Thermo Fisher, A21203, 1:500), and Alexa Fluor 633 goat anti-rabbit IgG (H + L) (Thermo Fisher, A21070, 1:500). Protein fractionation Cells grown in a 6-well plate were rinsed with PBS to remove residual medium and harvested in 500 µL PBS. After centrifugation at 500 g for 5 minutes at 4°C, the supernatant was discarded, and the cell pellet was lysed in 150 µL NP-40 lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1 mM MgCl2, 0.5% NP-40, 1x Protease Inhibitor, 50 U/mL DNArase, MilliQ). The lysates were vortexed thoroughly and incubated on ice for 30 minutes with periodic vortexing. Protein content was measured using the DC Protein Assay (Bio-Rad) and adjusted to a final concentration of 1–2 µg/µL. For fractionation, 15 µL of the lysate was taken as the whole-cell lysate (WCL). The remaining lysate was centrifuged at 14,000 g for 10 minutes at 4°C, and 15 µL of the supernatant was collected as the 0.5% NP-40 soluble fraction (S1). The leftover supernatant was discarded, and the pellet was washed by adding 150 µL Wash buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1 mM MgCl2, MilliQ) and centrifuged at 14,000 g for 5 minutes at 4°C. The supernatant was discarded, and the pellet was resuspended in 100 µL of 0.1% SDS buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1 mM MgCl2, 0.1% SDS, 1x Protease Inhibitor, MilliQ). This suspension was incubated in a ThermoMixer (Eppendorf) at 1,000 RPM and RT for 30–60 minutes, followed by centrifugation at 14,000 g for 10 minutes at RT. A 15 µL supernatant aliquot was collected as the 0.1% SDS soluble fraction (S2). The remaining supernatant was discarded, and the pellet was washed again with 150 µL Wash buffer and centrifuged as before. The pellet was then resuspended in 50 µL of 2% SDS buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1 mM MgCl2, 2% SDS, 1x Protease Inhibitor, MilliQ) and incubated in the ThermoMixer at 1,000 RPM and RT for 30–60 minutes. After centrifugation at 14,000 g for 10 minutes at RT, a 30 µL supernatant aliquot was collected as the 2% SDS soluble fraction (P1). The pellet was washed again with 150 µL Wash buffer, centrifuged as before, and resuspended in 30 µL of 8M urea buffer to obtain the SDS-insoluble fraction (P2). This mixture was incubated overnight in the ThermoMixer at 1,000 RPM at RT. All aliquots (WCL, S1, S2, P1, and P2) were treated with 4X Laemmli buffer containing 10% β-mercaptoethanol, boiled at 99°C for 5 minutes, and loaded into TGX FastCast acrylamide gel (Bio-Rad, 1610172) for SDS-PAGE and western blot analysis. Evolutionary analysis To assess the conservation of DNAJB6-IDR, two B’ JDP proteins (DNAJB2 and DNAJB8), and one unrelated IDP (P53) were used for a comparative analysis. A manually curated dataset of DNAJB6, DNAJB2, and DNAJB8 was utilized for subsequent steps, encompassing approximately 150 mammalian species. The UniProt IDs were uploaded to https://www.uniprot.org/peptide-search and separate files containing (i) length, (ii) region, (iii) organism (ID), (iv) sequence, (v) domain [FT], (vi) protein existence, and (vii) geneID were downloaded for DNAJB6, DNAJB2, and DNAJB8. A custom-made Python script was used to individually assess the Alphafold ( Jumper et al , 2021 ) files containing the per-residue pLDTT scores to identify the IDRs (pLDTT ≤ 65.0). For the JDPs, the end of the annotated J-domain (folded domain) served as a reference to identify their respective IDR1. Small, ordered regions (length ≤ 5) situated between IDRs were merged into the IDR, resulting in IDR1 sequences starting after the regulatory helix and ending just before the C-terminal β-sheet. Sequences without a predicted Alphafold structure, or with IDRs outside the length criterion (30 ≤ length ≤ 200), were excluded from the analysis. Subsequently, only species with an identified IDR1 for the three JDPs were retained (n=57). In these species, the C-terminal β-sheet was utilized as a reference to identify IDR2 from DNAJB6 (n=51) and DNAJB2 (n=57). A dataset for P53 in these 57 species was compiled manually, and IDR1 was identified using the beginning of the protein and the annotated DNA-binding domain (folded domain) as boundaries. Multiple sequence alignments (MSAs) of the whole protein, folded domain, IDR1, and IDR2 were generated using the default parameters of Clustal and visualized in the Jalview software. Conservation scores were calculated using the information_content (Chang et al ) function from Bio.Align.AlignInfo module in Python, which returns a score per MSA column with a maximum number of 4.32 bits considering an equal probability for all 20 amino acids. A custom-made Python script was used to generate the IDR sequence logos using the logomaker package. Clustermap analysis of human IDRs All human protein entries were retrieved from MobiDB databse (version 6.2; Release: 2024_07) ( Piovesan et al , 2025 ). A custom Python script was used to analyze the corresponding AlphaFold structure files, extracting per-residue pLDDT scores to identify intrinsically disordered regions (IDRs), defined as contiguous segments with pLDDT ≤ 65.0. Short, ordered regions (≤ 5 residues) flanked by IDRs were merged to form continuous disordered segments. The NARDINI algorithm was applied to each identified IDR to compute z-scores for binary amino acid distribution and compositional fraction. Clustering of z-scores—either across the entire IDRome or within specific query sets—was performed using a custom Python script to generate clustermaps for comparative analysis. Statistical analysis Graph preparation and statistical tests were performed using Prism10 software (GraphPad). The number of biological and technical replicates are indicated for all experiments in the figure legends. A color gradient was applied to highlight the individual datapoints belonging to each of the independent replicates. Data was tested for normality using the Shapiro-Wilk and D’Agostino-Pearson tests. For normal distributed data, a parametric unpaired t test (two groups) ( Fig 7L ) or one-way ANOVA (three or more groups) ( Fig 2I,J , Fig 3D , Fig 4F,G ,H, Fig 5F-H , Fig 7D,E ,G,H,J,K ) was performed. For non-normal distributed data, a nonparametric Mann Whitney test (two groups) ( Appendix Fig S6D ) or Kruskal-Wallis test (three or more groups) ( Fig 1B-D , Appendix Fig S1D,E, Appendix Fig S2A-F, Fig 3I-K , Appendix Fig S3B-D, Appendix Fig S4B-D, Appendix Fig S5B-G, Fig 8B , Appendix Fig S6A-C ) with Dunn’s multiple comparisons test was performed. Two-tailed p values; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, ****p ≤ 0.0001. Author contributions TB (conceptualization, formal analysis, investigation, methodology, visualization, writing original draft and review and editing); MKM (conceptualization, formal analysis, investigation, methodology, visualization, writing original draft and review and editing); PG (formal analysis, investigation, methodology, visualization, review and editing); MER (data curation, review and editing); JF (formal analysis, investigation); SMYF (formal analysis, investigation); AS (supervision, review and editing); HHK (conceptualization, supervision, funding acquisition, review and editing); LMV (conceptualization, supervision, funding acquisition, review and editing). Disclosure and competing interests statement The authors declare that they have no conflict of interest. Download figure Open in new tab Appendix Figure S1. DNAJB6 can modulate a range of NupFG condensates. (A) Representative images showing DNAJB6-A594 particles, formed in the absence or presence of 10% PEG3350 for 1h or 24h. Scale bar, 1 µm. (B) Although a sharp reduction in the number of DNAJB6 particles was observed for the Nup100FG-5MF:DNAJB6-A594 mixture formed in the absence of 10% PEG3350 for 24h (as visualized in Fig. 1A ), in rare cases DNAJB6 particles remained and interacted with Nup100FG as exemplified in the image. (C) Table showing overview of median particle size, intensity and circularity for each of the conditions exemplified in ( Fig. 4A ), sorted in the same order. A colour-gradient was applied to each of the assessed particle properties, with lower values indicated in blue and higher values indicated in red. (D-E) Particle size, mean fluorescence intensity and circularity of Nup60FG-5MF, Nup100FG-5MF, Nup116FG-5MF, Nup145FG-5MF and Nup153FG-5MF particles, formed in the absence (D) or presence of 10% PEG3350 (E) for 1h or 24h, in the absence or presence of DNAJB6-A594 (molar ratio 1:1), exemplified in ( Fig. 4A ). Graphs show median ± interquartile range of 300 particles per condition (n=3). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Download figure Open in new tab Appendix Figure S2. Quantification of changing Nup100FG particle properties upon preforming NupFG and DNAJB6 particles. (A-C) Mean fluorescence intensity (A,D), size (B,E) and circularity (C,F) of Nup100FG-5MF particles exemplified in ( Fig. 2F ), in which proteins were either mixed from the start or either Nup100FG (top) or DNAJB6 (bottom) was pre-assembled for either 30 or 60 min prior to mixing (molar ratio 1:1). The mean fluorescence intensity and size data was plotted relative to the median of the control. Graphs show median ± interquartile range of ≥300 particles per condition (n=3). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Download figure Open in new tab Appendix Figure S3 The phenylalanine residues in the S/T-rich region of DNAJB6 are essential for its ability to self-associate and impact FG-Nup particle properties in vitro. ( A ) Representative images showing Nup100FG-5MF particles in the absence or presence of either DNAJB6 WT , DNAJB6 18xS/T>A or DNAJB6 12xF>A particles, formed in the presence of 10% PEG3350 (1h) (molar ratio 1:1). Scale bar, 1 µm. ( B-D ) Mean fluorescence intensity (B), size (C) and circularity (D) (relative to the median of the control) of Nup100FG-5MF particles exemplified in (A). Graphs show median ± interquartile range of ≥300 particles per condition (n=4). ****P<0.0001. Download figure Open in new tab Appendix Figure S4 Clustermap of B’ JDPs and FG-Nups, and microscopy assessment of DNAJB6, DNAJB8, and DNAJB2 self-association tendencies. ( A ) Clustermap of z-scores from NARDINI algorithm for binary amino acid distributions and amino acid fractions in the human IDRs of DNAJB2, DNAJB6, DNAJB7, DNAJB8, and FG-Nups. Rectangles indicate the IDRs of B’ JDPs located between the regulatory helix and the C-terminal β-sheets. ( B-D ) Mean fluorescence intensity, size and circularity of Nup100FG-5MF, Nup116FG-5MF and Nup153FG-5MF particles exemplified in ( Fig 5I-K ). Graphs show median ± interquartile range of 300 particles per condition (n=3). The mean fluorescence intensity and size data was plotted relative to the median of the control. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. ( E,F ) Line scan profiles showing interaction profiles for representative images of Nup100FG (E) and Nup153FG (F) in the presence of DNAJB6, DNAJB8 and DNAJB2, respectively. Download figure Open in new tab Appendix Figure S5 Alterations in the DNAJB6-IDR sequence impact DNAJB6 its ability to form stable gel-like assemblies. ( A ) Representative images of in vitro purified DNAJB6 WT and DNAJB6 rL1 , DNAJB6 rL2 , DNAJB6 SCR1 , DNAJB6 SCR2 and DNAJB6 SCR3 particles, formed in the presence of 10% PEG3350 (1h). After 1 hour incubation, samples were treated with either 5% 1.6-hexanediol or 0.5% SDS for 10 minutes. Numbers indicate the percentage of particles remaining after chemical treatment relative to the control sample, total number of particles counted and median particle size (n=2). ( B-G ) Mean fluorescence intensity, size and circularity of Nup100FG-5MF (B-D) and Nup153FG-5MF (E-G) particles exemplified in ( Fig 7A ). The mean fluorescence intensity and size data was plotted relative to the median of the control. Graphs show median ± interquartile range of 300 particles per condition (n=3)). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Download figure Open in new tab Appendix Figure S6 Evolutionary analysis of the intrinsically disordered regions from DNAJB2, DNAJB6, DNAJB8 and P53. ( A-D ) Conservation scores of residues from the whole protein (A), folded domain (B), IDR1 (C), or IDR2 (D) from DNAJB2, DNAJB6, DNAJB8, and P53. Graphs show median with interquartile range. **P<0.01, ***P<0.001, ****P<0.0001. ( E-H ) Z-scores for the binary amino acid distributions in the IDR1 of DNAJB2 (E), DNAJB6 (F), DNAJB8 (G), and P53 (H) across the 57 analyzed mammal species. Acknowledgements We thank Johan Zijlstra for his initial characterization of the different FG-Nup fragments. We thank Michael Chang and all members of the Veenhoff, Kampinga, and Chang laboratories for their valuable suggestions. This work was financially supported by the Dutch Research Council (NWO) grant no. VI.C.192.031 to L.M.V. 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