Nuclear Pore Proteins Research Articles

Sigma-1 receptor chaperones rescue nucleocytoplasmic transport deficit seen in cellular and Drosophila ALS/FTD models

ABSTRACTIn a subgroup of patients with amyotrophic lateral sclerosis (ALS)/Frontotemporal dementia (FTD), the (G4C2)-RNA repeat expansion from C9orf72 chromosome binds to the Ran-activating protein (RanGAP) at the nuclear pore, resulting in nucleocytoplasmic transport deficit and accumulation of Ran in the cytosol. Here, we found that the sigma-1 receptor (Sig-1R), a molecular chaperone, reverses the pathological effects of (G4C2)-RNA repeats in cell lines and in Drosophila. The Sig-1R colocalizes with RanGAP and nuclear pore proteins (Nups) and stabilizes the latter. Interestingly, Sig-1Rs directly bind (G4C2)-RNA repeats. Overexpression of Sig-1Rs rescues, whereas the Sig-1R knockout exacerbates, the (G4C2)-RNA repeats-induced aberrant cytoplasmic accumulation of Ran. In Drosophila, Sig-1R (but not the Sig-1R-E102Q mutant) overexpression reverses eye necrosis, climbing deficit, and firing discharge caused by (G4C2)-RNA repeats. These results on a molecular chaperone at the nuclear pore suggest that Sig-1Rs may benefit patients with C9orf72 ALS/FTD by chaperoning the nuclear pore assembly and sponging away deleterious (G4C2)-RNA repeats.

Genomic identification and expression analysis of nuclear pore proteins in Malus domestica

The nuclear pore complex (NPC), comprised of individual nucleoporin (Nup) proteins, controls nucleo-cytoplasmic transport of RNA and protein, and is important for regulating plant growth and development. However, there are no reports on this complex in fruit tree species. In this study, we identified 38 apple Nups and named them based on the known Arabidopsis thaliana homologs. We also completed bioinformatics analyses of the intron and exon structural data for apple Nups. The proteins encoded by the apple Nups lacked a universally conserved domain. Moreover, a phylogenetic analysis separated the apple and A. thaliana Nups into three groups. The phylogenetic tree indicated that MdNup54 and MdNup62 are most closely related to genes in other Rosaceae species. To characterize the 38 candidate Malus domestica Nups, we measured their stage-specific expression levels. Our tests revealed these proteins were differentially expressed among diverse tissues. We analyzed the expression levels of seven apple Nups in response to an indole-3-acetic acid (IAA) treatment. The phytohormone treatment significantly inhibited apple flowering. A qRT-PCR analysis proved that an IAA treatment significantly inhibited the expression of these seven genes. A preliminary study regarding two members of the Nup62 subcomplex, MdNup54 and MdNup62, confirmed these two proteins can interact with each other. A yeast two-hybrid assay verified that MdNup54 can interact with MdKNAT4 and MdKNAT6. On the basis of the study results, we identified apple NPC and predicted its structure and function. The data generated in this investigation provide important reference material for follow-up research.

Site-specific proximity ligation provides molecular insights into biologically relevant interfaces of protein-protein interaction

Dynamic protein-protein interactions (PPIs) are fundamental to spatiotemporal control of protein functions in biological systems. Dissecting binding interfaces in aqueous solution (i.e., biological interfaces) is of great importance for identifying molecular determinants that contribute to the affinity and specificity of PPIs. Herein, we describe a biochemical method, termed site-specific proximity ligation (SPL), that enables the identification and reconstruction of native binding interfaces distinct from those present in crystal structures and models from computational prediction. SPL involves the strategic incorporation of an aryl azide-containing unnatural amino acid (AZF) into residues of interest in a particular protein that forms a multiprotein complex. Depending on the interfacial role of a targeted residue, a photo-inducible highly reactive incorporated AZF moiety may react with neighboring functional groups to covalently capture an otherwise non-covalent or weak interaction with a specific partner protein, thereby revealing the landscape of biological interfaces. Using a heterotrimeric nuclear pore protein as a model, we show that the biological interfaces of the complex mapped by SPL provide new insight into dynamic molecular recognition that is missed by, or even in conflict with, static models.

Dissecting the roles of Cse1 and Nup2 in classical NLS-cargo release in vivo.

The importin α/β transport machinery mediates the nuclear import of cargo proteins that bear a classical nuclear localization sequence (cNLS). These cargo proteins are linked to the major nuclear protein import factor, importin‐β, by the importin‐α adapter, after which cargo/carrier complexes enter the nucleus through nuclear pores. In the nucleus, cargo is released by the action of RanGTP and the nuclear pore protein Nup2, after which the importins are recycled to the cytoplasm for further transport cycles. The nuclear export of importin‐α is mediated by Cse1/CAS. Here, we exploit structures of functionally important complexes to identify residues that are critical for these interactions and provide insight into how cycles of protein import and recycling of importin‐α occur in vivo using a Saccharomyces cerevisiae model. We examine how these molecular interactions impact protein localization, cargo import, function and complex formation. We show that reversing the charge of key residues in importin‐α (Arg44) or Cse1 (Asp220) results in loss of function of the respective proteins and impairs complex formation both in vitro and in vivo. To extend these results, we show that basic residues in the Nup2 N‐terminus are required for both Nup2 interaction with importin‐α and Nup2 function. These results provide a more comprehensive mechanistic model of how Cse1, RanGTP and Nup2 function in concert to mediate cNLS‐cargo release in the nucleus.

NUP62 is required for the maintenance of the spindle assembly checkpoint and chromosomal stability.

The nuclear pore protein NUP62 localizes to spindle poles in mitosis and plays a role in maintaining centrosome homeostasis. In this study, we found that NUP62-depleted cells exhibited a defective spindle assembly checkpoint (SAC) and that depletion of NUP62 caused a slight decrease in MAD2 protein levels after nocodazole treatment. However, depletion of NUP62 did not cause a failure in kinetochore localization of the SAC proteins BUBR1, MAD1, and MAD2 in prometaphase. NUP62 depletion slightly prolonged mitotic timing but did not affect cell doubling time. In addition, NUP62 depletion caused a SAC defect and induced aneuploidy in human neural stem cells. Furthermore, overexpression of NUP62Q391P, a mutant protein causing autosomal recessive infantile bilateral striatal necrosis, resulted in a defect in the SAC, indicating that the amino acid residue Q391 in NUP62 is crucial for its effect on the SAC. Overall, we conclude that NUP62 maintains the SAC downstream of kinetochores and thereby ensures maintenance of chromosomal stability.

The autophagy protein Ambra1 regulates gene expression by supporting novel transcriptional complexes

Ambra1 is considered an autophagy and trafficking protein with roles in neurogenesis and cancer cell invasion. Here, we report that Ambra1 also localizes to the nucleus of cancer cells, where it has a novel nuclear scaffolding function that controls gene expression. Using biochemical fractionation and proteomics, we found that Ambra1 binds to multiple classes of proteins in the nucleus, including nuclear pore proteins, adaptor proteins such as FAK and Akap8, chromatin-modifying proteins, and transcriptional regulators like Brg1 and Atf2. We identified biologically important genes, such as Angpt1, Tgfb2, Tgfb3, Itga8, and Itgb7, whose transcription is regulated by Ambra1-scaffolded complexes, likely by altering histone modifications and Atf2 activity. Therefore, in addition to its recognized roles in autophagy and trafficking, Ambra1 scaffolds protein complexes at chromatin, regulating transcriptional signaling in the nucleus. This novel function for Ambra1, and the specific genes impacted, may help to explain the wider role of Ambra1 in cancer cell biology.

Comprehensive nucleosome interactome screen establishes fundamental principles of nucleosome binding.

Nuclear proteins bind chromatin to execute and regulate genome-templated processes. While studies of individual nucleosome interactions have suggested that an acidic patch on the nucleosome disk may be a common site for recruitment to chromatin, the pervasiveness of acidic patch binding and whether other nucleosome binding hot-spots exist remain unclear. Here, we use nucleosome affinity proteomics with a library of nucleosomes that disrupts all exposed histone surfaces to comprehensively assess how proteins recognize nucleosomes. We find that the acidic patch and two adjacent surfaces are the primary hot-spots for nucleosome disk interactions, whereas nearly half of the nucleosome disk participates only minimally in protein binding. Our screen defines nucleosome surface requirements of nearly 300 nucleosome interacting proteins implicated in diverse nuclear processes including transcription, DNA damage repair, cell cycle regulation and nuclear architecture. Building from our screen, we demonstrate that the Anaphase-Promoting Complex/Cyclosome directly engages the acidic patch, and we elucidate a redundant mechanism of acidic patch binding by nuclear pore protein ELYS. Overall, our interactome screen illuminates a highly competitive nucleosome binding hub and establishes universal principles of nucleosome recognition.

Calcium-Mediated Interactions Regulate the Subcellular Localization of Extracellular Signal-Regulated Kinases (ERKs).

The subcellular localization of ERK1 and ERK2 (ERKs) in cells, which is important for proper signaling, may be regulated through protein-protein interactions. However, the proteins involved and the way they are regulated to affect localization is not entirely understood. In order to identify the interacting proteins upon varying conditions, we used co-immunoprecipitation of ERK, active ERK and its binding CRS mutant. In addition, we examined the effect of intracellular calcium on the binding using calcium chelators and ionophores, analyzing the binding using silver stain, mass spectrometry and immunoblotting. The effect of calcium on ERK localization was examined using immunofluorescent staining and Western blotting. We found that inactive ERK2 interacts with a large number of proteins through its CRS/CD domain, whereas the phospho-ERK2 interacts with only few substrates. Varying calcium concentrations significantly modified the repertoire of ERK2-interacting proteins, of which many were identified. The effect of calcium on ERKs' interactions influenced also the localization of ERKs, as calcium chelators enhanced nuclear translocation, while elevated calcium levels prevented it. This effect of calcium was also apparent upon the physiological lysophosphatidic acid stimulation, where ERKs translocation was delayed compared to that induced by EGF in a calcium-dependent manner. In vitro translocation assay revealed that high calcium concentrations affect ERKs' translocation by preventing the shuttling machinery through the nuclear envelope, probably due to higher binding to nuclear pore proteins such as NUP153. These results are consistent with a model in which ERKs in quiescent cells are bound to several cytoplasmic proteins. Upon stimulation, ERKs are phosphorylated and released from their cytoplasmic anchors to allow shuttling into the nucleus. This translocation is delayed when calcium levels are increased, and this modifies the localization of ERKs and therefore also their spatiotemporal regulation. Thus, calcium regulates ERKs localization, which is important for the compartmentalization of ERKs with their proper substrates, and thereby their signaling specificity.

Intrinsically disordered nuclear pore proteins show ideal-polymer morphologies and dynamics.

In the nuclear pore complex, intrinsically disordered nuclear pore proteins (FG Nups) form a selective barrier for transport into and out of the cell nucleus, in a way that remains poorly understood. The collective FG Nup behavior has long been conceptualized either as a polymer brush, dominated by entropic and excluded-volume (repulsive) interactions, or as a hydrogel, dominated by cohesive (attractive) interactions between FG Nups. Here we compare mesoscale computational simulations with a wide range of experimental data to demonstrate that FG Nups are at the crossover point between these two regimes. Specifically, we find that repulsive and attractive interactions are balanced, resulting in morphologies and dynamics that are close to those of ideal polymer chains. We demonstrate that this property of FG Nups yields sufficient cohesion to seal the transport barrier, and yet maintains fast dynamics at the molecular scale, permitting the rapid polymer rearrangements needed for transport events.

Preparation of Drosophila Polytene Chromosomes, Followed by Immunofluorescence Analysis of Chromatin Structure by Multi-fluorescence Correlations.

Drosophila larval salivary gland polytene chromosome squashes have been used for decades to analyze genome-wide protein-binding patterns, transcriptional activation processes, and changes in chromatin structure at specific genetic loci. There have been many evolutions of the squashing protocol over the years, with sub-optimal reproducibility and low sample success rate as accepted caveats. However, low sample success rates are an obvious disadvantage when polytene chromosomes are used for more high-throughput approaches, such as genetic or antibody screens, or for experiments requiring high-quality chromosome structure preservation. Here we present an exceptionally reproducible squashing and fluorescence staining protocol, which generates high-quality fluorescence images on well-spread chromosomes. This is followed by our novel, semi-automated MATLAB analysis program used to determine correlations between fluorescence signals of interest at a single site on polytene chromosomes, in a pixel-by-pixel manner. In our case, we have used this approach to assess chromatin changes at genomic sites, ectopically targeted by nuclear pore proteins. The use of our analysis program increases the ability to make unbiased conclusions on changes in chromatin structure, or in protein recruitment to chromatin, regardless of sample variation in immunofluorescence staining. As it is simply based upon differences in fluorescence intensity at a defined location, the provided analysis program is not limited to analysis of polytene chromosome, and could be applied to many different contexts where correlation between fluorescent signals at any particular location is of interest.

Adapter Proteins for Opposing Motors Interact Simultaneously with Nuclear Pore Protein Nup358.

Nup358 is a protein subunit of the nuclear pore complex that recruits the opposing microtubule motors kinesin-1 and dynein [via the dynein adaptor Bicaudal D2 (BicD2)] to the nuclear envelope. This pathway is important for positioning of the nucleus during the early steps of mitotic spindle assembly and also essential for an important process in brain development. It is unknown whether dynein and kinesin-1 interact with Nup358 simultaneously or whether they compete. Here, we have reconstituted and characterized a minimal complex of kinesin-1 light chain 2 (KLC2) and Nup358. The proteins interact through a W-acidic motif in Nup358, which is highly conserved among vertebrates but absent in insects. While Nup358 and KLC2 form predominantly monomers, their interaction results in the formation of 2:2 complexes, and the W-acidic motif is required for the oligomerization. In active motor complexes, BicD2 and KLC2 each form dimers. Notably, we show that the dynein adaptor BicD2 and KLC2 interact simultaneously with Nup358, resulting in the formation of 2:2:2 complexes. Mutation of the W-acidic motif results in the formation of 1:1:1 complexes. On the basis of our data, we propose that Nup358 recruits simultaneously one kinesin-1 motor and one dynein motor via BicD2 to the nucleus. We hypothesize that the binding sites are close enough to promote direct interactions between these motor recognition domains, which may be important for the regulation of the motility of these opposing motors. Our data provide important insights into a nuclear positioning pathway that is crucial for brain development and faithful chromosome segregation.

Nuclear Pore Proteins in Regulation of Chromatin State.

Nuclear pore complexes (NPCs) are canonically known to regulate nucleocytoplasmic transport. However, research efforts over the last decade have demonstrated that NPCs and their constituent nucleoporins (Nups) also interact with the genome and perform important roles in regulation of gene expression. It has become increasingly clear that many Nups execute these roles specifically through regulation of chromatin state, whether through interactions with histone modifiers and downstream changes in post-translational histone modifications, or through relationships with chromatin-remodeling proteins that can result in physical changes in nucleosome occupancy and chromatin compaction. This review focuses on these findings, highlighting the functional connection between NPCs/Nups and regulation of chromatin structure, and how this connection can manifest in regulation of transcription.

Nuclear pore protein Nup98 is involved in replication of Rift Valley fever virus and nuclear import of virulence factor NSs.

The non-structural protein NSs is the main virulence factor of Rift Valley fever virus, a major zoonotic pathogen in Africa. NSs forms large aggregates in the nucleus and impairs induction of the antiviral type I IFN system by several mechanisms, including degradation of subunit p62 of the general RNA polymerase II transcription factor TFIIH. Here, we show that depletion of the nuclear pore protein Nup98 affects the nuclear import of NSs. Nonetheless, NSs was still able to degrade TFIIH-p62 under these conditions. Depletion of Nup98, however, had a negative effect on Rift Valley fever virus multiplication. Our data thus indicate that NSs utilizes Nup98 for import into the nucleus, but also plays a general role in the viral replication cycle.

Thioredoxin-related transmembrane protein 2 (TMX2) regulates the Ran protein gradient and importin-\u03b2-dependent nuclear cargo transport

TMX2 is a thioredoxin family protein, but its functions have not been clarified. To elucidate the function of TMX2, we explored TMX2-interacting proteins by LC-MS. As a result, importin-β, Ran GTPase (Ran), RanGAP, and RanBP2 were identified. Importin-β is an adaptor protein which imports cargoes from cytosol to the nucleus, and is exported into the cytosol by interaction with RanGTP. At the cytoplasmic nuclear pore, RanGAP and RanBP2 facilitate hydrolysis of RanGTP to RanGDP and the disassembly of the Ran-importin-β complex, which allows the recycling of importin-β and reentry of Ran into the nucleus. Despite its interaction of TMX2 with importin-β, we showed that TMX2 is not a transport cargo. We found that TMX2 localizes in the outer nuclear membrane with its N-terminus and C-terminus facing the cytoplasm, where it co-localizes with importin-β and Ran. Ran is predominantly distributed in the nucleus, but TMX2 knockdown disrupted the nucleocytoplasmic Ran gradient, and the cysteine 112 residue of Ran was important in its regulation by TMX2. In addition, knockdown of TMX2 suppressed importin-β-mediated transport of protein. These results suggest that TMX2 works as a regulator of protein nuclear transport, and that TMX2 facilitates the nucleocytoplasmic Ran cycle by interaction with nuclear pore proteins.

Pleiotropic requirements for human TDP-43 in the regulation of cell and organelle homeostasis.

TDP-43 is an RNA-binding protein that forms cytoplasmic aggregates in multiple neurodegenerative diseases. Although the loss of normal TDP-43 functions likely contributes to disease pathogenesis, the cell biological consequences of human TDP-43 depletion are not well understood. We, therefore, generated human TDP-43 knockout (KO) cells and subjected them to parallel cell biological and transcriptomic analyses. These efforts yielded three important discoveries. First, complete loss of TDP-43 resulted in widespread morphological defects related to multiple organelles, including Golgi, endosomes, lysosomes, mitochondria, and the nuclear envelope. Second, we identified a new role for TDP-43 in controlling mRNA splicing of Nup188 (nuclear pore protein). Third, analysis of multiple amyotrophic lateral sclerosis causing TDP-43 mutations revealed a broad ability to support splicing of TDP-43 target genes. However, as some TDP-43 disease-causing mutants failed to fully support the regulation of specific target transcripts, our results raise the possibility of mutation-specific loss-of-function contributions to disease pathology.

The nuclear pore proteins Nup88/214 and T-cell acute lymphatic leukemia–associated NUP214 fusion proteins regulate Notch signaling

The Notch receptor is a key mediator of developmental programs and cell-fate decisions. Imbalanced Notch signaling leads to developmental disorders and cancer. To fully characterize the Notch signaling pathway and exploit it in novel therapeutic interventions, a comprehensive view on the regulation and requirements of Notch signaling is needed. Notch is regulated at different levels, ranging from ligand binding, stability to endocytosis. Using an array of different techniques, including reporter gene assays, immunocytochemistry, and ChIP-qPCR we show here, to the best of our knowledge for the first time, regulation of Notch signaling at the level of the nuclear pore. We found that the nuclear pore protein Nup214 (nucleoporin 214) and its interaction partner Nup88 negatively regulate Notch signaling in vitro and in vivo in zebrafish. In mammalian cells, loss of Nup88/214 inhibited nuclear export of recombination signal-binding protein for immunoglobulin κJ region (RBP-J), the DNA-binding component of the Notch pathway. This inhibition increased binding of RBP-J to its cognate promoter regions, resulting in increased downstream Notch signaling. Interestingly, we also found that NUP214 fusion proteins, causative for certain cases of T-cell acute lymphatic leukemia, potentially contribute to tumorigenesis via a Notch-dependent mechanism. In summary, the nuclear pore components Nup88/214 suppress Notch signaling in vitro, and in zebrafish, nuclear RBP-J levels are rate-limiting factors for Notch signaling in mammalian cells, and regulation of nucleocytoplasmic transport of RBP-J may contribute to fine-tuning Notch activity in cells.

Chromatin targeting of nuclear pore proteins induces chromatin decondensation.

Nuclear pore complexes have emerged in recent years as chromatin-binding nuclear scaffolds, able to influence target gene expression. However, how nucleoporins (Nups) exert this control remains poorly understood. Here we show that ectopically tethering Drosophila Nups, especially Sec13, to chromatin is sufficient to induce chromatin decondensation. This decondensation is mediated through chromatin-remodeling complex PBAP, as PBAP is both robustly recruited by Sec13 and required for Sec13-induced decondensation. This phenomenon is not correlated with localization of the target locus to the nuclear periphery, but is correlated with robust recruitment of Nup Elys. Furthermore, we identified a biochemical interaction between endogenous Sec13 and Elys with PBAP, and a role for endogenous Elys in global as well as gene-specific chromatin decompaction. Together, these findings reveal a functional role and mechanism for specific nuclear pore components in promoting an open chromatin state.

Cleavage and Sub-Cellular Redistribution of Nuclear Pore Protein 98 by Coxsackievirus B3 Protease 2A Impairs Cardioprotection.

Myocarditis, inflammation of the heart muscle, affects all demographics and is a major cause of sudden and unexpected death in young people. It is most commonly caused by viral infections of the heart, with coxsackievirus B3 (CVB3) being among the most prevalent pathogens. To understand the molecular pathogenesis of CVB3 infection and provide strategies for developing treatments, we examined the role of a key nuclear pore protein 98 (NUP98) in the setting of viral myocarditis. NUP98 was cleaved as early as 2 h post-CVB3 infection. This cleavage was further verified through both the ectopic expression of viral proteases and in vitro using purified recombinant CVB3 proteases (2A and 3C), which demonstrated that CVB3 2A but not 3C is responsible for this cleavage. By immunostaining and confocal imaging, we observed that cleavage resulted in the redistribution of NUP98 to punctate structures in the cytoplasm. Targeted siRNA knockdown of NUP98 during infection further increased viral protein expression and viral titer, and reduced cell viability, suggesting a potential antiviral role of NUP98. Moreover, we discovered that expression levels of neuregulin-1 (NRG1), a cardioprotective gene, and presenilin-1 (PSEN1), a cellular protease processing the tyrosine kinase receptor ERBB4 of NRG1, were reliant upon NUP98 and were downregulated during CVB3 infection. In addition, expression of these NUP98 target genes in myocardium tissue not only occurred at an earlier phase of infection, but also appeared in areas away from the initial inflammatory regions. Collectively, CVB3-induced cleavage of NUP98 and subsequent impairment of the cardioprotective NRG1-ERBB4/PSEN1 signaling cascade may contribute to increased myocardial damage in the context of CVB3-induced myocarditis. To our knowledge, this is the first study to demonstrate the link between NUP98 and the NRG1 signaling pathway in viral myocarditis.

Mapping the native organization of the yeast nuclear pore complex using nuclear radial intensity measurements

Selective transport across the nuclear envelope (NE) is mediated by the nuclear pore complex (NPC), a massive ∼100-MDa assembly composed of multiple copies of ∼30 nuclear pore proteins (Nups). Recent advances have shed light on the composition and structure of NPCs, but approaches that could map their organization in live cells are still lacking. Here, we introduce an in vivo method to perform nuclear radial intensity measurements (NuRIM) using fluorescence microscopy to determine the average position of NE-localized proteins along the nucleocytoplasmic transport axis. We apply NuRIM to study the organization of the NPC and the mobile transport machinery in budding yeast. This reveals a unique snapshot of the intact yeast NPC and identifies distinct steady-state localizations for various NE-associated proteins and nuclear transport factors. We find that the NPC architecture is robust against compositional changes and could also confirm that in contrast to Chlamydomonas reinhardtii, the scaffold Y complex is arranged symmetrically in the yeast NPC. Furthermore, NuRIM was applied to probe the orientation of intrinsically disordered FG-repeat segments, providing insight into their roles in selective NPC permeability and structure.

Liquid-liquid phase separation and biomolecular condensates

Eukaryotic cells contain many membrane-less compartments, such as P granules, stress granules and p62 bodies, which are composed of highly concentrated biomacromolecules. These compartments are involved in various cellular processes, including germ cell specification, stress response, and selective autophagy. They are called “biomolecular condensates” and function in organizing cellular biochemical reactions, to make them relatively independent, highly ordered, and highly efficient. According to recent studies, biomolecular condensates exhibit liquid-like properties, such as a spherical shape, fusion upon contact and high dynamics. They arise from “liquid-liquid phase separation” (LLPS) driven by multivalent interactions among macromolecules. There are many ways to achieve macromolecular multivalence: Linear modular domains with similar function, like PRM repeats in N-WASP; oligomerization domains, like the PB1 domain in the selective autophagy receptor p62; similar post-translational modifications at multiple sites; poly-ubiquitination at one site; degenerate binding of RNA to proteins, etc. Additionally, intrinsically disordered regions (IDRs), sometimes called low complexity domains (LCDs), which contain high frequencies of a few specific amino acid types, are a common way to mediate multivalent interactions and drive LLPS. Many IDR-containing proteins, such as the P granule component PGL-3, the nuclear pore protein Nup98, and the RNA-binding protein FUS, were shown to undergo phase separation in vitro and localize to specific biomolecular condensates in vivo . RNAs with disease-related nucleotide repeat expansions, such as CUG repeats, CAG repeats, and GGGGCC repeats, can also form phase-separated puncta in vitro and in patients’ cells. Mechanisms for regulating phase separation include modulating the relationship between the concentration and saturation concentration of biomacromolecules, and altering biomolecular interactions. Regulation of transcription, translation and degradation can change biomacromolecule concentrations. Temperature, crowding agents, and ATP (which functions as a hydrotrope), can impact protein solubility. Protein modifications and novel interactions with other biomacromolecules can impact inter-molecular or/and intra-molecular interactions, thus disrupting phase separation. A special example is the nuclear transport receptor Kapβ2, which suppresses normal and aberrant phase separation of some RNA-binding proteins with IDRs, including FUS, by interacting with their PY-NLS region. The components that are essential for establishing phase separation are called “scaffolds”, and they enrich their “clients” via interaction through their free binding sites. The composition of clients can be altered by changing the stoichiometries of scaffolds. Droplets formed by different biomacromolecules, such as FIB1 and NPM, can co-exist to form immiscible multi-phase condensates, which are analogous to multi-layered membrane-less organelles in vivo , such as nucleoli and stress granules. Many in vitro phase-separation systems have been established, which can induce receptor clustering, activate actin or tubulin polymerization, facilitate enzymatic reactions, etc. Some studies have suggested a link between phase separation and heterochromatin formation or gene control. Based on reconstituted phase separation systems in vitro , and their links to biomolecular condensates in vivo , we can gain deep understanding of the assembly, dissolution, composition, physical properties, biochemical activities and cellular functions of biomolecular condensates.