Cytoplasmic Assembly Research Articles

RAB14 GTPase is essential for actin-based asymmetric division during mouse oocyte maturation.

ObjectivesRAB14 is a member of small GTPase RAB family which localizes at the endoplasmic reticulum (ER), Golgi apparatus and endosomal compartments. RAB14 acts as molecular switches that shift between a GDP‐bound inactive state and a GTP‐bound active state and regulates circulation of vesicles between the Golgi and endosomal compartments. In present study, we investigated the roles of RAB14 during oocyte meiotic maturation.Materials and methodsMicroinjection with siRNA and exogenous mRNA for knock down and rescue, and immunofluorescence staining, Western blot and real‐time RT‐PCR were utilized for the study.ResultsOur results showed that RAB14 localized in the cytoplasm and accumulated at the cortex during mouse oocyte maturation, and it was also enriched at the spindle periphery. Depletion of RAB14 did not affect polar body extrusion but caused large polar bodies, indicating the failure of asymmetric division. We found that absence of RAB14 did not affect spindle organization but caused the spindle migration defects, and this might be due to the regulation on cytoplasmic actin assembly via the ROCK‐cofilin signalling pathway. We also found that RAB14 depletion led to aberrant Golgi apparatus distribution. Exogenous Myc‐Rab14 mRNA supplement could significantly rescue these defects caused by Rab14 siRNA injection.ConclusionsTaken together, our results suggest that RAB14 affects ROCK‐cofilin pathway for actin‐based spindle migration and Golgi apparatus distribution during mouse oocyte meiotic maturation.

Kinase-mediated RAS signaling via membraneless cytoplasmic protein granules

SummaryReceptor tyrosine kinase (RTK)-mediated activation of downstream effector pathways such as the RAS GTPase/MAP kinase (MAPK) signaling cascade is thought to occur exclusively from lipid membrane compartments in mammalian cells. Here, we uncover a membraneless, protein granule-based subcellular structure that can organize RTK/RAS/MAPK signaling in cancer. Chimeric (fusion) oncoproteins involving certain RTKs including ALK and RET undergo de novo higher-order assembly into membraneless cytoplasmic protein granules that actively signal. These pathogenic biomolecular condensates locally concentrate the RAS activating complex GRB2/SOS1 and activate RAS in a lipid membrane-independent manner. RTK protein granule formation is critical for oncogenic RAS/MAPK signaling output in these cells. We identify a set of protein granule components and establish structural rules that define the formation of membraneless protein granules by RTK oncoproteins. Our findings reveal membraneless, higher-order cytoplasmic protein assembly as a distinct subcellular platform for organizing oncogenic RTK and RAS signaling.

Defects in the cytoplasmic assembly of axonemal dynein arms cause morphological abnormalities and dysmotility in sperm cells leading to male infertility.

Axonemal protein complexes, such as outer (ODA) and inner (IDA) dynein arms, are responsible for the generation and regulation of flagellar and ciliary beating. Studies in various ciliated model organisms have shown that axonemal dynein arms are first assembled in the cell cytoplasm and then delivered into axonemes during ciliogenesis. In humans, mutations in genes encoding for factors involved in this process cause structural and functional defects of motile cilia in various organs such as the airways and result in the hereditary disorder primary ciliary dyskinesia (PCD). Despite extensive knowledge about the cytoplasmic assembly of axonemal dynein arms in respiratory cilia, this process is still poorly understood in sperm flagella. To better define its clinical relevance on sperm structure and function, and thus male fertility, further investigations are required. Here we report the fertility status in different axonemal dynein preassembly mutant males (DNAAF2/ KTU, DNAAF4/ DYX1C1, DNAAF6/ PIH1D3, DNAAF7/ZMYND10, CFAP300/C11orf70 and LRRC6). Besides andrological examinations, we functionally and structurally analyzed sperm flagella of affected individuals by high-speed video- and transmission electron microscopy as well as systematically compared the composition of dynein arms in sperm flagella and respiratory cilia by immunofluorescence microscopy. Furthermore, we analyzed the flagellar length in dynein preassembly mutant sperm. We found that the process of axonemal dynein preassembly is also critical in sperm, by identifying defects of ODAs and IDAs in dysmotile sperm of these individuals. Interestingly, these mutant sperm consistently show a complete loss of ODAs, while some respiratory cilia from the same individual can retain ODAs in the proximal ciliary compartment. This agrees with reports of solely one distinct ODA type in sperm, compared to two different ODA types in proximal and distal respiratory ciliary axonemes. Consistent with observations in model organisms, we also determined a significant reduction of sperm flagellar length in these individuals. These findings are relevant to subsequent studies on the function and composition of sperm flagella in PCD patients and non-syndromic infertile males. Our study contributes to a better understanding of the fertility status in PCD-affected males and should help guide genetic and andrological counselling for affected males and their families.

Cryo-Em Studies of Phosphomimetic and Phospho-Null Triple Mutants of Cardiac Ryanodine Receptor (RyR2)

The activity of cardiac ryanodine receptor type 2 (RyR2), a key player in cardiac contractility, is modulated by various post-translational modifications including phosphorylation at three key residues: S2808, S2814, and S2030. Out of these, two residues (S2808 and S2814) lie in a highly flexible loop of the phosphorylation (P2) domain which has remained unresolved in full-length RyR and in crystal structures of the P2 domain alone. This domain faces the inter-subunit interface near the corner of the square prism-shaped cytoplasmic assembly.

Cytoplasmic virion assembly compartment of betaherpesviruses

During their life cycle, betaherpesviruses extensively reorganize the membranous system of the cell in order to develop a unique cytoplasmic environment of virion production (virus factory), known as cytoplasmic virion assembly compartment (cVAC). The betaherpesvirus cVAC has been studied both in human and murine cytomegalovirus (CMV) infected cells as they are considered to be models for betaherpesviruses biology. Studies of the cVAC were mainly based on electron microscopy and immunofluorescence tools, and in the previous decade, they have expanded to the use of new technologies and systems approaches (i.e., transcriptomics, proteomics, lipidomics), which led to the acquisition of a vast amount of data. Despite the immense expansion of knowledge about membranous organelle system, including a large amount of data derived from CMV infected cells, the organization and biogenesis of the cVAC remain unclear. Accordingly, very little is known about the processes of a final stage of CMV maturation and the virion egress. This minireview summarizes current knowledge about the biogenesis of the betaherpesvirus factory from the already existent endosomal compartments.

The structure and mechanism of the bacterial type II secretion system.

The type II secretion system (T2SS) is a multi-protein complex used by many bacteria to move substrates across their cell membrane. Substrates released into the environment serve as local and long-range effectors that promote nutrient acquisition, biofilm formation, and pathogenicity. In both animals and plants, the T2SS is increasingly recognized as a key driver of virulence. The T2SS spans the bacterial cell envelope and extrudes substrates through an outer membrane secretin channel using a pseudopilus. An inner membrane assembly platform and a cytoplasmic motor controls pseudopilus assembly. This microreview focuses on the structure and mechanism of the T2SS. Advances in cryo-electron microscopy are enabling increasingly elaborate sub-complexes to be resolved. However, key questions remain regarding the mechanism of pseudopilus extension and retraction, and how this is coupled with the choreography of the substrate moving through the secretion system. The T2SS is part of an ancient type IV filament superfamily that may have been present within the last universal common ancestor (LUCA). Overall, mechanistic principles that underlie T2SS function have implication for other closely related systems such as the type IV and tight adherence pilus systems.

Spatiotemporal Proteomic Analysis of Stress Granule Disassembly Using APEX Reveals Regulation by SUMOylation and Links to ALS Pathogenesis

Stress granules (SGs) are cytoplasmic assemblies of proteins and non-translating mRNAs. Whereas much has been learned about SG formation, a major gap remains in understanding the compositional changes SGs undergo during normal disassembly and under disease conditions. Here, we address this gap by proteomic dissection of the SG temporal disassembly sequence using multi-bait APEX proximity proteomics. We discover 109 novel SG proteins and characterize distinct SG substructures. We reveal dozens of disassembly-engaged proteins (DEPs), some of which play functional roles in SG disassembly, including small ubiquitin-like modifier (SUMO) conjugating enzymes. We further demonstrate that SUMOylation regulates SG disassembly and SG formation. Parallel proteomics with amyotrophic lateral sclerosis (ALS)-associated C9ORF72 dipeptides uncovered attenuated DEP recruitment during SG disassembly and impaired SUMOylation. Accordingly, SUMO activity ameliorated C9ORF72-ALS-related neurodegeneration in Drosophila. By dissecting the SG spatiotemporal proteomic landscape, we provide an in-depth resource for future work on SG function and reveal basic and disease-relevant mechanisms of SG disassembly.

A prion-like domain of Tpk2 catalytic subunit of protein kinase A modulates P-body formation in response to stress in budding yeast

Low complexity regions are involved in the assembly and disassembly of P-bodies (PBs). Saccharomyces cerevisiae contains three genes encoding the protein kinase A (PKA) catalytic subunit: TPK1, TPK2 and TPK3. Tpk2 and Tpk3 isoforms localize to PBs upon glucose starvation showing different mechanisms and kinetics of accumulation. In contrast to the other two isoforms, Tpk2 harbors a glutamine-rich prion-like domain (PrLD) at the N-terminus. Here we show that the appearance of Tpk2 foci in response to glucose starvation, heat stress or stationary phase was dependent on its PrLD. Moreover, the PrLD of Tpk2 was necessary for efficient PB and stress granule aggregation during stress conditions and in quiescent cells. Deletion of PrLD does not affect the in vitro and in vivo kinase activity of Tpk2 or its interaction with the regulatory subunit Bcy1. We present evidence that the PrLD of Tpk2 serves as a scaffold domain for PB assembly in a manner that is independent of Pat1 phosphorylation by PKA. In addition, a mutant strain where Tpk2 lacks PrLD showed a decrease of turnover of mRNA during glucose starvation. This work therefore provides new insight into the mechanism of stress-induced cytoplasmic mRNP assembly, and the role of isoform specific domains in the regulation of PKA catalytic subunit specificity and dynamic localization to cytoplasmic RNPs granules.

Human Cytomegalovirus Envelope Protein gpUL132 Regulates Infectious Virus Production through Formation of the Viral Assembly Compartment.

The human cytomegalovirus (HCMV) UL132 open reading frame encodes a 270-amino-acid type I envelope glycoprotein, gpUL132. The deletion of UL132 (ΔUL132) from the HCMV genome results in a pronounced deficit in virus yield, with an approximately 2-log decrease in the production of infectious virus compared to the wild-type (WT) virus. Characterization of the ΔUL132 mutant virus indicated that it was less infectious with a high particle-to-infectious unit ratio and an altered composition of virion proteins compared to the WT virus. In addition, the viral assembly compartment (AC) failed to form in cells infected with the ΔUL132 mutant virus. The expression of gpUL132 in trans rescued the defects in the morphogenesis of the AC in cells infected with the ΔUL132 mutant virus and in infectious virus production. Furthermore, using cell lines expressing chimeric proteins, we demonstrated that the cytosolic domain of gpUL132 was sufficient to rescue AC formation and WT levels of virus production. Progeny virions from ΔUL132-infected cells expressing the cytosolic domain of gpUL132 exhibited particle-to-infectious unit ratios similar to those of the WT virus. Together, our findings argue that gpUL132 is essential for HCMV AC formation and the efficient production of infectious particles, thus highlighting the importance of this envelope protein for the virus-induced reorganization of intracellular membranes and AC formation in the assembly of infectious virus.IMPORTANCE Following infection of permissive cells, human cytomegalovirus (HCMV) induces the reorganization of intracellular membranes resulting in the formation of a distinctive membranous compartment in the cytoplasm of infected cells. This compartment has been designated the viral assembly compartment (AC) and is thought to be a site for cytoplasmic virion assembly and envelopment. In this study, we have demonstrated that a single virion envelope glycoprotein is essential for AC formation in infected cells, and in its absence, there is a significant decrease in the production of infectious virions. These findings are consistent with those from other studies that have demonstrated the importance of host cell proteins in the formation of the AC and demonstrate a critical role of a single virion protein in AC formation and the efficient assembly of infectious virus.

Articles of Significant Interest in This Issue

Study of alphaherpesvirus cytoplasmic envelopment has been complicated by its formation of multiple, small, dispersed assembly centers in most cells. White et al. (e00900-20) found that in cell lines of neuronal origin and in primary mouse neurons, herpes simplex virus (HSV) reorganizes cellular membranes to form a major cytoplasmic virus assembly center (cVAC) around the centrosome. Virus capsids and structural proteins accumulate at this site in a manner dependent on microtubules and on the viral protein pUL51. This provides a powerful system for defining roles for viral and cellular factors in alphaherpesvirus assembly.

The prion-like domain of Fused in Sarcoma is phosphorylated by multiple kinases affecting liquid- and solid-phase transitions.

Fused in Sarcoma (FUS) is a ubiquitously expressed protein that can phase-separate from nucleoplasm and cytoplasm into distinct liquid-droplet structures. It is predominantly nuclear and most of its functions are related to RNA and DNA metabolism. Excessive persistence of FUS within cytoplasmic phase-separated assemblies is implicated in the diseases amyotrophic lateral sclerosis and frontotemporal dementia. Phosphorylation of FUS’s prion-like domain (PrLD) by nuclear phosphatidylinositol 3-kinase-related kinase (PIKK)-family kinases following DNA damage was previously shown to alter FUS’s liquid-phase and solid-phase transitions in cell models and in vitro. However, proteomic data suggest that FUS’s PrLD is phosphorylated at numerous additional sites, and it is unknown if other non-PIKK and nonnuclear kinases might be influencing FUS’s phase transitions. Here we evaluate disease mutations and stress conditions that increase FUS accumulation into cytoplasmic phase-separated structures. We observed that cytoplasmic liquid-phase structures contain FUS phosphorylated at novel sites, which occurred independent of PIKK-family kinases. We engineered phosphomimetic substitutions within FUS’s PrLD and observed that mimicking a few phosphorylation sites strongly inhibited FUS solid-phase aggregation, while minimally altering liquid-phase condensation. These effects occurred independent of the exact location of the phosphomimetic substitutions, suggesting that modulation of PrLD phosphorylation may offer therapeutic strategies that are specific for solid-phase aggregation observed in disease.

I Will Survive: How NPR1 Condensation Promotes Plant Cell Survival

The plant immune response regulator NPR1 resides in either the nucleus or in cytoplasmic puncta, depending on levels of the plant hormone salicylic acid. NPR1 nuclear roles include pathogenesis response (PR) gene regulation. In this issue of Cell, Zavaliev etal. determine that cytoplasmic NPR1-containing assemblies are consistent with multi-component protein condensates with roles to promote cell survival.

Structural mechanism of two gain-of-function cardiac and skeletal RyR mutations at an equivalent site by cryo-EM.

Mutations in ryanodine receptors (RyRs), intracellular Ca2+ channels, are associated with deadly disorders. Despite abundant functional studies, the molecular mechanism of RyR malfunction remains elusive. We studied two single-point mutations at an equivalent site in the skeletal (RyR1 R164C) and cardiac (RyR2 R176Q) isoforms using ryanodine binding, Ca2+ imaging, and cryo-electron microscopy (cryo-EM) of the full-length protein. Loss of the positive charge had greater effect on the skeletal isoform, mediated via distortion of a salt bridge network, a molecular latch inducing rotation of a cytoplasmic domain, and partial progression to open-state traits of the large cytoplasmic assembly accompanied by alteration of the Ca2+ binding site, which concur with the major "hyperactive" feature of the mutated channel. Our cryo-EM studies demonstrated the allosteric effect of a mutation situated ~85 Å away from the pore and identified an isoform-specific structural effect.

MRNA localization mediates maturation of cytoplasmic cilia in Drosophila spermatogenesis.

Cytoplasmic cilia, a specialized type of cilia in which the axoneme resides within the cytoplasm rather than within the ciliary compartment, are proposed to allow for the efficient assembly of very long cilia. Despite being found diversely in male gametes (e.g., Plasmodium falciparum microgametocytes and human and Drosophila melanogaster sperm), very little is known about cytoplasmic cilia assembly. Here, we show that a novel RNP granule containing the mRNAs for axonemal dynein motor proteins becomes highly polarized to the distal end of the cilia during cytoplasmic ciliogenesis in Drosophila sperm. This allows for the incorporation of these axonemal dyneins into the axoneme directly from the cytoplasm, possibly by localizing translation. We found that this RNP granule contains the proteins Reptin and Pontin, loss of which perturbs granule formation and prevents incorporation of the axonemal dyneins, leading to sterility. We propose that cytoplasmic cilia assembly requires the precise localization of mRNAs encoding key axonemal constituents, allowing these proteins to incorporate efficiently into the axoneme.

Phosphorylation of tegument protein pp28 contributes to trafficking to the assembly compartment in human cytomegalovirus infection.

Human cytomegalovirus (HCMV) UL99 encodes a late tegument protein pp28 that is essential for envelopment and production of infectious virus. This protein is localized to the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) in transfected cells but it localizes to the cytoplasmic assembly compartment (AC) in HCMV-infected cells. Trafficking of pp28 to the AC is required for the assembly of infectious virus. The N-terminal domain (aa 1-61) of pp28 is sufficient for trafficking and function of the wild type protein during viral infection. However, residues required for authentic pp28 trafficking with the exception of the acidic cluster in the N-terminal domain of pp28 remain undefined. Monitoring protein migration on SDS-PAGE, we found that pp28 is phosphorylated in the virus-infected cells and dephosphorylated in the viral particles. By generating substitution mutants of pp28, we showed that three serine residues (aa 41-43) and a tyrosine residue (aa 34) account for its phosphorylation. The mutant forms of pp28 were localized to the plasma membrane as well as the ERGIC in transfected cells. Likewise, these mutant proteins were localized to the plasma membrane as well as the AC in virus-infected cells. These results suggested that phosphorylation of pp28 contributes to its intracellular trafficking and efficient viral assembly and incorporation.

Cytomegalovirus Strain TB40/E Restrictions and Adaptations to Growth in ARPE-19 Epithelial Cells.

Despite displaying broad tropism in vivo, human cytomegalovirus (CMV) contained in bodily fluids replicates inefficiently in most cultured cell types except fibroblasts. As propagation in fibroblasts leads to the accumulation of genomic changes, a number of strains were generated by serial passaging on endothelial cells. One of these, TB40/E, was shown to contain a mixture of genetically distinct virus variants, and to retain tropism for fibroblasts, endothelial and epithelial cells. Cloning of an endotheliotropic subpopulation produced the TB40-BAC4 variant, extensively used in CMV tropism studies. Because TB40-BAC4 represents only one of the different variants comprising TB40/E, we generated a series of epithelial-cell adapted stocks derived from a TB40/E mixed stock, rather than from TB40-BAC4. Within two passages on ARPE-19 cells, virus populations were produced with the ability to enter and initiate replication with similar efficiencies in both epithelial cells and fibroblasts. Although the ability to release progeny also increased, cell-free virus yields from ARPE-19 cells remained consistently two to three-logs lower than from fibroblasts, hinting at the existence of a post-entry and post-genome synthesis block in epithelial cells. Multinucleated syncytia also rapidly appeared exclusively in ARPE-19 cell cultures, where their numbers and dimensions increased with virus passage. Irrespective of the number of infected nuclei comprising each syncytium, however, only one cytoplasmic virion assembly compartment was consistently observed, leading us to speculate that improvements in entry efficiency associated with ARPE-19 cell adaptation lead to the development of syncytia, which may negatively affect progeny release by limiting the amount of resources available to maturing virions.

Identifying the compositional features facilitating recruitment of prion‐like domains to stress granules

Stress granules are highly dynamic nonmembranous cytoplasmic RNA‐protein assemblies that form via interactions between mRNAs stalled in translation initiation and RNA‐binding proteins. Many of the RNA‐binding proteins in stress granules contain prion‐like domains (PrLDs). PrLDs are intrinsically disordered protein domains that compositionally resemble yeast prion domains; some PrLDs have been identified as playing a key role in stress granule formation. PrLDs are thought to be recruited to stress granules in part by liquid‐liquid phase separation (LLPS). Mutations in various RNA binding proteins containing PrLDs are associated with degenerative diseases, including Amyotrophic Lateral Sclerosis. These mutations are associated with the formation of cytoplasmic inclusions that share common components with stress granules, suggesting that the mutations perturb stress granule dynamics. However, the sequence features that drive recruitment of PrLDs into stress granules have yet to be completely defined. We recently demonstrated that many PrLDs are sufficient for stress granule recruitment in yeast, and that this recruitment is driven largely by amino acid composition. Here, we utilize synthetic prion‐like domains to rigorously examine the compositional features driving stress granule recruitment. We show that based solely on amino acid composition, we can rationally design synthetic PrLDs that are recruited to stress‐induced assemblies. Surprisingly, although aromatic amino acids are widely believed to play a key role in recruitment of PrLDs to stress granules, we find that aliphatic amino acids can functionally replace aromatic amino acids in supporting recruitment.

G3BP1 Is a Tunable Switch that Triggers Phase Separation to Assemble Stress Granules

The mechanisms underlying ribonucleoprotein (RNP) granule assembly, including the basis for establishing and maintaining RNP granules with distinct composition, are unknown. One prominent type of RNP granule is the stress granule (SG), a dynamic and reversible cytoplasmic assembly formed in eukaryotic cells in response to stress. Here, we show that SGs assemble through liquid-liquid phase separation (LLPS) arising from interactions distributed unevenly across a core protein-RNA interaction network. The central node of this network is G3BP1, which functions as a molecular switch that triggers RNA-dependent LLPS in response to a rise in intracellular free RNA concentrations. Moreover, we show that interplay between three distinct intrinsically disordered regions (IDRs) in G3BP1 regulates its intrinsic propensity for LLPS, and this is fine-tuned by phosphorylation within the IDRs. Further regulation of SG assembly arises through positive or negative cooperativity by extrinsic G3BP1-binding factors that strengthen or weaken, respectively, the core SG network.

Autophagy interferes with human cytomegalovirus genome replication, morphogenesis, and progeny release

ABSTRACT Viral infections are often accompanied by the induction of autophagy as an intrinsic cellular defense mechanism. Herpesviruses have developed strategies to evade autophagic degradation and to manipulate autophagy of the host cells to their benefit. Here we addressed the role of macroautophagy/autophagy in human cytomegalovirus replication and for particle morphogenesis. We found that proteins of the autophagy machinery localize to cytoplasmic viral assembly compartments and enveloped virions in the cytoplasm. Surprisingly, the autophagy receptor SQSTM1/p62 was also found to colocalize with HCMV capsids in the nucleus of infected cells. This finding indicates that the autophagy machinery interacts with HCMV already at the early nuclear stages of particle morphogenesis. The membrane-bound form of LC3 and several autophagy receptors were packaged into extracellular HCMV virions. This suggested that autophagic membranes were included during secondary envelopment of HCMV virions. To further address the importance of autophagy in HCMV infection, we generated an HCMV mutant that expressed a dominant-negative version of the protease ATG4B (BAD-ATG4BC74A). The proteolytic activity of ATG4B is required for LC3 cleavage, priming it for membrane conjugation. Surprisingly, both genome replication and virus release were enhanced in cells infected with BAD-ATG4BC74A, compared to control strains. These results show that autophagy operates as an antiviral process during HCMV infection but is dispensable for secondary HCMV particle envelopment. Abbreviations: ATG: autophagy-related; BAC: bacterial artificial chromosome; BECN1: beclin 1; CPE: cytopathic effect; cVACs: cytoplasmic viral assembly compartments; d.p.i.: days post-infection; DB: dense body; EBV: Epstein-Barr virus; galK: galactokinase; HCMV: human cytomegalovirus; HFF: human foreskin fibroblasts; IE: immediate-early; IRS: internal repeat short; LC3: MAP1LC3A/B; m.o.i.; multiplicity of infection; MCP: major capsid protein; Pp: phosphoprotein; sCP/UL48a: smallest capsid protein; TRS: terminal repeat short; UL: unique long; US: unique short

UsnRNP trafficking is regulated by stress granules and compromised by mutant ALS proteins

Activation of the integrated stress response (ISR), alterations in nucleo-cytoplasmic (N/C) transport and changes in alternative splicing regulation are all common traits of the pathogenesis of Amyotrophic Lateral Sclerosis (ALS). However, whether these processes act independently from each other, or are part of a coordinated mechanism of gene expression regulation that is affected in pathogenic conditions, is still rather undefined. To answer these questions, in this work we set out to characterise the functional connections existing between ISR activation and nucleo-cytosol trafficking and nuclear localization of spliceosomal U-rich small nuclear ribonucleoproteins (UsnRNPs), the core constituents of the spliceosome, and to study how ALS-linked mutant proteins affect this interplay. Activation of the ISR induces a profound reorganization of nuclear Gems and Cajal bodies, the membrane-less particles that assist UsnRNP maturation and storage. This effect requires the cytoplasmic assembly of SGs and is associated to the disturbance of the nuclear import of UsnRNPs by the snurportin-1/importin-β1 system. Notably, these effects are reversed by both inhibiting the ISR or upregulating importin-β1. This indicates that SGs are major determinants of Cajal bodies assembly and that the modulation of N/C trafficking of UsnRNPs might control alternative splicing in response to stress. Importantly, the dismantling of nuclear Gems and Cajal bodies by ALS-linked mutant FUS or C9orf72-derived dipeptide repeat proteins is halted by overexpression of importin-β1, but not by inhibition of the ISR. This suggests that changes in the nuclear localization of the UsnRNP complexes induced by mutant ALS proteins are uncoupled from ISR activation, and that defects in the N/C trafficking of UsnRNPs might play a role in ALS pathogenesis.