Assembly Of Membraneless Organelles Research Articles

Transcription templated assembly of the nucleolus in the C. elegans embryo.

The nucleolus is a multicomponent structure made of RNA and proteins that serves as the site of ribosome biogenesis within the nucleus. It has been extensively studied as a prototype of a biomolecular condensate whose assembly is driven by phase separation. While the steady-state size of the nucleolus is quantitatively accounted for by the thermodynamics of phase separation, we show that experimental measurements of the assembly dynamics are inconsistent with a simple model of a phase-separating system relaxing to its equilibrium state. Instead, we show that the dynamics are well described by a model in which the transcription of ribosomal RNA actively drives nucleolar assembly. We find that our model of active transcription-templated assembly quantitatively accounts for the rapid kinetics observed in early embryos at different developmental stages, and for different RNAi perturbations of embryo size. Our model predicts a scaling of the time to assembly with the volume of the nucleus to the one-third power, which is confirmed by experimental data. Our study highlights the role of active processes such as transcription in controlling the placement and timing of assembly of membraneless organelles.

In Vivo Optogenetic Phase Transition of an Intrinsically Disordered Protein.

Proteins containing intrinsically disordered regions (IDRs) control a wide variety of cellular processes by assembly of membrane-less organelles via IDR-mediated liquid-liquid phase separation. Dysregulated IDR-mediated phase transition has been implicated in the pathogenesis of diseases characterized by deposition of abnormal protein aggregates. Here, we describe a method to enhance interactions between the IDRs of the RNA/DNA-binding protein and TAR DNA-binding protein 43 (TDP-43) by light to drive its phase transition in the motor neurons of zebrafish. The optically controlled TDP-43 phase transition in motor neurons, in vivo, provides a unique opportunity to evaluate the impact of dysregulated TDP-43 phase transition on the physiology of motor neurons. This will help to address the etiology of neurodegenerative diseases associated with abnormal TDP-43 phase transition and aggregation, including amyotrophic lateral sclerosis (ALS).

Synthetic Proteins behind the Plasma Barrier: Molecular Spies.

ConspectusThere is a continuous demand to improve our understanding of fundamental processes that underlie human health and disease. Therefore, novel strategies that can assist in these efforts are required. For example, molecular biology and genetic approaches have revolutionized our understanding of protein-mediated processes by facilitating their direct visualization and analyses in living cells. Despite these developments, genetic manipulation has limitations in controlling events that occur after translation such as posttranslational modifications (PTMs), which are imperative regulatory elements. As a result, developing new methods to study PTMs in live cells is a major bottleneck in deciphering their exact roles in the myriad cellular processes.Synthetic and semisynthetic proteins are prepared by combining solid phase peptide synthesis (SPPS) and chemoselective ligation approaches with synthetic or recombinant peptides. Employing protein synthesis allows chemists to incorporate natural and unnatural modifications with virtually unlimited number of functional groups into the protein’s sequence, such as PTMs and their mimics. In addition, synthetic proteins can include additional elements such as fluorescent tags, reactive groups, caged units, and enrichment handles. Therefore, harnessing the power of chemical protein synthesis offers great opportunities to study fundamental biological processes.Unfortunately, the low cell permeability of proteins limits their applications mainly to in vitro settings, excluding live cell studies. As a result, chemical biologists have been attempting to overcome these limitations by developing protein delivery methods that would enable the study of custom-made proteins in a biological context. Success with these strategies should enable accurate determination of protein localization, degradation, folding, interactions, and involvement in the assembly of membrane-less organelles formed by liquid–liquid phase separation inside cells. Importantly, protein delivery approaches are complementary to genetic manipulations, and combining these approaches should pave the way to new discoveries.In this Account, we describe recent developments in protein delivery methods, with emphasis on those most compatible with synthetic proteins. We highlight experimental approaches and conceptual adaptations required to design and study synthetic proteins in live cells, with or without genetic manipulation. In addition, we highlight the strength and weakness of these approaches for both the delivery and the subsequent studies. We also describe our endeavors to deliver synthetic proteins to cells via cell penetrating peptides (CPPs) and multiplexed bead loading (MBL), as showcases of the applications of these methods to shed light on biological processes. Lastly, we contemplate other future applications of synthetic proteins to answer questions that are currently unapproachable.

A Framework to Assess Liquid‐Liquid‐Phase‐Separation in Bacterial Cells

Liquid Liquid Phase Separation (LLPS) has emerged as a mechanism for the assembly of membraneless organelles in eukaryotes, but little is known about this process in bacteria. LLPS refers to the ability of macromolecules to demix into a dilute phase and a dense phase, called a ‘biomolecular condensate’, which can be observed as clusters or foci in the cell. The major challenge for the study of LLPS in bacteria is the poor spatial resolution of foci in such tiny cells. As a result, it is difficult to demonstrate the liquid‐like nature of a focus in bacterial cells using the conventional approaches for studying large condensates in eukaryotic cells. Here, we developed a rigorous experimental framework for the characterization of LLPS in bacteria, using Escherichia coli as the host organism and the intrinsically disordered protein McdB, which robustly forms liquid‐like droplets in vitro. McdB is a protein that coats a bacterial organelle called the carboxysome. This coating demarcates the carboxysome as cargo for its positioning system, which equally distributes carboxysomes along the cell length of rod‐shaped cyanobacteria. We developed a suite of experiments to investigate the LLPS activity of McdB in vivo, based on the ability of biomolecular condensates to tune their size and shape, fuse, dissolve, and transition between phase states. We used both overexpression and tunable promoters to express fluorescent fusions of McdB and cIEP8, a well‐known aggregator protein. We found that fluorescent fusions of McdB formed nucleoid‐excluded foci in E. coli, but also maintained a soluble phase in the cytoplasm, consistent with LLPS theory. The aggregator protein cIEP8, on the other hand, lacked a soluble fraction in the cytoplasm. Condensates form at a saturation concentration threshold, called Csat. A hallmark of LLPS is that condensates will dissolve if the concentration drops below Csat, while insoluble aggregates should remain as stable foci even after dilution. We decreased protein concentration in vivo by increasing cell volume and by generational dilution via cell division. In both methods, McdB foci dissolved while cIEP8 foci remained intact as insoluble aggregates in response to decreased concentration in the cell. Finally, we also discovered that a well‐established marker for insoluble protein aggregates in vivo, IbpA, does not colocalize with McdB foci. The result suggests that the colocalization of IbpA foci can be used as a broad‐use sensor for the material state of protein complexes in bacterial cells. Our results provide multiple lines of evidence in support of LLPS of McdB in vivo. More broadly, our experimental framework for studying LLPS in bacteria overcomes current limitations in the field and can be used to assess the LLPS activity of other proteins of interest in bacterial cells.

Myosin 1D and the branched actin network control the condensation of p62 bodies.

Biomolecular condensation driven by liquid-liquid phase separation (LLPS) is key to assembly of membraneless organelles in numerous crucial pathways. It is largely unknown how cellular structures or components spatiotemporally regulate LLPS and condensate formation. Here we reveal that cytoskeletal dynamics can control the condensation of p62 bodies comprising the autophagic adaptor p62/SQSTM1 and poly-ubiquitinated cargos. Branched actin networks are associated with p62 bodies and are required for their condensation. Myosin 1D, a branched actin-associated motor protein, drives coalescence of small nanoscale p62 bodies into large micron-scale condensates along the branched actin network. Impairment of actin cytoskeletal networks compromises the condensation of p62 bodies and retards substrate degradation by autophagy in both cellular models and Myosin 1D knockout mice. Coupling of LLPS scaffold to cytoskeleton systems may represent a general mechanism by which cells exert spatiotemporal control over phase condensation processes.

GraPES: The Granule Protein Enrichment Server for prediction of biological condensate constituents.

Phase separation-based condensate formation is a novel working paradigm in biology, helping to rationalize many important cellular phenomena including the assembly of membraneless organelles. Uncovering the functional impact of cellular condensates requires a better knowledge of these condensates’ constituents. Herein, we introduce the webserver GraPES (Granule Protein Enrichment Server), a user-friendly online interface containing the MaGS and MaGSeq predictors, which provide propensity scores for proteins’ localization into cellular condensates. Our webpage contains models trained on human (Homo sapiens) and yeast (Saccharomyces cerevisiae) stress granule proteins. MaGS utilizes experimentally-based protein features for prediction, whereas MaGSeq is an entirely protein sequence-based implementation. GraPES is implemented in HTML/CSS and Javascript and is freely available for public use at https://grapes.msl.ubc.ca/. Documentation for using the provided webtools, descriptions of their methodology, and implementation notes can be found on the webpage.

Global mapping of RNA homodimers in living cells.

RNA homodimerization is important for various physiological processes, including the assembly of membraneless organelles, RNA subcellular localization, and packaging of viral genomes. However, understanding RNA dimerization has been hampered by the lack of systematic in vivo detection methods. Here, we show that CLASH, PARIS, and other RNA proximity ligation methods detect RNA homodimers transcriptome-wide as “overlapping” chimeric reads that contain more than one copy of the same sequence. Analyzing published proximity ligation data sets, we show that RNA:RNA homodimers mediated by direct base-pairing are rare across the human transcriptome, but highly enriched in specific transcripts, including U8 snoRNA, U2 snRNA, and a subset of tRNAs. Mutations in the homodimerization domain of U8 snoRNA impede dimerization in vitro and disrupt zebrafish development in vivo, suggesting an evolutionarily conserved role of this domain. Analysis of virus-infected cells reveals homodimerization of SARS-CoV-2 and Zika genomes, mediated by specific palindromic sequences located within protein-coding regions of N gene in SARS-CoV-2 and NS2A gene in Zika. We speculate that regions of viral genomes involved in homodimerization may constitute effective targets for antiviral therapies.

Mapping the regulatory landscape of membraneless organelle assembly and dynamics

Membraneless organelles (MLOs) are an elusive class of cellular structures that intersect diverse cellular pathways and have been implicated in multiple pathologies, including neurodegeneration and cancer. A defining feature of MLOs is their assembly through molecular condensation, which creates a dynamic range of material properties. Understanding how molecular condensation is regulated within a living system is essential for defining how these unique cellular structures function in homeostasis and how their dysregulation is linked to disease.

Liquid-Liquid Phase Separation in Biology: Specific Stoichiometric Molecular Interactions vs Promiscuous Interactions Mediated by Disordered Sequences.

Extensive studies in the past few years have shown that nonmembrane bound organelles are likely assembled via liquid-liquid phase separation (LLPS), a process that is driven by multivalent protein-protein and/or protein-nucleic acid interactions. Both stoichiometric molecular interactions and intrinsically disordered region (IDR)-driven interactions can promote the assembly of membraneless organelles, and the field is currently dominated by IDR-driven biological condensate formation. Here we discuss recent studies that demonstrate the importance of specific biomolecular interactions for functions of diverse physiological condensates. We suggest that phase separation based on combinations of specific interactions and promiscuous IDR-driven interactions is likely a general feature of biological condensation under physiological conditions.

Assembly of higher-order SMN oligomers is essential for metazoan viability and requires an exposed structural motif present in the YG zipper dimer.

Protein oligomerization is one mechanism by which homogenous solutions can separate into distinct liquid phases, enabling assembly of membraneless organelles. Survival Motor Neuron (SMN) is the eponymous component of a large macromolecular complex that chaperones biogenesis of eukaryotic ribonucleoproteins and localizes to distinct membraneless organelles in both the nucleus and cytoplasm. SMN forms the oligomeric core of this complex, and missense mutations within its YG box domain are known to cause Spinal Muscular Atrophy (SMA). The SMN YG box utilizes a unique variant of the glycine zipper motif to form dimers, but the mechanism of higher-order oligomerization remains unknown. Here, we use a combination of molecular genetic, phylogenetic, biophysical, biochemical and computational approaches to show that formation of higher-order SMN oligomers depends on a set of YG box residues that are not involved in dimerization. Mutation of key residues within this new structural motif restricts assembly of SMN to dimers and causes locomotor dysfunction and viability defects in animal models.

Karyopherin-βs play a key role as a phase separation regulator.

Recent studies have revealed that cells utilize liquid-liquid phase separation (LLPS) as a mechanism in assembly of membrane-less organelles, such as RNP granules. The nucleus is a well-known membrane-bound organelle surrounded by the nuclear envelope; the nuclear pore complex on the nuclear envelope likely applies LLPS in the central channel to facilitate selective biological macromolecule exchange. Karyopherin-β family proteins exclusively pass through the central channel with cargos by dissolving the phase separated hydrogel formed by the phenylalanine-glycine (FG) repeats-containing nucleoporins. Karyopherin-βs also exhibit dissolution activity for the phase separation of cargo proteins. Many cargos, including RNA-binding proteins containing intrinsically disordered regions (IDRs), undergo phase separation; however, aberrant phase separation is linked to fatal neurodegenerative diseases. Multiple weak interactions between karyopherin-βs and phase separation-prone proteins, such as FG repeats-containing nucleoporins or IDR-containing karyopherin-β cargos, are likely to be important for passing through the nuclear pore complex and maintaining the soluble state of cargo, respectively. In this review, we discuss how karyopherin-βs regulate phase separation to function.

A global analysis of low-complexity regions in the Trypanosoma brucei proteome reveals enrichment in the C-terminus of nucleic acid binding proteins providing potential targets of phosphorylation.

Background: Low-complexity regions (LCRs) on proteins have attracted increasing attention recently due to their role in the assembly of membraneless organelles or granules by liquid-liquid phase separation. Several examples of such granules have been shown to sequester RNA and proteins in an inactive state, providing an important mechanism for dynamic post-transcriptional gene regulation. In trypanosome parasites, post-transcriptional control overwhelmingly dominates gene regulation due to the organisation of their genome into polycistronic transcription units. The purpose of the current study was to generate a substantially more comprehensive genome-wide survey of LCRs on trypanosome proteins than currently available . Methods: Using the Shannon's entropy method, provided in the R package 'entropy', we identified LCRs in the proteome of Trypanosoma brucei. Our analysis predicts LCRs and their positional enrichment in distinct protein cohorts and superimposes on this a range of post-translational modifications derived from available experimental datasets. Results: We have identified 8162 LCRs present on 4914 proteins, representing 42% of the proteome, placing Trypanosoma brucei among the eukaryotes with the highest percentage of LCRs . Our results highlight the enrichment of LCRs in the C-terminal region of predicted nucleic acid binding proteins, these acting as favoured sites for potential phosphorylation. Phosphorylation represents 51% of the post-translational modifications present on LCRs compared to 16% on the rest of the proteome. Conclusions: The post-translational modifications of LCRs, and in particular phosphorylation events, could contribute to post-transcriptional gene expression control and the dynamics of protein targeting to membraneless organelles in kinetoplastid parasites.

A global analysis of low-complexity regions in the Trypanosoma brucei proteome reveals enrichment in the C-terminus of nucleic acid binding proteins providing potential targets of phosphorylation

Background: Low-complexity regions (LCRs) on proteins have attracted increasing attention recently due to their role in the assembly of membraneless organelles or granules by liquid-liquid phase separation. Several examples of such granules have been shown to sequester RNA and proteins in an inactive state, providing an important mechanism for dynamic post-transcriptional gene regulation. In trypanosome parasites, post-transcriptional control overwhelmingly dominates gene regulation due to the organisation of their genome into polycistronic transcription units. The purpose of the current study was to generate a substantially more comprehensive genome-wide survey of LCRs on trypanosome proteins than currently available . Methods: Using the Shannon’s entropy method, provided in the R package ‘entropy’, we identified LCRs in the proteome of Trypanosoma brucei. Our analysis predicts LCRs and their positional enrichment in distinct protein cohorts and superimposes on this a range of post-translational modifications derived from available experimental datasets. Results: We have identified 8162 LCRs present on 4914 proteins, representing 42% of the proteome, placing Trypanosoma brucei among the eukaryotes with the highest percentage of LCRs . Our results highlight the enrichment of LCRs in the C-terminal region of predicted nucleic acid binding proteins, these acting as favoured sites for potential phosphorylation. Phosphorylation represents 51% of the post-translational modifications present on LCRs compared to 16% on the rest of the proteome. Conclusions: The post-translational modifications of LCRs, and in particular phosphorylation events, could contribute to post-transcriptional gene expression control and the dynamics of protein targeting to membraneless organelles in kinetoplastid parasites.

A global analysis of low-complexity regions in the Trypanosoma brucei proteome reveals enrichment in the C-terminus of nucleic acid binding proteins providing potential targets of phosphorylation

Background: Low-complexity regions (LCRs) on proteins have attracted increasing attention recently due to their role in the assembly of membraneless organelles or granules by liquid-liquid phase separation. Several examples of such granules have been shown to sequester RNA and proteins in an inactive state, providing an important mechanism for dynamic post-transcriptional gene regulation. In trypanosome parasites, post-transcriptional control overwhelmingly dominates gene regulation due to the organisation of their genome into polycistronic transcription units. The purpose of the current study was to generate a substantially more comprehensive genome-wide survey of LCRs on trypanosome proteins than currently available. Methods: Using the Shannon’s entropy method, provided in the R package ‘entropy’, we identified LCRs in the proteome of Trypanosoma brucei. Our analysis predicts LCRs and their positional enrichment in distinct protein cohorts and superimposes on this a range of post-translational modifications derived from available experimental datasets. Results: Our results highlight the enrichment of LCRs in the C-terminal region of predicted nucleic acid binding proteins, these acting as favoured sites for potential phosphorylation. Conclusions: The post-translational modifications of LCRs, and in particular the phosphorylation events, could contribute to post-transcriptional gene expression control and the dynamics of protein targeting to membraneless organelles in kinetoplastid parasites.

Hsp27 chaperones FUS phase separation under the modulation of stress-induced phosphorylation.

Protein phase separation drives the assembly of membraneless organelles, but little is known about how these membraneless organelles are maintained in a metastable liquid- or gel-like phase rather than proceeding to solid aggregation. Here, we find that human small heat-shock protein 27 (Hsp27), a canonical chaperone that localizes to stress granules (SGs), prevents FUS from undergoing liquid-liquid phase separation (LLPS) via weak interactions with the FUS low complexity (LC) domain. Remarkably, stress-induced phosphorylation of Hsp27 alters its activity, leading Hsp27 to partition with FUS LC to preserve the liquid phase against amyloid fibril formation. NMR spectroscopy demonstrates that Hsp27 uses distinct structural mechanisms for both functions. Our work reveals a fine-tuned regulation of Hsp27 for chaperoning FUS into either a polydispersed state or a LLPS state and suggests an essential role for Hsp27 in stabilizing the dynamic phase of stress granules.

Clinical and neuropathological features of ALS/FTD with TIA1 mutations

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) represent a disease continuum with common genetic causes and molecular pathology. We recently identified mutations in the T-cell restricted intracellular antigen-1 (TIA1) protein as a cause of ALS +/− FTD. TIA1 is an RNA-binding protein containing a low complexity domain (LCD) that promotes the assembly of membrane-less organelles, such as stress granules (SG). Whole exome sequencing of two family members with fALS/FTD revealed a novel missense mutation in the TIA1 LCD (P362L). Subsequent screening identified five more TIA1 mutations in six additional ALS patients, but none in controls. All mutation carriers presented with weakness, behavioral abnormalities or language impairments and had a final diagnosis of ALS +/− FTD. Autopsy on five TIA1 mutation carriers showed widespread neurodegeneration with TDP-43 pathology. Round eosinophilic inclusions in lower motor neurons were a consistent feature. Cellular assays revealed abnormal SG dynamics in the presence of TIA1 mutations. In summary, missense mutations in the LCD of TIA1 are a newly recognized cause of ALS/FTD with TDP-43 pathology and strengthen the role of RNA metabolism in the pathogenesis in this disease.

Optochemical Control of Protein Localization and Activity within Cell-like Compartments.

We report inducible dimerization strategies for controlling protein positioning, enzymatic activity, and organelle assembly inside synthetic cell-like compartments upon photostimulation. Using a photocaged TMP-Haloligand compound, we demonstrate small molecule and light-induced dimerization of DHFR and Haloenzyme to localize proteins to a compartment boundary and reconstitute tripartite sfGFP assembly. Using photocaged rapamycin and fragments of split TEV protease fused to FRB and FKBP, we establish optical triggering of protease activity inside cell-size compartments. We apply light-inducible protease activation to initiate assembly of membraneless organelles, demonstrating the applicability of these tools for characterizing cell biological processes in vitro. This modular toolkit, which affords spatial and temporal control of protein function in a minimal cell-like system, represents a critical step toward the reconstitution of a tunable synthetic cell, built from the bottom up.

Neuronal Synapses: Microscale Signal Processing Machineries Formed by Phase Separation?

The organization principles underlying non-membrane-bound organelles have started to unravel in the past 10 years. A new biophysical model known as biomolecular condensates has been proposed to explain many aspects of membraneless organelle assembly and regulation. Neurons are extremely complex, and each neuron can contain tens of thousands of synapses, building an extensive neuronal circuit. Intriguingly, neuronal synapses are characterized by specialized compartmentalization, where highly enriched supramolecular complexes are semi-membrane-enclosed into submicrometer-sized signal processing compartments. Recent findings have demonstrated that this postsynaptic density may be driven by phase separation, and an increasing number of studies of membraneless compartments have shed light on the important molecular features shared by these organelles. Here, we discuss the unique morphology and composition of synapses and consider how synaptic assembly might be driven by phase separation. Understanding the molecular behavior of this semi-membrane-bound compartment could ultimately help to explain the mechanistic details underlying synaptic transmission and plasticity, as well as the numerous brain disorders caused by synaptic defects.

C9orf72 Dipeptide Repeats Impair the Assembly, Dynamics, and Function of Membrane-Less Organelles

Expansion of a hexanucleotide repeat GGGGCC (G4C2) in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Transcripts carrying (G4C2) expansions undergo unconventional, non-ATG-dependent translation, generating toxic dipeptide repeat (DPR) proteins thought to contribute to disease. Here, we identify the interactome of all DPRs and find that arginine-containing DPRs, polyGly-Arg (GR) and polyPro-Arg (PR), interact with RNA-binding proteins and proteins with low complexity sequence domains (LCDs) that often mediate the assembly of membrane-less organelles. Indeed, most GR/PR interactors are components of membrane-less organelles such as nucleoli, the nuclear pore complex and stress granules. Genetic analysis in Drosophila demonstrated the functional relevance of these interactions to DPR toxicity. Furthermore, we show that GR and PR altered phase separation of LCD-containing proteins, insinuating into their liquid assemblies and changing their material properties, resulting in perturbed dynamics and/or functions of multiple membrane-less organelles.