Biology subjects

Ranganathan, S.

Publications and source records attributed to Ranganathan, S..

34 records · Page 2Linked to original sources

bioRxiv2023

Pyro: A Comprehensive Pipeline for Eukaryotic Genome Assembly

1The assembly of reference-quality, chromosome-level genomes for both model and novel eukaryotic organisms is an increasingly achievable task for single research teams. However, the broad variety of sequencing technologies, assembly algorithms, and post-assembly processing tools currently available means that there is no clear consensus on a best-practice computational protocol for eukaryotic de novo genome assembly. An ever-increasing field of algorithms and packages with unique parameters, setup requirements, and environments makes it difficult for groups to pick up and test new tools, despite potential benefits. Here, we present a comprehensive Snakemake-based pipeline for eukaryotic genome assembly, Pyro, to further assist future de novo assembly and benchmarking projects. Pyro combines 20 assembly and eight polishing packages, comprising 30 different assembly approaches and up to 48 different polishing approaches in combination. These are available across Illumina short-read, Nanopore and PacBio CLR long-read technologies in one container, complete with data preparation, quality metric calculation and result reporting. We demonstrate Pyro effectiveness by running Pyro on publicly available Illumina, Nanopore and PacBio CLR read sets for Arabidopsis thaliana, producing 12 candidate assembly options with minimal initial input or configuration, each with extremely high contiguity and completeness. Pyro is highly customizable to expert needs, while also providing an accessible suggested set of tools for more casual users based on simple inputs. Pyro is available as a Singularity container suitable for execution on any Unix-compatible OS, and is freely available on GitHub (https://github.com/genomeassembler/pyro). This pipeline provides a one-stop solution for a variety of de novo eukaryotic genome assembly needs, and will also assist in the assessment of new tools as a convenient benchmark-generating platform.

bioinformatics↗

bioRxiv2022

Benchmarking single-cell hashtag oligo demultiplexing methods

Sample multiplexing is often used to reduce cost and limit batch effects in single-cell RNA sequencing (scRNA-seq) experiments. A commonly used multiplexing technique involves tagging cells prior to pooling with a hashtag oligo (HTO) that can be sequenced along with the cells RNA to determine their sample of origin. Several tools have been developed to demultiplex HTO sequencing data and assign cells to samples. In this study, we critically assess the performance of seven HTO demultiplexing tools: hashedDrops, HTODemux, GMM-Demux, demuxmix, deMULTIplex, BFF and HashSolo. The comparison uses data sets where each sample has also been demultiplexed using genetic variants from the RNA, enabling comparison of HTO demultiplexing techniques against complementary data from the genetic "ground truth". We find that all methods perform similarly where HTO labelling is of high quality, but methods that assume a bimodal counts distribution perform poorly on lower quality data. We also suggest heuristic approaches for assessing the quality of HTO counts in a scRNA-seq experiment.

bioinformatics↗

bioRxiv2022

Identification of novel PGRP3 protein -protein interactions using yeast two hybrid system

BackgroundPeptidoglycan recognition protein-3 (PGRP3) is a pattern recognition receptor that binds peptidoglycan to elicit an immune response. How PGRP3 mediates these immunomodulatory effects remains unknown. Identifying the proteins that interact with PGRP3 will help begin to address this important knowledge gap. Therefore, in this study our objective was to identify and validate novel PGRP3-protein interactions. MethodsPGRP3-protein interactions were identified using a yeast two-hybrid system. PGRP3 cloned into a pGBKT7 DNA-BD vector and transformed into a Y2HGold yeast strain served as the bait. A normalized universal human cDNA library cloned in a pGADT7 AD vector and pre-transformed into an Y187 yeast strain served as the prey. Y2HGold and Y187 yeast strains were mated and diploids were plated on selective media. True positive clones were sent in for DNA sequencing and corresponding proteins were identified through NCBI Blast. Two proteins were selected for validation. His-tagged PGRP3 expressing 293T cells were then co-transfected with expression vectors containing the identified proteins tagged with c-Myc. Lysates from co-transfected cells were subjected to anti-His and anti-Myc co-immunoprecipitation and analyzed with western blots. ResultsFour unique proteins-- MBNL3, RBP5, ATXN2, and GPATCH8--were identified from our yeast two-hybrid screen. Co-transfection and co-immunoprecipitation assays successfully confirmed MBNL3 and RBP5 as positive interactors with PGRP3. ConclusionsBased on the known functions of MBNL3, RBP5, ATXN2, and GPATCH8, we hypothesize that PGRP3 may be involved in RNA processing, endocytic trafficking, and PPAR{gamma} pathways related to innate immunity.

immunology↗

bioRxiv2022

Phosphorylation sites are evolutionary checkpoints against liquid-solid transition in protein condensates

Assemblies of multivalent RNA-binding protein FUS can exist in the functional liquid-like state as well as less dynamic and potentially toxic amyloid- and hydrogel-like states. How could then cells form liquid-like condensates while avoiding their transformation to amyloids? Here we show how post-translational phosphorylation can provide a "handle" that prevents liquid-solid transition of intracellular condensates containing FUS. Using residue-specific coarse-grained simulations, for 85 different mammalian FUS sequences, we show how the number of phosphorylation sites and their spatial arrangement affect intracluster dynamics preventing conversion to amyloids. All atom simulations further confirm that phosphorylation can effectively reduce the {beta}-sheet propensity in amyloid-prone fragments of FUS. A detailed evolutionary analysis shows that mammalian FUS PLDs are enriched in amyloid-prone stretches compared to control neutrally evolved sequences suggesting that mammalian FUS proteins evolved to self-assemble. However, in stark contrast to proteins that do not phase-separate for their function, mammalian sequences have phosphosites in close proximity to these amyloid-prone regions. These results suggest that evolution uses amyloid-prone sequences in prion-like domains to enhance phase-separation of condensate proteins while enriching phosphorylation sites in close proximity to safe-guard against liquid-solid transitions. Significance StatementIntrinsically disordered regions and prion-like domains are widely observed in proteins that get enriched in membrane-less organelles (MLOs). Mammalian Fused in Sarcoma (FUS) sequences are significantly enriched in amyloid-prone sequences suggesting that they have evolved to self-assemble. While the amyloid-prone stretches promote self-assembly of these proteins at lower threshold concentrations, these assemblies are vulnerable to aberrant liquid-solid phase transitions. Molecular simulations and bioinformatics analyses show that evolution overcomes this challenge by placing phosphosites specifically close to amyloid-prone stretches. Introduction of negatively charged residues at phosphosite locations results in fewer amyloid-prone contacts and thereby lower beta-sheet propensity. Phosphorylation can thus allow cells to utilize amyloid-prone stretches to promote biogenesis of MLOs while protecting against liquid-solid transitions.

biophysics↗

bioRxiv2022

A CRISPR/Cas9-based enhancement of high-throughput single-cell transcriptomics

Single-cell transcriptomics suffers from lapses in coverage of the full transcriptome, providing an incomplete gene expression profile of the cell. Here, we introduce single-cell CRISPRclean (scCLEAN), an in vitro molecular method that can be universally inserted into any single-cell RNA-seq workflow to improve the sensitivity of the assay. Utilizing CRISPR/Cas9, scCLEAN works to selectively remove highly abundant uninformative molecules, redistributing ~50% of reads to enrich for lowly expressed transcripts. Utilizing immune cells, we describe a validation of scCLEAN showing a 2.1-fold enrichment in library complexity with negligible off-target effects. Subsequently, applying scCLEAN to single-cell iso-seq samples results in a 4.6-fold improvement in unique isoform detection. Thus, demonstrating a benefit in short and long read sequencing applications. Finally, we illustrate the ability of scCLEAN to elucidate biological insights by applying it to two participant cohorts of cardiovascular samples, bringing to light novel molecular characteristics including inflammatory signatures.

genetics↗

bioRxiv2022

Multimodal single cell analysis of the paediatric lower airway reveals novel immune cell phenotypes in early life health and disease

Inflammation is a key driver of cystic fibrosis (CF) lung disease, not addressed by current standard care. Improved understanding of the mechanisms leading to aberrant inflammation may assist the development of effective anti-inflammatory therapy. Single-cell RNA sequencing (scRNA-seq) allows profiling of cell composition and function at previously unprecedented resolution. Herein, we seek to use multimodal single-cell analysis to comprehensively define immune cell phenotypes, proportions and functional characteristics in preschool children with CF. We analyzed 42,658 cells from bronchoalveolar lavage of 11 preschool children with CF and a healthy control using scRNA-seq and parallel assessment of 154 cell surface proteins. Validation of cell types identified by scRNA-seq was achieved by assessment of samples by spectral flow cytometry. Analysis of transcriptome expression and cell surface protein expression, combined with functional pathway analysis, revealed 41 immune and epithelial cell populations in BAL. Spectral flow cytometry analysis of over 256,000 cells from a subset of the same patients revealed high correlation in major cell type proportions across the two technologies. Macrophages consisted of 13 functionally distinct sub populations, including previously undescribed populations enriched for markers of vesicle production and regulatory/repair functions. Other novel cell populations included CD4 T cells expressing inflammatory IFN/{beta} and NF{kappa}B signalling genes. Our work provides a comprehensive cellular analysis of the pediatric lower airway in preschool children with CF, reveals novel cell types and provides a reference for investigation of inflammation in early life CF.

systems biology↗

bioRxiv2021

TRPA1: A Potential Prognostic Indicator for Oral Squamous Cell Carcinoma

IntroductionTransient receptors are related to oral cancer pain. Previously capsaicin (TRPV1 agonist) was shown to induce cell death in oral cancer cells. We hypothesised that these receptors are present in oral cancer. MethodWe examined the presence of cannabinoid receptors (CB1 and CB2) and targets (TRPV1, TRPA1, CaV 3.1, CaV 3.2, CaV 3.3) via quantitative polymerase chain reaction (qPCR) in oral cancer cells SCC4, SCC9, SCC25, Cal27, and normal oral cell line OKF6. ResultCannabinoid receptors are absent in all the cell lines, while TRPA1 is only present in normal cells, but absent in all the oral cancer cell lines. Voltage-gated calcium channels are present in all the cell lines. Conclusion and Future AspectsTRPA1 could be the possible future prognostic indicator of oral squamous cell carcinoma. Future functionality assays could use precancerous cell lines to follow the loss of TRPA1.

cell biology↗

bioRxiv2021

Emergence of order in condensates composed of multi-valent, multi-domain proteins

Many RNA-binding proteins (RBPs) that assemble into membraneless organelles, have a common architecture including disordered prion-like domain (PLD) and folded RNA-binding domain (RBD). An enrichment of PLD within the condensed phase gives rise to formation, on longer time scales, amyloid-like fibrils (aging). In this study, we employ coarse-grained Langevin dynamics simulations to explore the physical basis for the structural diversity in condensed phases of multi-domain RBPs. We discovered a highly cooperative first order transition between disordered structures and an ordered phase whereby chains of PLD organize in fibrils with high nematic orientational order. An interplay between homo-domain (PLD-PLD) and hetero-domain (PLD-RBD) interactions results in variety of structures with distinct spatial architectures. Interestingly, the different structural phases also exhibit vastly different intra-cluster dynamics of proteins, with diffusion coefficients 5 (disordered structures) to 50 times (ordered structures) lower than that of the dilute phase. Cooperativity of this liquid-solid transition makes fibril formation highly malleable to mutations or post-translational modifications. Our results provide a mechanistic understanding of how multi-domain RBPs could form assemblies with distinct structural and material properties. Significance StatementAssembly of proteins and nucleic acids into dense, liquid-like pockets is associated with several key functions including stress response, gene-regulation, DNA-repair and RNA processing. Several RNA binding proteins such as FUS are known to form liquid-like condensates that progressively harden into more dynamically, solid-like structures, a phenomenon that gets accelerated by disease mutations. In this study, we discover the mechanistic origins of this transition and show that small mutational or posttranslational modifications could result in sharp disorder-order transitions that could characterize accelerated liquid-solid transition in disease mutants.

biophysics↗

bioRxiv2021

A Developmentally Prometastatic Niche to Hepatoblastoma in Neonatal Liver mediated by the Cxcl1/Cxcr2 Axis

Background and RationaleHepatoblastoma (HB) is the most common pediatric liver cancer. Its predominant occurrence in very young children led us to investigating whether the neonatal liver provides a protumorigenic niche to HB development. MethodsHB development was compared between orthotopic transplantation models established in postnatal day 5 and 60 mice (P5Tx and P60Tx models). Single-cell RNA-sequencing was performed using tumor and liver tissues from both models and the top candidate cell types and genes identified are investigated for their roles in HB cell growth, migration, and survival. ResultsWe found that various HB cell lines including HepG2 cells were consistently and considerably more tumorigenic and metastatic in the P5Tx model than in the P60Tx models. Sc-RNAseq of the P5Tx and P60Tx HepG2 models revealed that the P5Tx tumor was more hypoxic and had a larger number of activated hepatic stellate cells (aHSCs) in the tumor-surrounding liver which express significantly higher levels of Cxcl1 than those from the P60Tx model. We found these differences were developmentally present in normal P5 and P60 liver. We showed that the Cxcl1/Cxcr2 axis mediated HB cell migration and was critical to HB cell survival under hypoxia. Treating HepG2 P60Tx model with recombinant CXCL1 protein induced intrahepatic and pulmonary metastasis and CXCR2 knockout in HepG2 cells abolished their metastatic potential in the P5Tx model. Lastly, we showed that in metastatic HB patient tumors there was a similar larger population of aHSCs in the tumor-surrounding liver than in localized tumors, and tumor hypoxia was uniquely associated with HB patient prognosis among pediatric cancers. ConclusionWe demonstrated that the neonatal liver provides a prometastatic niche to HB development via the Cxcl1/Cxcr2 axis.

cancer biology↗

bioRxiv2021

Coordinated Cross-Talk Between the Myc and Mlx Networks in Liver Regeneration and Neoplasia

Background & AimsThe c-Myc (Myc) bHLH-ZIP transcription factor is deregulated in most cancers. In association with Max, Myc controls target genes that supervise metabolism, ribosome biogenesis, translation and proliferation. This "Myc Network" cross-talks with the "Mlx Network", which consists of the Myc-like proteins MondoA and ChREBP and Max-like Mlx. Together, this "Extended Myc Network" regulates both common and distinct genes targets. Here we studied the consequence of Myc and/or Mlx ablation in the liver, particularly those pertaining to hepatocyte proliferation, metabolism and spontaneous tumorigenesis. MethodsWe examined the ability of hepatocytes lacking Mlx (MlxKO) or Myc+Mlx (double KO or DKO) to repopulate the liver over an extended period of time in a murine model of Type I tyrosinemia. We also compared this and other relevant behaviors, phenotypes and transcriptomes of the livers to those from previously characterized MycKO, ChrebpKO and MycKO x ChrebpKO mice. ResultsHepatocyte regenerative potential deteriorated as the Extended Myc Network was progressively dismantled. Genes and pathways dysregulated in MlxKO and DKO hepatocytes included those pertaining to translation, mitochondrial function and non-alcoholic fatty liver disease (NAFLD). The Myc and Mlx Networks were shown to cross-talk, with the latter playing a disproportionate role in target gene regulation. All cohorts also developed NAFLD and molecular evidence of early steatohepatitis. Finally, MlxKO and DKO mice displayed extensive hepatic adenomatosis. ConclusionsIn addition to demonstrating cooperation between the Myc and Mlx Networks, this study revealed the latter to be more important in maintaining proliferative, metabolic and translational homeostasis, while concurrently serving as a suppressor of benign tumorigenesis. SynopsisThe Myc and Mlx Networks exhibit extensive cross-talk and regulate distinct but overlapping sets of transcriptional targets. The current work demonstrates the cooperation between these two Networks in supporting the regenerative capabilities of normal hepatocytes while also revealing that the Mlx Network serves as a suppressor of spontaneous hepatic adenomatosis

cancer biology↗

bioRxiv2021

Role of non-specific interactions in the phase-separation and maturation of macromolecules

Phase separation of biomolecules could be mediated by both specific and non-specific interactions. How the interplay between non-specific and specific interactions along with polymer entropy influences phase separation is an open question. We address this question by simulating self-associating molecules as polymer chains with a short core stretch that forms the specifically interacting functional interface, and longer non-core regions that participate in non-specific/promiscuous interactions. Our results show that the interplay of specific (strength,{epsilon} sp) and non-specific interactions (strength,{epsilon} ns) could result in phase separation of polymers and its transition to solid-like aggregates (mature state). In the absence of{epsilon} ns, the polymer chains do not dwell long enough in the vicinity of each other to undergo phase separation and transition into a mature state. On the other hand, in the limit of strong{epsilon} ns, the assemblies cannot transition into the mature state and form a non-specific assembly, suggesting an optimal range of interactions favoring mature multimers. In the scenario where only a fraction (Nfrac) of the non-core regions participate in attractive interactions, we find that a slight modifications to either{epsilon} ns or Nfrac can result in a dramatically altered self-assembled states. Using a combination of heterogeneous and homogeneous mix of polymers, we establish how this interplay between interaction energies dictates the propensity of biomolecules to find the correct binding partner at dilute concentrations in crowded environments. Author SummaryBiological function relies on the ability of biomolecules to bind to specific interaction partners. In the crowded cellular milieu, the process of biomolecules binding to specific interaction partners to carry out a function is non-trivial. A mere diffusion-limited meeting of interaction partners in space does not ensure biomolecular function. Rather, even when in contact these molecules have to find the correct orientations to carry out function. Further, for dynamic biomolecules such as polymers, the functional configuration is often one of several possible configurations. In this scenario, assuming the functional configuration after binding involves overcoming of a significant entropic barrier. Therefore, the interacting biomolecules have to dwell in contact long enough before they reorganize to find the functional orientation! While functional contacts offer an enthalpic gain, they are often only a small fraction of all protein-protein interactions. Therefore, in this paper, we study the role played by non-functional, promiscuous interactions in shaping the thermodynamics and kinetics of formation of specific interactions. Our results suggest that there exists an optimal range of non-specific interaction strengths which promotes the process of biomolecular complexes finding the functional configurations.

biophysics↗

bioRxiv2021

Effect of RNA on morphology and dynamics of membraneless organelles.

Membraneless organelles (MLOs) are spatiotemporally regulated structures that concentrate multi-valent proteins or RNA, often in response to stress. The proteins enriched within MLOs are often classified as high-valency "scaffolds" or low valency "clients", with the former being associated with a phase-separation promoting role. In this study, we employ a minimal model for P-body components, with a defined protein-protein interaction network, to study their phase-separation at biologically realistic low protein concentrations. Without RNA multivalent proteins can assemble into solid-like clusters only in the regime of high concentration and stable interactions. RNA molecules promote cluster formation in an RNA-length dependent manner, even in the regime of weak interactions and low protein volume fraction. Our simulations reveal that long RNA chains act as super-scaffolds that stabilize large RNA-protein clusters by recruiting low-valency proteins within them while also ensuring functional "liquid-like" turnover of components. Our results suggest that RNA-mediated phase separation could be a plausible mechanism for spatiotemporally regulated phase-separation in the cell.

biophysics↗

bioRxiv2021

A UPF1 variant drives conditional remodeling of nonsense-mediated mRNA decay

The nonsense-mediated mRNA decay (NMD) pathway monitors translation termination to degrade transcripts with premature stop codons and regulate thousands of human genes. Here we show that an alternative mammalian-specific isoform of the core NMD factor UPF1, termed UPF1LL, enables condition-dependent remodeling of NMD specificity. Previous studies indicate that the extension of a conserved regulatory loop in the UPF1LL helicase core confers a decreased propensity to dissociate from RNA upon ATP hydrolysis relative to the major UPF1 isoform, designated UPF1 SL. Using biochemical and transcriptome-wide approaches, we find that UPF1LL overcomes the protective RNA binding proteins PTBP1 and hnRNP L to preferentially bind and down-regulate long 3UTRs normally shielded from NMD. Unexpectedly, UPF1LL supports induction of NMD on new populations of substrate mRNAs in response to activation of the integrated stress response and impaired translation efficiency. Thus, while canonical NMD is abolished by moderate translational repression, UPF1LL activity is enhanced, providing a mechanism to rapidly rewire NMD specificity in response to cellular stress.

molecular biology↗

bioRxiv2020

Genomic diversity and antimicrobial resistance of Haemophilus colonising the airways of young children with cystic fibrosis

Respiratory infection during childhood is a key risk factor in early cystic fibrosis (CF) lung disease progression. Haemophilus influenzae (Hi) and Haemophilus parainfluenzae (Hpi) are routinely isolated from the lungs of children with CF, however little is known about the frequency and characteristics of Haemophilus colonisation in this context. Here, we describe detection, antimicrobial resistance (AMR) and genome sequencing of Hi/Hpi isolated from sputum, cough swab, and bronchoalveolar lavage samples regularly collected from 147 participants aged [≤]12 years enrolled in the Australian Respiratory Early Surveillance Team for Cystic Fibrosis (AREST CF) program. The frequency of colonisation per visit was 4.6% for Hi and 32.1% for Hpi, 80.3% of participants had Hi and/or Hpi detected on at least one visit, and using genomic data we estimate 15.6% of participants had persistent colonisation with the same strain for at least two consecutive visits. Colonising strains were genetically highly diverse and AMR was common, with 52% of Hi and 82% of Hpi displaying resistance to at least one drug. The genetic basis for AMR could be identified in most cases; novel determinants include a new plasmid encoding blaTEM-1 (ampicillin resistance), a new inhibitor-resistant blaTEM allele (augmentin resistance), and previously unreported mutations in chromosomally-encoded genes (pbp3, ampicillin resistance; folA and folP, co-trimoxazole resistance; rpoB, rifampicin resistance). Acquired AMR genes were significantly more common in Hpi than Hi (51% vs 21%, p=0.0107) and were mostly associated with the ICEHin mobile element carrying blaTEM-1, resulting in higher rates of ampicillin resistance in Hpi (73% vs 30%, p=0.0004). The genome data identified six potential instances of Haemophilus transmission between participants, three of which involved participant pairs who attended the clinic on the same day. The high prevalence of Haemophilus colonisation and high burden of antimicrobial use in children with CF likely provides a reservoir for emergence and spread of AMR as well as a source of infections.

microbiology↗

bioRxiv2020

Patient-Derived Mutant Forms of NFE2L2/NRF2 Drive Aggressive Murine Hepatoblastomas

Background and AimsHepatoblastoma (HB), the most common pediatric liver cancer, often bears {beta}-catenin mutations and deregulates the Hippo tumor suppressor pathway. Murine HBs can be generated by co-expressing {beta}-catenin mutants and the constitutively active Hippo effector YAPS127A. Some HBs and other cancers also express mutants of NFE2L2/NRF2 (NFE2L2), a transcription factor that tempers oxidative and electrophilic stress. In doing so, NFE2L2 either suppresses or facilitates tumorigenesis. MethodsWe evaluated NFE2L2s role in HB pathogenesis by co-expressing all combinations of mutant {beta}-catenin, YAPS127A and the patient-derived NFE2L2 mutants L30P and R34P in murine livers. We evaluated growth, biochemical and metabolic profiles and transcriptomes of the ensuing tumors. ResultsIn association with {beta}-catenin+YAPS127A, L30P and R34P markedly accelerated HB growth and generated widespread cyst formation and necrosis, which are otherwise uncommon features. Surprisingly, any two members of the mutant {beta}-catenin-YAPS127A-L30P/R34P triad were tumorigenic, thus directly establishing NFE2L2s oncogenicity. Each tumor group displayed distinct features but shared 22 similarly deregulated transcripts, 10 of which perfectly correlated with survival in human HBs and 17 of which correlated with survival in multiple adult cancers. One highly up-regulated transcript encoded serpin E1, a serine protease inhibitor that regulates fibrinolysis, growth and extracellular matrix. The combination of mutant {beta}-catenin, YAPS127A and Serpin E1, while not accelerating cystogenic tumor growth, did promote the wide-spread necrosis associated with mutant {beta}-catenin-YAPS127A-L30P/R34P tumors. ConclusionsOur findings establish the direct oncogenicity of NFE2L2 mutants and key transcripts, including serpin E1, that drive specific HB features.

cancer biology↗

bioRxiv2019

Liquid-liquid microphase separation leads to formation of membraneless organelles

Proteins and nucleic acids can spontaneously self-assemble into membraneless droplet-like compartments, both in vitro and in vivo. A key component of these droplets are multi-valent proteins that possess several adhesive domains with specific interaction partners (whose number determines total valency of the protein) separated by disordered regions. Here, using multi-scale simulations we show that such proteins self-organize into micro-phase separated droplets of various sizes as opposed to the Flory-like macro-phase separated equilibrium state of homopolymers or equilibrium physical gels. We show that the micro-phase separated state is a dynamic outcome of the interplay between two competing processes: a diffusion-limited encounter between proteins, and the dynamics within small clusters that results in exhaustion of available valencies whereby all specifically interacting domains find their interacting partners within smaller clusters, leading to arrested phase separation. We first model these multi-valent chains as bead-spring polymers with multiple adhesive domains separated by semi-flexible linkers and use Langevin Dynamics (LD) to assess how key timescales depend on the molecular properties of associating polymers. Using the time-scales from LD simulations, we develop a coarse-grained kinetic model to study this phenomenon at longer times. Consistent with LD simulations, the macro-phase separated state was only observed at high concentrations and large interaction valencies. Further, in the regime where cluster sizes approach macro-phase separation, the condensed phase becomes dynamically solid-like, suggesting that it might no longer be biologically functional. Therefore, the micro-phase separated state could be a hallmark of functional droplets formed by proteins with the sticker-spacer architecture. Significance statementMembraneless organells (MO) are ubiquitous in healthy living cells, with an altered state in disease. Their formation is likened to liquid-liquid phase separation (LLPS) between MO-forming proteins. However most models of LLPS predict complete macrophase separation while in reality MOs are small droplets of various sizes, which are malleable to rapid morphological changes. Here we present a microscopic multiscale theoretical study of thermodynamics and kinetics of formation of MO. We show that MOs are long-living dynamic structures formed as a result of arrested macrophase separation. Our study provides a direct link beween the molecular properies of MO-forming proteins and the morphology and dynamics of MO paving a path to rational design and control of MO.

biophysics↗