Biology subjects

Venkatesan, S.

Publications and source records attributed to Venkatesan, S..

35 records · Page 2Linked to original sources

bioRxiv2023

Enhanced prefrontal nicotinic signaling as evidence of active compensation in Alzheimer's disease models

BackgroundCognitive reserve allows for resilience to neuropathology, potentially through active compensation. Here, we examine ex vivo electrophysiological evidence for active compensation in Alzheimers disease (AD) focusing on the cholinergic innervation of layer 6 in prefrontal cortex. Cholinergic pathways are vulnerable to neuropathology in AD and its preclinical models, and their modulation of deep layer prefrontal cortex is essential for attention and executive function. MethodsWe functionally interrogate cholinergic modulation of prefrontal layer 6 pyramidal neurons in two preclinical models: a compound transgenic AD mouse that permits optogenetically-triggered release of endogenous acetylcholine and a transgenic AD rat that closely recapitulates the human trajectory of AD. We then tested the impact of therapeutic interventions to further amplify the compensated responses and preserve the typical kinetic profile of cholinergic signaling. ResultsIn two AD models, we find a potentially-compensatory upregulation of functional cholinergic responses above non-transgenic controls after onset of pathology. To identify the locus of this enhanced cholinergic signal, we dissect key pre- and post-synaptic components with pharmacological strategies. We identify a significant and selective increase in post-synaptic nicotinic receptor signalling on prefrontal cortical neurons. To probe the additional impact of therapeutic intervention on the adapted circuit, we test cholinergic and nicotinic-selective pro-cognitive treatments. The inhibition of acetylcholinesterase further enhances endogenous cholinergic responses but greatly distorts their kinetics. Positive allosteric modulation of nicotinic receptors, by contrast, enhances endogenous cholinergic responses and retains their rapid kinetics. ConclusionsWe demonstrate that functional nicotinic upregulation occurs within the prefrontal cortex in two AD models. Promisingly, this nicotinic signal can be further enhanced while preserving its rapid kinetic signature. Taken together, our work suggests compensatory mechanisms are active within the prefrontal cortex that can be harnessed by nicotinic receptor positive allosteric modulation, highlighting a new direction for cognitive treatment in AD neuropathology.

neuroscience↗

bioRxiv2023

Cell and tissue-specific glycosylation pathways and transcriptional regulation informed by single-cell transcriptomics

While single cell studies have made significant impacts in various subfields of biology, they lag in the Glycosciences. To address this gap, we analyzed single-cell glycogene expressions in the Tabula Sapiens dataset of human tissues and cell types using a recent glycosylation-specific gene ontology (GlycoEnzOnto). At the median sequencing (count) depth, [~]40-50 out of 400 glycogenes were detected in individual cells. Upon increasing the sequencing depth, the number of detectable glycogenes saturates at [~]200 glycogenes, suggesting that the average human cell expresses about half of the glycogene repertoire. Hierarchies in glycogene and glycopathway expressions emerged from our analysis: nucleotide-sugar synthesis and transport exhibited the highest gene expressions, followed by genes for core enzymes, glycan modification and extensions, and finally terminal modifications. Interestingly, the same cell types showed variable glycopathway expressions based on their organ or tissue origin, suggesting nuanced cell- and tissue-specific glycosylation patterns. Probing deeper into the transcription factors (TFs) of glycogenes, we identified distinct groupings of TFs controlling different aspects of glycosylation: core biosynthesis, terminal modifications, etc. We present webtools to explore the interconnections across glycogenes, glycopathways, and TFs regulating glycosylation in human cell/tissue types. Overall, the study presents an overview of glycosylation across multiple human organ systems.

bioinformatics↗

bioRxiv2023

A fluorescence-based sensor screen identifies MED12 as a potential microsatellite instability regulator in colon cancer

Inactivation of the DNA mismatch repair (MMR) system, due to (epi)genetic alterations of MMR genes, increases the frequency of mutations across the genome, creating a phenotype known as microsatellite instability (MSI). Cancers with this phenotype have been associated with a better prognosis for some time, but only since recently it has been recognised as a predictive biomarker of response to immunotherapy. Because MSI tumours accumulate more insertions and/or deletions in coding regions of the genome containing microsatellites, there is an increase in neoantigens resulting from reading frame shifts, which promotes immunogenicity. To investigate if additional genes exist that can cause an MSI phenotype, we developed a fluorescence-based sensor to identify genes whose inactivation increases the rate of frameshift mutations on microsatellite sequences in cancer cells. Using genome-scale CRISPR/Cas9 screens, we identified MED12 as a potential new regulator of microsatellite instability. Consistent with this, we found that MED12 mutant colon cancers that lack mutations in the known MMR genes are more likely to be of the MSI phenotype.

cancer biology↗

bioRxiv2023

Pathologic polyglutamine aggregation begins with a self-poisoning polymer crystal

A long-standing goal of amyloid research has been to characterize the structural basis of the rate-determining nucleating event. However, the ephemeral nature of nucleation has made this goal unachievable with existing biochemistry, structural biology, and computational approaches. Here, we addressed that limitation for polyglutamine (polyQ), a polypeptide sequence that causes Huntingtons and other amyloid-associated neurodegenerative diseases when its length exceeds a characteristic threshold. To identify essential features of the polyQ amyloid nucleus, we used a direct intracellular reporter of self-association to quantify frequencies of amyloid appearance as a function of concentration, conformational templates, and rational polyQ sequence permutations. We found that nucleation of pathologically expanded polyQ involves segments of three glutamine (Q) residues at every other position. We demonstrate using molecular simulations that this pattern encodes a four-stranded steric zipper with interdigitated Q side chains. Once formed, the zipper poisoned its own growth by engaging naive polypeptides on orthogonal faces, in a fashion characteristic of polymer crystals with intramolecular nuclei. We further show that self-poisoning can be exploited to block amyloid formation, by genetically oligomerizing polyQ prior to nucleation. By uncovering the physical nature of the rate-limiting event for polyQ aggregation in cells, our findings elucidate the molecular etiology of polyQ diseases.

biophysics↗

bioRxiv2023

Protein supersaturation powers innate immune signaling

How minute pathogenic signals trigger decisive immune responses is a fundamental question in biology. Classical signaling often relies on ATP-driven enzymatic cascades, but innate immunity frequently employs death fold domain (DFD) self-assembly. The energetic basis of this assembly is unknown. Here, we show that specific DFDs function as energy reservoirs through metastable supersaturation. Characterizing all 109 human DFDs, we identified sequence-encoded nucleation barriers specifically in the central adaptors of inflammatory signalosomes, allowing them to accumulate far above their saturation concentration while remaining soluble and poised for activation. We demonstrate that the inflammasome adaptor ASC is constitutively supersaturated in vivo, retaining energy that powers on-demand cell death. Swapping a non-supersaturable DFD in the apoptosome with a supersaturable one sensitized cells to sublethal stimuli. Mapping all DFD nucleating interactions revealed that supersaturated adaptors are triggered to polymerize specifically by other DFDs in their respective pathways, limiting potentially deleterious crosstalk. Across human cell types, adaptor supersaturation strongly correlates with cell turnover, implicating this thermodynamic principle in the trade-off between immunity and longevity. Profiling homologues from fish and sponge, we find nucleation barriers to be conserved across metazoa. These findings reveal DFD adaptors as biological phase change materials in thermal batteries to power cellular life-or-death decisions on demand.

immunology↗

bioRxiv2023

Persistence of SARS-CoV-2 and its surrogate, bacteriophage Phi6, on surfaces and in water

The COVID-19 pandemic has motivated research on the persistence of infectious SARS-CoV-2 in environmental reservoirs such as surfaces and water. Viral persistence data has been collected for SARS-CoV-2 and its surrogates, including bacteriophage Phi6. Despite its wide use, no side-by-side comparisons between Phi6 and SARS-CoV-2 exist. Here, we quantified the persistence of SARS-CoV-2 and Phi6 on surfaces (plastic and metal) and in water and evaluated the influence that the deposition solution has on viral persistence by using four commonly used deposition solutions: two culture media (DMEM and Tryptone Soya Broth (TSB)), Phosphate Buffered Saline (PBS), and human saliva. Phi6 remained infectious in water significantly longer than SARS-CoV-2, having a half-life of 27 hours as compared with 15 hours for SARS-CoV-2. The persistence of viruses on surfaces was significantly influenced by the virus used and the deposition solution, but not by the surface material. Phi6 remained infectious significantly longer than SARS-CoV-2 when the inoculation solution was culture media (DMEM, TSB) and saliva. Using culture media and saliva led to half-lives between 9 hours and 2 weeks for Phi6, as compared to 0.5 to 2 hours for SARS-CoV-2. Using PBS as a deposition solution led to half-lives shorter than 4 hours for both viruses on all surfaces. Our results showed that, although it has been frequently used as a surrogate for coronaviruses, bacteriophage Phi6 is not an adequate surrogate for studies quantifying SARS-CoV-2 persistence, as it over-estimates infectiousness. Additionally, our findings reveal the need of using adequate deposition solutions when evaluating viral persistence on surfaces.

microbiology↗

bioRxiv2022

Deficits in integrative NMDA receptors caused by Grin1 disruption can be rescued in adulthood

Glutamatergic NMDA receptors (NMDAR) are critical for cognitive function, and their reduced expression leads to intellectual disability. Since subpopulations of NMDARs exist in distinct subcellular environments, their functioning may be unevenly vulnerable to genetic disruption. Here, we investigate synaptic and extrasynaptic NMDARs on the major output neurons of the prefrontal cortex in mice deficient for the obligate NMDAR subunit encoded by Grin1 and wild-type littermates. With whole-cell recording in brain slices, we find that single, low-intensity stimuli elicit surprisingly-similar glutamatergic synaptic currents in both genotypes. By contrast, clear genotype differences emerge with manipulations that recruit extrasynaptic NMDARs, including stronger, repetitive, or pharmacological stimulation. These results reveal a disproportionate functional deficit of extrasynaptic NMDARs compared to their synaptic counterparts. To probe the repercussions of this deficit, we examine an NMDAR-dependent phenomenon considered a building block of cognitive integration, basal dendrite plateau potentials. Since we find this phenomenon is readily evoked in wild-type but not in Grin1-deficient mice, we ask whether plateau potentials can be restored by an adult intervention to increase Grin1 expression. This genetic manipulation, previously shown to restore cognitive performance in adulthood, successfully rescues electrically-evoked basal dendrite plateau potentials after a lifetime of NMDAR compromise. Taken together, our work demonstrates NMDAR subpopulations are not uniformly vulnerable to the genetic disruption of their obligate subunit. Furthermore, the window for functional rescue of the more-sensitive integrative NMDARs remains open into adulthood.

neuroscience↗

bioRxiv2022

Xanomeline restores endogenous nicotinic acetylcholine receptor signaling in mouse prefrontal cortex

Cholinergic synapses in prefrontal cortex are vital for attention, but this modulatory system undergoes substantial pre- and post-synaptic alterations during adulthood. To examine the integrated impact of these changes, we optophysiologically probe cholinergic synapses ex vivo, revealing a clear decline in neurotransmission in middle adulthood. Pharmacological dissection of synaptic components reveals a selective reduction in postsynaptic nicotinic receptor currents. Other components of cholinergic synapses appear stable, by contrast, including acetylcholine autoinhibition, metabolism, and excitation of postsynaptic muscarinic receptors. Pursuing strategies to strengthen cholinergic neurotransmission, we find that positive allosteric modulation of nicotinic receptors with NS9283 is effective in young adults but wanes with age. To boost nicotinic receptor availability, we harness the second messenger pathways of the preserved excitatory muscarinic receptors with xanomeline. This muscarinic agonist and cognitive-enhancer restores nicotinic signaling in older mice significantly, in a muscarinic- and PKC-dependent manner. The rescued nicotinic component regains youthful sensitivity to allosteric enhancement: treatment with xanomeline and NS9283 restores cholinergic synapses in older mice to the strength, speed, and receptor mechanism of young adults. Our results reveal a new and efficient strategy to rescue age-related nicotinic signaling deficits, demonstrating a novel pathway for xanomeline to restore cognitively-essential endogenous cholinergic neurotransmission.

neuroscience↗

bioRxiv2022

Chrna5 is a marker of acetylcholine super-responder subplate neurons with specialized expression of nicotinic modulator proteins

Attention depends on cholinergic excitation of prefrontal neurons but is sensitive to perturbation of 5-containing nicotinic receptors encoded by Chrna5. However, Chrna5-expressing (Chrna5+) neurons remain enigmatic, despite their potential as a target to improve attention. Here, we generate complex transgenic mice to probe Chrna5+ neurons and their sensitivity to endogenous acetylcholine. Through opto-physiological experiments, we discover that Chrna5+ neurons contain a distinct population of acetylcholine super-responders. Leveraging single-cell transcriptomics, we discover molecular markers conferring subplate identity on this subset. We determine that Chrna5+ super-responders express a unique complement of GPI-anchored lynx prototoxin genes (Lypd1, Ly6g6e, and Lypd6b), predicting distinct nicotinic receptor regulation. To manipulate lynx regulation of endogenous nicotinic responses, we developed a pharmacological strategy guided by transcriptomic predictions. Overall, we reveal Chrna5-Cre mice as a transgenic tool to target the diversity of subplate neurons in adulthood, yielding new molecular strategies to manipulate their cholinergic activation relevant to attention disorders.

neuroscience↗

bioRxiv2022

The coordination between penicillin-binding protein 1a (PBP1a) and the hydrolytic peptidase DacB determines the integrity of bacterial cell poles

Peptidoglycan (PG) defines cell shape and protects bacteria against osmotic stress. The growth and integrity of PG require coordinated actions between synthases that insert new PG strands and hydrolases that generate openings to allow the insertion. However, the mechanisms of their coordination remain elusive. Here we show that moenomycin that inhibits a family of PG synthases known as Class-A penicillin-binding proteins (aPBPs), triggers cell lysis despite aPBPs being non-essential for cell integrity. We demonstrate that inhibited PBP1a2, an aPBP, accelerates the degradation of cell poles by DacB, a hydrolytic PG peptidase, in the bacterium Myxococcus xanthus. Moenomycin reduces the mobility of DacB molecules through PBP1a2, potentially promoting the binding between DacB and PG. Conversely, DacB also regulates the distribution and dynamics of aPBPs. These findings reveal the lethal action of moenomycin and suggest that disrupting the coordination between PG synthases and hydrolases could be more lethal than eliminating individual enzymes.

microbiology↗

bioRxiv2021

The polyglutamine amyloid nucleus in living cells is monomeric and has competing dimensions of order

A long-standing goal of the study of amyloids has been to characterize the structural basis of the rate-determining nucleating event. However, the ephemeral nature of that event has made it inaccessible to classical biochemistry, structural biology, and computational approaches. Here, we addressed that limitation by measuring the dependence of amyloid formation on concentration and conformational templates in living cells, whose volumes are sufficiently small to resolve the outcomes of independent nucleation events. We characterized numerous rationally designed sequence variants of polyglutamine (polyQ), a polypeptide that precipitates Huntingtons and other amyloid-associated neurodegenerative diseases when its length exceeds a characteristic threshold. This effort uncovered a pattern of approximately twelve Qs, only for polypeptides exceeding the clinical length threshold, that allow for amyloid nucleation to occur spontaneously within single polypeptides. Nucleation was inhibited by intermolecular phase separation. Using atomistic molecular dynamics simulations, we found that the pattern encodes a minimal steric zipper of interdigitated side chains. Lateral growth of the steric zipper competed with axial growth to produce "pre-amyloid" oligomers. By illuminating the structural mechanism of polyQ amyloid formation in cells, our findings reveal a potential molecular etiology for polyQ diseases, and may provide a roadmap for the design of new therapies.

biophysics↗

bioRxiv2020

Targeted cancer therapy induces APOBEC fuelling the evolution of drug resistance

Introductory paragraphThe clinical success of targeted cancer therapy is limited by drug resistance that renders cancers lethal in patients1-4. Human tumours can evolve therapy resistance by acquiring de novo genetic alterations and increased heterogeneity via mechanisms that remain incompletely understood1. Here, through parallel analysis of human clinical samples, tumour xenograft and cell line models and murine model systems, we uncover an unanticipated mechanism of therapy-induced adaptation that fuels the evolution of drug resistance. Targeted therapy directed against EGFR and ALK oncoproteins in lung cancer induced adaptations favoring apolipoprotein B mRNA-editing enzyme, catalytic polypeptide (APOBEC)-mediated genome mutagenesis. In human oncogenic EGFR-driven and ALK-driven lung cancers and preclinical models, EGFR or ALK inhibitor treatment induced the expression and DNA mutagenic activity of APOBEC3B via therapy-mediated activation of NF-{kappa}B signaling. Moreover, targeted therapy also mediated downregulation of certain DNA repair enzymes such as UNG2, which normally counteracts APOBEC-catalyzed DNA deamination events. In mutant EGFR-driven lung cancer mouse models, APOBEC3B was detrimental to tumour initiation and yet advantageous to tumour progression during EGFR targeted therapy, consistent with TRACERx data demonstrating subclonal enrichment of APOBEC-mediated mutagenesis. This study reveals how cancers adapt and drive genetic diversity in response to targeted therapy and identifies APOBEC deaminases as future targets for eliciting more durable clinical benefit to targeted cancer therapy.

cancer biology↗

bioRxiv2020

Evolutionary genetics of Drosophila melanogaster immunity: role of the X chromosome and sex-specific dominance

Intralocus Sexual Conflict (IaSC) ensues when males and females of the same species experience divergent selection on shared traits. A large number of traits have been implicated in IaSC and there is growing evidence for sexual antagonism associated with immunity. X chromosomes are thought to be hotspots of sexually antagonistic genetic variation and have been shown to harbour substantial immunity-related genetic variation. Here, using interpopulation crosses and cytogenetic cloning, we investigated the role of the X chromosome in improved immune response of laboratory populations of the fruit-fly Drosophila melanogaster selected against systemic infection by Pseudomonas entomophila. We could not detect any contribution of the X chromosome in the evolved immune response of our selected populations. However, we found strong evidence of sex-specific dominance related to immunity in our populations. Our results indicate that alleles that confer a superior immune response to the selected populations are, on average, partially dominant in females but partially recessive in males. We argue that sex-specific dominance over immunity evolved as a by-product of sexually antagonistic selection in the wild ancestors of our populations. We also highlight sex-specific dominance as a potential mechanism of sex differences in immunity, with population-level sex differences primarily driven by sex differences in heterozygotes.

evolutionary biology↗

bioRxiv2020

Spatiotemporal variation in drivers of parasitism in a wild wood mouse population

O_LIHost-parasite interactions in nature are driven by a range of factors across several ecological scales, so observed relationships are often context-dependent. Importantly, if these factors vary across space and time, practical sampling limitations can limit or bias inferences, and the relative importance of different drivers can be hard to discern. C_LIO_LIWe collected a replicated, longitudinal dataset of >1000 individual wood mice (Apodemus sylvaticus) encompassing 6 years of sampling across 5 different woodland sites to investigate how environmental, host and within-host factors determine infection intensity of a highly prevalent gastrointestinal nematode, Heligmosomoides polygyrus. C_LIO_LIWe used a Bayesian modelling approach to further quantify if and how each factor varied in space and time. Finally, we examined the extent to which a lack of spatially or temporally replication (i.e., within single years or single sites) and single (cross-sectional) versus repeated (longitudinal) sampling of individuals would affect which drivers were found to predict H. polygyrus infection. C_LIO_LISeason, host body condition, and sex were the three most important determinants of infection intensity; however, the strength and even direction of these effects varied in time, but not in space. Additionally, longitudinal datasets, in which we can control for within-individual variation through repeat observations, provided more robust estimates of the drivers of parasite intensity compared to cross-sectional data. C_LIO_LIThese results highlight the importance of accounting for spatiotemporal variation in drivers of disease dynamics and the need to incorporate spatiotemporal replication when designing sampling regimes. Furthermore, they suggest that embracing rather than simply controlling for spatiotemporal variation can reveal important insight into host-parasite relationships in the wild. C_LI

ecology↗

bioRxiv2020

Chrna5 is essential for a rapid and protected response to optogenetic release of endogenous acetylcholine in prefrontal cortex

Optimal attention performance requires cholinergic modulation of corticothalamic neurons in the prefrontal cortex. These pyramidal cells express specialized nicotinic acetylcholine receptors containing the 5 subunit encoded by Chrna5. Disruption of this gene impairs attention, but the advantage 5 confers for the detection of endogenous cholinergic signaling is unknown. To ascertain this underlying mechanism, we used optogenetics to stimulate cholinergic afferents in prefrontal cortex brain slices from compound-transgenic wild-type and Chrna5 knockout mice of both sexes. These electrophysiological experiments identify that Chrna5 is critical for the rapid onset of the postsynaptic cholinergic response. Loss of 5 slows cholinergic excitation and delays its peak, and these effects are observed in two different optogenetic mouse lines. Disruption of Chrna5 does not otherwise perturb the magnitude of the response, which remains strongly mediated by nicotinic receptors and tightly controlled by autoinhibition via muscarinic M2 receptors. However, when conditions are altered to promote sustained cholinergic receptor stimulation, it becomes evident that 5 also works to protect nicotinic responses against desensitization. Rescuing Chrna5 disruption thus presents the double challenge of improving the onset of cholinergic signaling without triggering desensitization. Here, we identify that an agonist for the unorthodox - nicotinic binding site can allosterically enhance this cholinergic pathway considered vital for attention. Minimal NS9283 treatment restores the rapid onset of the postsynaptic cholinergic response without triggering desensitization. Taken together, this work demonstrates the advantages of speed and resilience that Chrna5 confers on endogenous cholinergic signaling, defining a critical window of interest for cue detection and attentional processing. Significance statementThe 5 nicotinic receptor subunit (Chrna5) is important for attention, but its advantage in detecting endogenous cholinergic signals is unknown. Here, we show that 5 subunits permit rapid cholinergic responses in prefrontal cortex and protect these responses from desensitization. Our findings clarify why Chrna5 is required for optimal attentional performance under demanding conditions. To treat the deficit arising from Chrna5 disruption without triggering desensitization, we enhanced nicotinic receptor affinity using NS9283 stimulation at the unorthodox - nicotinic binding site. This approach successfully restored the rapid-onset kinetics of endogenous cholinergic neurotransmission. In summary, we reveal a previously unknown role of Chrna5 as well as an effective approach to compensate for genetic disruption and permit fast cholinergic excitation of prefrontal attention circuits.

neuroscience↗

bioRxiv2019

Supplemented nutrition increases immunity and drug efficacy in a natural host-helminth system

Gastrointestinal helminths are common parasites of humans, wildlife, and livestock, leading to chronic infections in large parts of the world. In humans, there is also an overlap in the incidence of malnutrition and helminth infections which can predispose individuals to higher infection burdens and reduced anthelmintic efficacy due to compromised immunity. This relationship has been well-studied in laboratory models by testing for the impact of dramatic reductions of specific nutrients on infection outcomes. However, much less is known about the benefits of whole-diet supplementation in natural host-helminth systems. We experimentally supplemented the diet of wood mice (Apodemus sylvaticus) and measured resistance to the gastrointestinal nematode Heligmosomoides polygyrus and anthelmintic treatment efficacy in both natural and captive populations. Supplemented wood mice were more resistant to H. polygyrus infection, cleared worms more efficiently after treatment, produced stronger general and parasite-specific antibody responses, and maintained better body condition. In addition, supplemented nutrition in conjunction with anthelminthic treatment significantly reduced H. polygyrus transmission potential. These large-scale improvements in condition and immunity of supplemented nutrition found in controlled and wild environments show the rapid and extensive benefits of a well-balanced diet and have important implications for using diet interventions to improve disease control programmes. Author SummaryGastrointestinal helminths are ubiquitous parasites which cause chronic infections and debilitating symptoms worldwide for human and animal populations alike. Efforts to control helminth infections rely primarily on deworming drugs. However, despite high availability and low cost of these drugs, reinfection is common and helminths remain a substantial problem in many areas of the world. One factor which contributes to the persistence of helminth infections is co-occurrence of malnutrition. Extensive work in model laboratory systems has shown that deficiencies in both macro- and micronutrients can impair host immune responses to helminth infections, but how this relationship translates to natural host-helminth systems and drug efficacy is still poorly understood. Here, we used a combination of experimental nutrition supplementation and deworming treatment in paired wood mouse populations in both wild and captive environments to determine the role of a well-balanced diet for infection control in a natural host-helminth system. We found that mice on a supplemented diet had lower burden of infection and increased drug efficacy, body condition, and immune responses. We also found that supplementation lowered transmission potential of hosts. These broad and rapid effects of increased nutrition quality have important implications for the benefits of diet interventions alongside deworming drugs.

ecology↗

bioRxiv2017

Quinary structure kinetically controls protein function and dysfunction

Protein self-assemblies modulate protein activities over biological time scales that can exceed the lifetimes of the proteins or even the cells that harbor them. We hypothesized that these time scales relate to kinetic barriers inherent to the nucleation of ordered phases. To investigate nucleation barriers in living cells, we developed Distributed Amphifluoric FRET (DAmFRET). DAmFRET exploits a photoconvertible fluorophore, heterogeneous expression, and large cell numbers to quantify via flow cytometry the extent of a proteins self-assembly as a function of cellular concentration. We show that kinetic barriers limit the nucleation of ordered self-assemblies, and that the persistence of the barriers with respect to concentration relates to structure. Supersaturation resulting from sequence-encoded nucleation barriers gave rise to prion behavior, and enabled a prion-forming protein, Sup35 PrD, to partition into dynamic intracellular condensates or to form toxic aggregates. Our results suggest that nucleation barriers govern cytoplasmic inheritance, subcellular organization, and proteotoxicity.\n\nHighlightsO_LIDistributed Amphifluoric FRET (DAmFRET) quantifies nucleation in living cells\nC_LIO_LIDAmFRET rapidly distinguishes prion-like from non-prion phase transitions\nC_LIO_LINucleation barriers allow switch-like temporal control of protein activity\nC_LIO_LISequence-intrinsic features determine the concentration-dependence of nucleation barriers\nC_LI

biophysics↗