Biology subjects

Ly, T.

Publications and source records attributed to Ly, T..

At least 19 recordsLinked to original sources

bioRxiv2026

Community assembly dynamics in a regulated stream: Insights from environmental DNA

Degradation of freshwater habitats, including the alteration of natural flow regimes by impoundment, is a major threat to native fishes. When dam removal is not possible, management actions to recover natural flow regimes can benefit native species, but monitoring community responses is crucial to show efficacy. We used eDNA metabarcoding to quantify spatiotemporal trends in fish assemblages along the longitudinal profile of a regulated stream where a court-ratified environmental flows framework is used to benefit native fishes. At a subset of sites, we also compared eDNA results with concurrent surveys conducted using long-term standardized fisheries monitoring techniques to evaluate concordance. From 59 eDNA samples collected over approximately 2 years, we detected 26 fish taxa, including 8 native species, with 25 taxa resolving to species level using a custom reference sequence database; one taxon could not be resolved between closely related species. Fish assemblages varied predictably along the longitudinal profile of the stream and by season (fall and spring). Diversity indices were higher at downstream sites and in fall, reflecting greater richness of warm-water non-native species. Evenness was similar among sites but shifts in species ranks suggests reordering processes drive spatiotemporal variation in assembly. Gradient forest analysis identified thirteen species associated with spatiotemporal gradients, including both native cold-water species and non-native warm-water species. Overall, eDNA results were concordant with conventional fisheries monitoring but eDNA detected significantly greater species richness through increased detection of rare species. eDNA results align with long-term monitoring that demonstrated assemblage shifts in upper, but not lower stream reaches, and extends the insights into spatial responses, species reordering, and ecology of rare species. These findings demonstrate the value of eDNA as a complementary tool for understanding community assembly dynamics, including responses to stream rehabilitation and ecosystem management.

ecology↗

bioRxiv2026

Reciprocal control of the stomach and intestine by viscerosensory neurons in caudal brainstem

Digestion requires precisely timed transit of food through the gastrointestinal tract. Here we show that emptying of food from the stomach occurs in discrete pulses, separated by pauses that allow the proximal duodenum to clear before the next bolus arrives. By combining X-ray fluoroscopy of the gut with calcium imaging of the brainstem, we identify a population of neurons defined by expression of neuropeptide FF (NPFF) that tracks these duodenal dynamics. Stimulation of NPFF neurons induces a coordinated response that inhibits gastric emptying and accelerates intestinal transit, thereby clearing the proximal intestine for the next bolus of food. These neurons also bidirectionally control food intake through cholinergic signaling in the gut. These findings reveal a mechanism by which brainstem transforms the sensory detection of nutrients in the duodenum into the opposing control of the stomach and intestine. They also establish an experimental approach for dissecting gut-brain signaling in behaving animals.

neuroscience↗

bioRxiv2026

Meiotic kinetochore reconstitution identifies chromosome segregation factors

Kinetochores are complex protein assemblies at centromeres that direct and regulate chromosome segregation1. During gamete formation by meiosis, kinetochore specialisations drive two sequential and distinct segregation events to partition half the genome to the offspring2,3. Age-dependent segregation errors in human oocytes are a major cause of reproductive failure and are linked to kinetochore dysfunction4-6, however the molecular composition and function of mammalian meiotic centromeric chromatin and kinetochores remains poorly defined. Here, we reconstitute vertebrate meiosis I and II centromeric chromatin using meiosis I and II endogenous proteins to uncover conserved regulators of chromosome segregation in mammalian meiosis. We find that, in addition to canonical kinetochore proteins, a specific subset of chromatin regulators is enriched on meiotic centromeric chromatin. We discover previously unrecognized kinetochore proteins conserved from frogs to mouse and humans and demonstrate their localisation at mammalian oocyte centromeres. Among these, we show that the newly identified centromeric protein HSPBAP1, is required to maintain chromosome integrity and ensure faithful segregation in both mouse and human oocytes. These findings define the composition of vertebrate meiotic kinetochores and uncover conserved factors that safeguard mammalian meiotic chromosome segregation, a highly error-prone process and major cause of aneuploidy and reproductive failure in humans7.

cell biology↗

bioRxiv2025

Cellular coding of ingestion in the caudal brainstem

The termination of a meal is triggered by sensory feedback from the stomach and intestines that reports on ingested food1-10 and is relayed to the caudal nucleus of the solitary tract (cNTS) in the brainstem11-14. This sensory feedback is thought to gradually intensify as a meal progresses, resulting in the progressive activation of cNTS circuits that promote satiety15,16, but this idea has never been tested by recording the single-cell activity of cNTS neurons while animals eat. Here, we have used a preparation for calcium imaging in the caudal brainstem of behaving animals17 to characterize how food ingestion is encoded in the cNTS. We find that when food is delivered directly to the stomach, most cNTS neurons exhibit ramping activation over many minutes that tracks cumulative food consumed and depends on canonical gut-brain pathways. However, when the same food is consumed by mouth, this widespread ramping activation is gone, and most cNTS neurons instead exhibit phasic, seconds-timescale responses to oral contact with food. We show that these rapid responses are driven by a combination of mechanical, gustatory and nutritive signals from the mouth and throat and do not require traditional gut-brain pathways, including gut-innervating vagal afferents, although GI feedback modulates their duration. We show that one source of this rapid input is descending projections from the paraventricular hypothalamus, which track ingestion dynamics and are required for proper meal termination. These findings reveal that sensory feedback from the stomach and intestines, which is directly transmitted to the cNTS and thought to be critical for satiety, is not the major driver of cNTS activity during ingestion. Instead, these circuits make extensive use of rapid, pregastric signals that report on the dynamics of behavior.

neuroscience↗

bioRxiv2025

Ingestion-activated CGRP neurons control learning but not satiety

Calcitonin Gene-Related Peptide (CGRP) neurons in the parabrachial nucleus are critical for sickness and malaise but have also been proposed to control non-aversive (rewarding) satiation. How one cell type can coordinate these opposing processes has not been explained. Here we reinvestigate the function of these cells using single-cell imaging as well as optical and chemogenetic manipulations. Contrary to current models, we show that CGRP neurons do not track cumulative food consumption, and their activity is not necessary for meal termination. Instead, we identify two distinct populations of CGRP cells, one of which responds rapidly to appetitive signals during ingestion and the other of which responds slowly to aversive visceral cues. Surprisingly, the ingestion-activated CGRP neurons are important for learning about post-ingestive effects but have little effect on food consumption. This reveals two populations of CGRP neurons that are sequentially engaged during, and responsible for, the distinct stages of post-ingestive learning.

neuroscience↗

bioRxiv2025

CD8 T lymphocytes redeploy embryonic cell cycle control mechanisms to facilitate rapid cell proliferation

Rapid proliferation and expansion of cytotoxic CD8 T lymphocytes is crucial for adaptive immunity against viral infection. CD8 T cell cycles complete cell division cycles in <6 hours, representing a physiological extreme for somatic mammalian cells. Embryonic stem cells also rapidly divide and have been shown to utilize specialized cell cycle control mechanisms that differ from somatic fibroblasts, including subdued periodicity of cyclin proteins. CD8 T cell cycle control remains poorly understood compared to embryonic and other somatic cell types. Here, we tested whether CD8 T cells utilize similar control mechanisms to embryonic stem cells to promote rapid cell cycles. We used mass spectrometry-based proteomics to comprehensively measure protein abundances in G1, S and G2&M phases somatic mouse CD8 T cells, mouse embryonic stem cells (mESC) and mouse fibroblasts (NIH3T3). We isolated cell cycle phases using PRIMMUS, thereby avoiding potential artefacts due to arrest-based synchronization. We discovered striking similarities between mESC and CD8 T cells. Similar to mESC, Cyclin E1 was expressed at high levels and constitutively across the cell cycle in CD8 T cells. Overall, cyclins and cell cycle regulated proteins were present in higher abundance in CD8 T cells and mESC as compared with NIH3T3 cells. Additionally, CD8 T cells express high levels of Emi1/Fbxo5 to promote S-phase entry. Interestingly, Emi1 deletion unexpectedly resulted in changes in markers of CD8 T cell phenotype, suggesting an association between cell cycle control and immune function. Thus, we show several adaptations of cell cycle control of embryonic stem cells are redeployed in somatic T cells to promote rapid cell cycles.

cell biology↗

bioRxiv2025

Anionic polymers stabilize Cas9 ribonucleoprotein nanoparticles to improve direct protein delivery and genome editing efficiencies in plant protoplasts

The application of CRISPR-based genome editing tools in plants is often challenged by low editing efficiencies, requiring most plant editing workflows to proceed through the delivery of pre-formed ribonucleoprotein (RNP) complexes to protoplasts. Here, we report increases in protoplast-based RNP delivery and genome editing efficiencies through the addition of anionic polymers to standard protoplast transfection protocols. We test addition of various polymers and peptides for their ability to increase genome editing efficiencies in both Nicotiana benthamiana and Arabidopsis thaliana protoplasts, by adding these components to standard PEG-based protoplast transfection protocols: i) non-covalent addition of charged polymers, ii) non-covalent addition of amphiphilic peptide A5K, and iii) tyrosinase-mediated covalent conjugation of various relevant peptide motifs directly to the RNP. Incorporation of the amphiphilic peptide A5K or covalent attachment of peptides to the RNP had no positive effect on editing efficiencies. However, we found that addition of anionic polymer polyglutamic acid to standard PEG transfection protocols significantly improved editing efficiencies in both Nicotiana benthamiana and Arabidopsis thaliana protoplasts relative to RNPs alone without negatively impacting protoplast viability. Our results suggest anionic polymers stabilize the RNP and increase the colloidal stability of the protoplast transfection workflow. This simple and straightforward method of stabilizing Cas9 RNPs can be easily adopted by others working on direct protein delivery to plant protoplasts to increase genome editing efficiencies. Key messageThe addition of anionic polymer polyglutamic acid to standard protoplast PEG transfection workflows enhance CRISPR-Cas9-mediated gene editing in plant protoplasts. Our results suggest the mechanism of increased transfection efficiency is due to colloidal stabilization of RNPs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/635105v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1101f0org.highwire.dtl.DTLVardef@964402org.highwire.dtl.DTLVardef@14afe61org.highwire.dtl.DTLVardef@82a982_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2024

A chemical-genetic system to rapidly inhibit the PP2A-B56 phosphatase

AO_SCPLOWBSTRACTC_SCPLOWSerine-threonine phosphatases have been challenging to study because of the lack of specific inhibitors. Their catalytic domains are druggable, but these are shared or very similar between individual phosphatase complexes, precluding their specific inhibition. Instead, phosphatase complexes achieve specificity by interacting with short-linear motifs (SLiMs) in substrates or their binding partners. We develop here a chemical-genetic system to rapidly inhibit these interactions within the PP2A-B56 family. Drug-inducible recruitment of ectopic SLiMs ("directSLiMs") is used to rapidly block the SLiM-binding pocket on the B56 regulatory subunit, thereby displacing endogenous interactors and inhibiting PP2A-B56 activity within seconds. We use this system to characterise PP2A-B56 substrates during mitosis and to identify a novel role for PP2A-B56 in maintaining kinetochore-microtubule attachments at metaphase. The directSLiMs approach can be used to inhibit any other phosphatase, enzyme or protein that uses a critical SLiM-binding interface, providing a powerful strategy to inhibit and characterise proteins once considered ."undruggable".

cell biology↗

bioRxiv2023

Negative feedback control of hunger circuits by the taste of food

The rewarding taste of food is critical for motivating animals to eat, but whether taste has a parallel function in promoting meal termination is not well understood. Here we show that hunger-promoting AgRP neurons are rapidly inhibited during each bout of ingestion by a signal linked to the taste of food. Blocking these transient dips in activity via closed-loop optogenetic stimulation increases food intake by selectively delaying the onset of satiety. We show that upstream leptin receptor-expressing neurons in the dorsomedial hypothalamus (DMHLepR) are tuned to respond to sweet or fatty tastes and exhibit time-locked activation during feeding that is the mirror image of downstream AgRP cells. These findings reveal an unexpected role for taste in the negative feedback control of ingestion. They also reveal a mechanism by which AgRP neurons, which are the primary cells that drive hunger, are able to influence the moment-by-moment dynamics of food consumption.

neuroscience↗

bioRxiv2023

Rewiring of the phosphoproteome executes two meiotic divisions

The cell cycle is ordered by a controlled network of kinases and phosphatases. To generate gametes via meiosis, two distinct and sequential chromosome segregation events occur without an intervening S phase. How canonical cell cycle controls are modified for meiosis is not well understood. Here, using highly synchronous budding yeast populations, we reveal how the global proteome and phosphoproteome changes during the meiotic divisions. While protein abundance changes are limited to key cell cycle regulators, dynamic phosphorylation changes are pervasive. Our data indicate that two waves of cyclin-dependent kinase and Polo (Cdc5Polo) kinase activity drive successive meiotic divisions. These two distinct waves of phosphorylation are ensured by the meiosis-specific Spo13Meikin protein, which rewires the phosphoproteome. Spo13Meikin binds to Cdc5Polo to promote phosphorylation of a subset of substrates in meiosis I containing a newly identified motif, which we define as the Spo13Meikin -Cdc5Polo consensus phosphorylation motif. Overall, our findings reveal that a master regulator of meiosis redirects the activity of a kinase to change the phosphorylation landscape and elicit a developmental cascade.

cell biology↗

bioRxiv2022

Cell overgrowth during G1 arrest triggers an osmotic stress response and chronic p38 activation to promote cell cycle exit

Cell size and the cell cycle are intrinsically coupled and abnormal increases in cell size are associated with senescence. The mechanism by which overgrowth primes cells to exit the cell cycle remains unclear. We investigate this using CDK4/6 inhibitors that arrest cell cycle progression in G0/G1 and are used to treat ER+/HER2-metastatic breast cancer. We demonstrate that long-term CDK4/6 inhibition promotes cellular overgrowth during the G0/G1 arrest, causing widespread proteome remodeling and p38-p53-p21-dependent cell cycle exit. Cell cycle exit is triggered by two waves of p21 induction. First, overgrowth during a G0/G1 arrest induces an osmotic stress response, producing the first wave of p21 induction. Second, when CDK4/6 inhibitors are removed, a fraction of cells escape G0/G1 arrest and enter S-phase where overgrowth-driven replication stress results in a second wave of p21 induction that causes cell cycle withdrawal from G2, or the subsequent G1. This could explain why cellular hypertrophy is associated with senescence and why CDK4/6 inhibitors have long-lasting anti-proliferative effects in patients.

cell biology↗

bioRxiv2022

Cyclin A and Cks1 promote kinase consensus switching to non-proline directed CDK1 phosphorylation

Ordered protein phosphorylation by CDKs is a key mechanism for regulating the cell cycle. How temporal order is enforced in mammalian cells remains unclear. Using a fixed cell kinase assay and phosphoproteomics, we show how CDK1 activity and non-catalytic CDK1 subunits contribute to the choice of substrate and site of phosphorylation. Increases in CDK1 activity alters substrate choice, with intermediate and low sensitivity CDK1 substrates enriched in DNA replication and mitotic functions, respectively. This activity dependence was shared between Cyclin A- and Cyclin B-CDK1. Cks1 has a proteome-wide role as an enhancer of multisite CDK1 phosphorylation. Contrary to the model of CDK1 as an exclusively proline-directed kinase, we show that Cyclin A and Cks1 promote non-proline directed phosphorylation, preferably on sites with a +3 lysine residue. Indeed, 70% of cell cycle regulated phosphorylations, where the kinase carrying out this modification has not been identified, are non-proline directed CDK1 sites.

biochemistry↗

bioRxiv2022

Hypoxia shapes the immune landscape in lung injury promoting inflammation persistence

Acute Respiratory Distress Syndrome (ARDS), an often-fatal complication of pulmonary or systemic inflammation, has no cure. Hypoxemia is a defining feature, yet its impact on inflammation is often neglected. Patients with ARDS are monocytopenic early in the onset of the disease. Endotoxin or Streptococcus pneumoniae acute lung injury (ALI) in the context of hypoxia replicates this finding, through hypoxia-driven suppression of type I interferon signalling. This results in failed lung monocyte-derived interstitial macrophages (IM) niche expansion and unchecked neutrophilic inflammation. Administration of colony stimulating factor 1 (CSF1) rescues the monocytopenia, alters the circulating classical monocyte phenotype in hypoxic endotoxin-driven ALI and enables lung IM population expansion, thus limiting lung injury in endotoxin- and virally-induced hypoxic ALI. Hypoxia directly alters immune dynamics to the detriment of the host and manipulation of this aberrant response offers new therapeutic strategies for ARDS.

cell biology↗

bioRxiv2021

Enteroendocrine cell types that drive food reward and aversion

Animals must learn through experience which foods are nutritious and should be consumed, and which are toxic and should be avoided. Enteroendocrine cells (EECs) are the principal chemosensors in the GI tract, but investigation of their role in behavior has been limited by the difficulty of selectively targeting these cells in vivo. Here we describe an intersectional genetic approach for manipulating EEC subtypes in behaving mice. We show that multiple EEC subtypes inhibit food intake but have different effects on learning. Conditioned flavor preference is driven by release of cholecystokinin whereas conditioned taste aversion is mediated by serotonin and substance P. These positive and negative valence signals are transmitted by vagal and spinal afferents, respectively. These findings establish a cellular basis for how chemosensing in the gut drives learning about food.

physiology↗

bioRxiv2021

Essential role of translation factor eIF5a in cytokine production and cell cycle regulation in primary CD8 T lymphocytes

Translational control adjusts protein production rapidly and facilitates local cellular responses to environmental conditions. Translation can be regulated through sequestration of mRNAs by regulatory proteins or RNAs, but also by the availability of ribosomes and translation factors which enable initiation and elongation of nascent polypeptides. Traditionally initiation of mRNA translation has been considered to be a major translational control point, however, control of peptide elongation can also play a role. Here we show that post-translational modification of the elongation factor, eIF5a, controls translation of subsets of proteins in naive T-cells upon activation. Sequencing of nascent polypeptides indicated that functional eIF5a was required for the production of proteins which regulate T-cell proliferation and effector function. Control of translation in multiple immune cell lineages is required to co-ordinate immune responses and these data illustrate that translational elongation can contribute to post-transcriptional regulons important for the control of inflammation.

molecular biology↗

bioRxiv2021

CDK4/6 inhibitors induce replication stress to cause long-term cell cycle withdrawal

AO_SCPLOWBSTRACTC_SCPLOWCDK4/6 inhibitors arrest the cell cycle in G1-phase. They are approved to treat breast cancer and are also undergoing clinical trials against a range of other tumour types. To facilitate these efforts, it is important to understand why a cytostatic arrest in G1 causes long-lasting effects on tumour growth. Here we demonstrate that a prolonged G1-arrest following CDK4/6 inhibition downregulates replisome components and impairs origin licencing. This causes a failure in DNA replication after release from that arrest, resulting in a p53-dependent withdrawal from the cell cycle. If p53 is absent, then cells bypass the G2-checkpoint and undergo a catastrophic mitosis resulting in excessive DNA damage. These data therefore link CDK4/6 inhibition to genotoxic stress; a phenotype that is shared by most other broad-spectrum anti-cancer drugs. This provides a rationale to predict responsive tumour types and effective combination therapies, as demonstrated by the fact that CDK4/6 inhibition induces sensitivity to chemotherapeutics that also cause replication stress.

cancer biology↗

bioRxiv2020

Cell cycle state proteomics and classification using in-cell protease digests and mass spectrometry

Comprehensive proteome analysis of rare cell phenotypes remains a significant challenge. We report a method for low cell number mass spectrometry (MS)-based proteomics using protease digestion of mildly formaldehyde-fixed cells in cellulo, which we call the in-cell digest. We combined this with AMPL (Averaged MS1 Precursor Library Matching) to quantitatively characterise proteomes from low cell numbers of human lymphoblasts. 4,500 proteins were detected from 2,000 cells and 2,500 proteins were quantitated from 200 lymphoblasts. The ease of sample processing and high sensitivity makes this method exceptionally suited for the proteomic analysis of rare cell states, including immune cell subsets and cell cycle subphases. To demonstrate the method, we characterised the proteome changes across 16 cell cycle states isolated from an asynchronous TK6 human lymphoblast culture, avoiding synchronization. States included late mitotic cells present at extremely low frequency. We identified 119 pseudoperiodic proteins (PsPs) that vary across the cell cycle. Clustering of the PsPs showed abundance patterns consistent with waves of protein degradation in late S, at the G2&M border, mid-mitosis and at mitotic exit. These clusters were distinguished by significant differences in predicted nuclear localization and interaction with the APC/C. The dataset also identifies putative APC/C substrates in mitosis and the temporal order in which they are targeted for degradation. We demonstrate that a protein signature made of these 119 high confidence cell cycle regulated proteins can be used to perform unbiased classification of proteomes into cell cycle states. We applied this signature to 296 proteomes that encompass a range of quantitation methods, cell types, and experimental conditions. The analysis confidently assigns a cell cycle state for 49 proteomes, including correct classification for proteomes from synchronized cells. We anticipate this robust cell cycle protein signature will be crucial for classifying cell states in single cell proteomes.

cell biology↗

bioRxiv2018

Cyclin A triggers Mitosis either via Greatwall or Cyclin B

Two mitotic Cyclins, A and B, exist in higher eukaryotes, but their specialised functions in mitosis are poorly understood. Using degron tags we analyse how acute depletion of these proteins affects mitosis. Loss of Cyclin A in G2-phase prevents the initial activation of Cdk1. Cells lacking Cyclin B can enter mitosis and phosphorylate most mitotic proteins, because of parallel PP2A:B55 phos-phatase inactivation by Greatwall kinase. The final barrier to mitotic establishment corresponds to nuclear envelope breakdown that requires a decisive shift in the balance of Cdk1 and PP2A:B55 activity. Beyond this point Cyclin B/Cdk1 is essential to phosphorylate a distinct subset mitotic Cdk1 substrates that are essential to complete cell division. Our results identify how Cyclin A, B and Greatwall coordinate mitotic progression by increasing levels of Cdk1-dependent substrate phos-phorylation.

cell biology↗