bluuebunny/crossref_metadata_2025_split huggingface dataset:
Epsin1 enforces a condensation-dependent checkpoint for ubiquitylated cargo during clathrin-mediated endocytosis
Authors: Sarkar Susovan , Liu Hao-Yang , Yuan Feng , Malady Brandon T. , Wang Liping , Perez Jessica , Lafer Eileen M. , Huibregtse Jon M. , Stachowiak Jeanne C. 
Published: February 2025

AbstractClathrin-mediated endocytosis internalizes proteins and lipids from the cell surface, supporting nutrient uptake, signaling, and membrane trafficking. Recent work has demonstrated that a flexible, liquid-like network of initiator proteins is responsible for catalyzing assembly of clathrin-coated vesicles in diverse organisms including yeast, mammals, and plants. How do cells regulate the assembly of this dynamic network to produce cargo-loaded vesicles? Here we reveal the ability of an endocytic adaptor protein, Epsin1, to conditionally stabilize the initiator protein network, creating a cargo-dependent checkpoint during clathrin-mediated endocytosis. Epsin1 is known to recruit ubiquitylated transmembrane proteins to endocytic sites. Usingin vitroassays, we demonstrate that Epsin1 uses competitive binding and steric repulsion to destabilize condensation of initiator proteins in the absence of ubiquitin. However, when polyubiquitin is present, Epsin1 binds to both ubiquitin and initiator proteins, creating attractive interactions that stabilize condensation. Similarly, in mammalian cells, endocytic dynamics and ligand uptake are disrupted by removal of either ubiquitin or Epsin1. Surprisingly, when Epsin1 and ubiquitin are removed simultaneously, endocytic defects are rescued to near wildtype levels, although endocytic sites lose the ability to distinguish between ubiquitylated and non-ubiquitylated cargos. Taken together, these results suggest that Epsin1 tunes protein condensation to ensure the presence of ubiquitylated cargo during assembly of clathrin-coated vesicles. More broadly, these findings illustrate how a balance of attractive and repulsive molecular interactions controls the stability of liquid-like protein networks, providing dynamic control over key cellular events.
Polo-like kinase 1 maintains transcription and chromosomal accessibility during mitosis
Authors: Shen Zhouyuan , Adams Kristin , Moreno Ryan , Lera Robert , Kaufman Emily , Lang Jessica D. , Burkard Mark 
Published: February 2025

AbstractTranscription persists at low levels in mitotic cells and plays essential roles in mitotic fidelity and chromosomal dynamics. However, the detailed regulatory network of mitotic transcription remains largely unresolved. Here, we report the novel role of Polo-like kinase 1 (Plk1) in maintaining mitotic transcription. Using 5-ethynyl uridine (5-EU) labeling of nascent RNAs, we found that Plk1 inhibition leads to significant downregulation of nascent transcription in prometaphase cells. Chromatin-localized Plk1 activity is required for transcription regulation and mitotic fidelity. Plk1 sustains global chromosomal accessibility in mitosis, especially at promoter and transcription start site (promoter-TSS) regions, facilitating transcription factor binding and ensuring proper transcriptional activity. We identified SMC4, a common subunit of condensin I and II, as a potential Plk1 substrate. Plk1 activity is fundamental to these processes across non-transformed and transformed cell lines, underscoring its critical role in cell cycle regulation. This study elucidates a novel regulatory mechanism of global mitotic transcription, advancing our understanding of cell cycle control.Significance StatementCells retain a low level of transcription during mitosis, while the regulatory network and specific contributions of mitotic transcription are not well understood.We identify Polo-like kinase 1 (Plk1) as a novel regulator of mitotic transcription, crucial for chromosome condensation, genome accessibility, and maintaining mitotic fidelity.This study enhances our understanding of Plk1’s multifaceted role in mitotic progression, advancing cell cycle regulation knowledge, and informing new cancer therapies’ development.
3D markerless tracking of speech movements with submillimeter accuracy
Authors: Lovell Austin , Liu James , Borovsky Arielle , Yeh Raymond A. , Kim Kwang S. 
Published: February 2025

AbstractSpeech movements are highly complex and require precise tuning of both spatial and timing of oral articulators to support intelligible communication. These properties also make measurement of speech movements challenging, often requiring extensive physical sensors placed around the mouth and face that are not easily tolerated by certain populations such as young children. Recent progress in machine learning-based markerless facial landmark tracking technology demonstrated its potential to provide lip tracking without the need for physical sensors, but whether such technology can provide submillimeter precision and accuracy in 3D remains unknown. Moreover, it is also unclear whether such technology can be applied to track speech movements in young children. Here, we developed a novel approach that integrates Shape Preserving Facial Landmarks with Graph Attention Networks (SPIGA), a facial landmark detector, and CoTracker, a transformer-based neural network model that jointly tracks dense points across a video sequence. We further examined and validated this novel approach by assessing its tracking precision and accuracy. The findings revealed that our approach that integrates SPIGA and CoTracker was more precise (≈ 0.15 mm in standard deviation) than SPIGA alone (≈ 0.35 mm). In addition, its 3D tracking performance was comparable to electromagnetic articulography (≈ 0.29 mm RMSE against simultaneously recorded articulograph data). Importantly, the approach performed similarly well across adults and young children (i.e., 3- and 4-year-olds). Because our framework is built upon open-source pretrained models that are fully trained, it promotes accessibility and open science while saving computing resources. Furthermore, given that this framework combines a landmark detection model (SPIGA) with a tracker model (CoTracker) to improve precision/accuracy, our novel approach serves as a proof-of-concept for enhancing the performance of a wide variety of commonly used markerless tracking applications in biology and neuroscience.Author summaryIn this work, we examined whether machine learning based markerless tracking is feasible for tracking 3D lip movements in adults and young children. We developed a novel approach that integrates a landmark detection model (SPIGA) with a tracker model (CoTracker). Our combined CoTracker-based approach demonstrated submillimeter precision and accuracy desired for speech kinematic recording. In addition, our approach does not involve training and validation for each population (e.g., young children vs. adults), saving time and computing resources. We foresee that the proposed general framework of fusing a landmark detection model with a tracker model can be generalized for a wide variety of tracking applications in biology and neuroscience that require high precision and accuracy, including studying cell behaviors, animal motions, and other types of human movements.
p70S6 kinase-dependent phosphorylation of the μ2 subunit of the AP2 adaptor complex is needed for clathrin-mediated endocytosis
Authors: Tempes Aleksandra , Brzozowska Agnieszka , Węgierski Tomasz , Olek Katarzyna , Jastrzębski Kamil , Liszewska Ewa , Misztal Katarzyna , Machnicka Katarzyna , Macias Matylda , Szybińska Aleksandra , Sitkiewicz Ewa , Gozdz Agata , Wróbel Antoni G. , Miaczynska Marta , Pokrzywa Wojciech , Jaworski Jacek , Malik Anna R 
Published: February 2025

AbstractClathrin-mediated endocytosis (CME) is a process in which ligands and their corresponding receptors at the cell surface are internalized via clathrin-coated invaginations of the plasma membrane. Both clathrin and endocytic cargo are recruited to the clathrin-coated pit by the adaptor protein complex AP2. AP2 resides in the cytoplasm in the closed conformation and opens upon interacting with the plasma membrane. Effective CME requires both pools of AP2, open and closed, and effective transition between these states but the mechanisms regulating this transition are only partially understood. Here, we report that serine 45 (S45) of the µ2 subunit of the AP2 complex is phosphorylated in a p70S6 kinase-dependent manner in HeLa cells both with hyperactivated mTOR signaling and under basal conditions. We demonstrate that the loss of S45-µ2 phosphorylation results in decreased internalization of canonical CME cargo such as transferrin and PDGF receptors. InCaenorhabditis elegans, the absence of S45-µ2 phosphorylation produces the dumpy phenotype characteristic of the loss of function of AP2. Our live imaging experiments further suggest that these phenotypes might arise because S45-μ2 phosphorylation is needed for conformational changes of the AP2 complex. These findings uncover a mechanism central to CME control and extend our knowledge on the role of post-translational modifications of AP2 components in regulating the function of this complex.
Live cell GLUT4 translocation assay revealsPer3as a novel regulator of circadian insulin sensitivity in skeletal muscle cells
Authors: Sivasengh Rashmi , Scott Andrew , Gabriel Brendan M. 
Published: February 2025

AbstractType 2 Diabetes (T2D) is a growing global health concern, with skeletal muscle playing a central role in its pathology due to its role in postprandial glucose disposal. Insulin resistance in skeletal muscle often precedes T2D diagnosis and is linked to impaired GLUT4 trafficking. Additionally, circadian disruptions are increasingly recognized as contributors to metabolic dysfunction. We have shown that skeletal muscle cells from T2D donors exhibit disrupted CLOCK/BMAL1 rhythms, impairing mitochondrial oxidative function. To investigate circadian insulin sensitivity, we developed a high-throughput, live-cell assay for GLUT4 translocation, allowing us to explore novel regulators of metabolic rhythmicity in skeletal muscle.In this study, we analysed circadian insulin sensitivity in skeletal muscle using transcriptomics, PheWAS, and GLUT4-HiBiT assays. L6 and human myocytes were cultured, transfected, and assessed via our novel live-cell, circadian GLUT4 translocation assay. Circadian rhythms were analysed (JTK_CYCLE), and siRNA knockdowns targeted key genes, with glucose uptake measured using the Uptake-Glo assay in human myocytes.Skeletal muscle cells from individuals with T2D exhibited disrupted circadian rhythms, with altered rhythmicity inARNTL, HOXB5, PER3, andTSSK6(p<0.05). Public database analysis revealed significant associations between these genes and T2D (p=0.0001–0.03). Our high-sensitivity, live-cell assay demonstrated a 120% increase in GLUT4 translocation at 40 nM insulin (p<0.001). Circadian rhythmicity in GLUT4 translocation was evident at physiological insulin levels (5 & 30 nM).PER3/Per3knockdown significantly reduced GLUT4 translocation (p<0.001) and glucose uptake in human muscle cells (p<0.01), whileArntlknockdown caused a moderate reduction (p<0.01) in GLUT4 translocation.Per3silencing also abolished circadian rhythmicity in GLUT4 translocation (p<0.01).These findings establishPER3/Per3as a key regulator of circadian GLUT4 translocation and insulin sensitivity, linking its dysregulation to metabolic impairments in T2D. Additionally, this study presents a novel live-cell GLUT4 translocation assay that is highly sensitive and capable of performing time-course or circadian experiments in the same group of cells. The sensitivity of this assay, coupled with reduced variation that is possible from using live-cells in time-course assays make it a powerful new tool for the discovery of GLUT4 translocation regulators. In this study, we have identifiedPer3as a novel regulator of GLUT4 translocation in skeletal muscle cells.
Inducible degradation of endogenous proteins by AlissAID system and development of a photoactivatable inducer
Authors: Ogawa Yoshitaka , Nishimura Kohei , Nambo Masakazu , Tsuchiya Yuichiro , Oka Sanae , Fujimori Toshihiko , Ichihara Kazuya , Matsumoto Akinobu , Obara Keisuke , Kamura Takumi 
Published: February 2025

AbstractProtein analysis strategies involving targeted protein degradation are powerful approaches to determine gene functions. Auxin-inducible degron (AID) is among the most widely used methods for target protein knockdown. This system enables the rapid depletion of AID-tagged target proteins in an auxin-dependent manner. Various improved AID methods have been developed to date; however, the requirement to tag the target protein remains a common challenge. Here, we demonstrated the efficiency of the affinity-linker-based super-sensitive AID system for condition-knockdown of target proteins in cultured animal cells and mouse embryos. This system combines the improved AID method with small-molecule antibodies, enabling the control of GFP and mCherry fusion proteins. Additionally, this system can be used to degrade endogenous targets, such as Ras proteins. We also developed a novel inducer, caged 5-adamantyl-IAA, that precisely controlled targeted protein degradation under light irradiation. This advanced technique aids in the degradation of endogenous proteins of interest and can be used to develop new technologies for localized protein degradation.
Crosstalk between Rac and Rap GTPases in migrating cells
Authors: Lüttig Ricarda , Nanda Suchet , Chandran Haritha T. , Dehmelt Leif 
Published: February 2025

AbstractTo enable effective cell migration, local cell protrusion has to be coordinated with local cell attachment. Here, we investigate spatio-temporal activity patterns of key regulators of cell protrusion and adhesion, the small GTPases Rac and Rap, in migrating cells. These analyses show that Rac activity correlates very tightly with instantaneous cell protrusion events, while the Rap activity stays elevated for prolonged time periods after protrusion and is also detectable before cell protrusion. Direct analysis of activity crosstalk in living cells via light-based perturbation methods revealed that Rap can efficiently activate Rac, however, reciprocal crosstalk from Rac to Rap was not detectable. These findings suggest that Rap plays an instructive role in the generation of cell protrusions by its ability to activate Rac. Furthermore, prolonged Rap activity suggests that this molecule also plays a role in maintenance or stabilization of cell protrusions. Indeed, morphological analysis of Rap1-depleted A431 cells revealed a significant reduction of the cell attachment area, suggesting that Rap stimulated cell adhesion might indeed stabilize newly formed protrusions. Taken together, our study suggests a mechanism, by which cell protrusion is coupled to cell adhesion via unidirectional crosstalk that connects the activity of the small GTPases Rap and Rac.
LDL receptor-mediated endocytosis ofEscherichia coliα-hemolysin mediates renal epithelial toxicity
Authors: Kuhn Hunter W. , Smither Madeleine R. , Jin Rachel J. , Collins Christina A. , Ma Hongming , Sina Jason , Gaut Joseph P. , Diamond Michael S. , Hunstad David A. 
Published: February 2025

ABSTRACTPore-forming toxins (PFTs) are secreted bacterial effector molecules that disrupt host cell membranes. The α-hemolysin (HlyA) of uropathogenicEscherichia coli(UPEC) can exert damage to various mammalian cell types. While a candidate toxin receptor (CD11a/CD18 [LFA-1] integrin) exists on myeloid cells, the mechanism of HlyA cytotoxicity to epithelial cells remains undefined. We show that HlyA secretion by UPEC exacerbates renal tubular epithelial injury during ascending pyelonephritis in mice. A CRISPR-Cas9 loss-of-function screen in renal collecting duct cells identified clathrin-mediated endocytosis as required for HlyA cytotoxicity. HlyA internalization induces lysosomal permeabilization, facilitating protease release, cytoplasmic acidification, and mitochondrial dysfunction leading to rapid cell death. This mechanism contrasts with the described actions of other PFTs (plasma membrane poration and osmotic cytolysis). We also identify the low-density lipoprotein receptor (LDLR) as an epithelial receptor for HlyA; genetic or competitive inhibition of the HlyA-LDLR interaction prevented cytotoxicity. Our studies define a new mechanism of action for HlyA, in which its toxicity to epithelial cells requires LDLR-mediated, clathrin-dependent internalization. These results suggest therapeutic avenues for mitigating HlyA-induced damage duringE. coliinfections.
Structure of endogenous Pfs230:Pfs48/45 in complex with potent malaria transmission-blocking antibodies
Authors: Bekkering Ezra T. , Yoo Randy , Hailemariam Sophia , Heide Fabian , Ivanochko Danton , Jackman Matthew , Proellochs Nicholas I. , Stoter Rianne , Wanders Oscar T. , van Daalen Renate C. , Inklaar Maartje R. , Andrade Carolina M. , Jansen Pascal W.T.C. , Vermeulen Michiel , Bousema Teun , Rubinstein John L. , Kooij Taco W.A. , Jore Matthijs M. , Julien Jean-Philippe 
Published: February 2025

SummaryThe Pfs230:Pfs48/45 complex is essential for malaria parasites to infect mosquitoes and forms the basis for current leading transmission-blocking vaccine candidates, yet little is known about its molecular assembly. Here, we used cryogenic electron microscopy to elucidate the structure of the endogenous Pfs230:Pfs48/45 complex bound to six potent transmission-blocking antibodies. Pfs230 consists of multiple domain clusters rigidified by interactions mediated through insertion domains. Membrane-anchored Pfs48/45 forms a disc-like structure and interacts with a short C-terminal peptide on Pfs230 that is critical for Pfs230 membrane-retentionin vivo. Analyses of Pfs48/45- and Pfs230-targeted antibodies identify conserved epitopes on the Pfs230:Pfs48/45 complex and provides a structural paradigm for complement-dependent activity of Pfs230-targeting antibodies. Altogether, the Pfs230:Pfs48/45 antibody-complex structure presented improves our understanding of malaria transmission biology and the mechanisms of action of transmission-blocking antibodies, informing the development of next-generation transmission-blocking interventions.
DNA damage checkpoints balance a tradeoff between diploid- and polyploid-derived arrest failures
Authors: Fujimaki Kotaro , Jambhekar Ashwini , Lahav Galit 
Published: February 2025

SUMMARYThe DNA damage checkpoint system ensures genomic integrity by preventing the division of damaged cells. This system operates primarily through the G1/S and G2/M checkpoints, which are susceptible to failure; how these checkpoints coordinate quantitatively to ensure optimal cellular outcomes remains unclear. In this study, we exposed non-cancerous human cells to exogenous DNA damage and used single-cell imaging to monitor spontaneous arrest failure. We discovered that cells fail to arrest in two major paths, resulting in two types with distinct characteristics, including ploidy, nuclear morphology, and micronuclei composition. Computational simulations and experiments revealed strengthening one checkpoint reduced one mode of arrest failure but increased the other, leading to a critical tradeoff for optimizing total arrest failure rates. Our findings suggest optimal checkpoint strengths for minimizing total error are inherently suboptimal for any single failure type, elucidating the systemic cause of genomic instability and tetraploid-like cells in response to DNA damageHighlightsArrest-failed cells result from two routes, each leading to a distinct typeThese types differ in nuclear morphology, size, ploidy and micronuclei compositionStrengthening one checkpoint reduces one arrest-failure type but increases the otherTotal error is minimized when checkpoint strengths are suboptimal for each typeeTOCSingle-cell quantitative analyses and computational simulations reveal that the DNA damage checkpoint system balances a tradeoff between reducing arrest failure from diploid cells and from damage-induced polyploid cells. The study suggests that to minimize total arrest failure, the checkpoint strengths need to be sub-optimal for each individual arrest-failure type.
Therapeutic Inhibition of Metalloproteases by Tetracyclines during Infection by Multi-Drug ResistantPseudomonas
Authors: Carr Mary , Chou Sophia R. , Sun Joshua , LaRock Doris L. , LaRock Christopher N. 
Published: February 2025

ABSTRACTIntroductionBoth host and microbial proteases contribute to the destruction of tissue duringPseudomonas aeruginosapulmonary infections. We previously reported that beneficial therapeutic effects of metalloprotease inhibitors inPseudomonas aeruginosapulmonary infections are not only from targeting the host matrix metalloproteases, the intended target, but also from cross-inhibition of the secreted bacterial metalloprotease LasB.MethodsWe used computation modeling and biochemical assays to investigate whether doxycycline and other structurally-related antibiotics of the tetracycline family inhibit thePseudomonasprotease, LasB. Modeling infectionin vitrowith human neutrophils andin vivowith a murine model of pulmonary infection, we investigate the effect of on tetracyclines inhibit LasB activity, bacterial survival, and inflammation.ResultsTetracycline, doxycycline, minocycline, and sanocycline all inhibit proteolysis byPseudomonasLasB. At sub-antimicrobial concentrations, this sensitizes the bacterium killing by neutrophils. During pulmonary infection, tetracyclines significantly reduce pathological inflammation in the lung.ConclusionsOur study shows that tetracycline-family antibiotics, known inhibitors of mammalian matrix metalloproteinases, also inhibit LasB and have beneficial anti-inflammatory activity during infections by extensively drug-resistantP. aeruginosa. By controlling pathological host inflammation, inhibiting bacterial virulence factors, and directly killing bacteria, tetracyclines can have tripartite therapeutic benefits.